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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2016 Feb 1;113(7):1889–1894. doi: 10.1073/pnas.1525665113

Myotubes derived from human-induced pluripotent stem cells mirror in vivo insulin resistance

Salvatore Iovino a,b, Alison M Burkart a,b, Laura Warren a,b, Mary Elizabeth Patti a,b, C Ronald Kahn a,b,1
PMCID: PMC4763759  PMID: 26831110

Significance

In this manuscript, we have exploited induced pluripotent stem cells (iPS cells) to generate an in vitro model of human skeletal muscle insulin resistance using cells from patients affected by a monogenic form of insulin resistance (Donohue syndrome/leprechaunism). Following differentiation into skeletal muscle cells, these cells show dramatic defects in insulin signaling, glucose uptake, and glycogen accumulation, as well as insulin-regulated gene expression. To our knowledge, this represents the first use of human iPS cells differentiated into muscle to study diabetes and demonstrates that these cells can robustly reproduce insulin resistance in vitro.

Keywords: iPS cells, insulin resistance, insulin signaling, skeletal muscle, genetic disease

Abstract

Induced pluripotent stem cells (iPS cells) represent a unique tool for the study of the pathophysiology of human disease, because these cells can be differentiated into multiple cell types in vitro and used to generate patient- and tissue-specific disease models. Given the critical role for skeletal muscle insulin resistance in whole-body glucose metabolism and type 2 diabetes, we have created a novel cellular model of human muscle insulin resistance by differentiating iPS cells from individuals with mutations in the insulin receptor (IR-Mut) into functional myotubes and characterizing their response to insulin in comparison with controls. Morphologically, IR-Mut cells differentiated normally, but had delayed expression of some muscle differentiation-related genes. Most importantly, whereas control iPS-derived myotubes exhibited in vitro responses similar to primary differentiated human myoblasts, IR-Mut myotubes demonstrated severe impairment in insulin signaling and insulin-stimulated 2-deoxyglucose uptake and glycogen synthesis. Transcriptional regulation was also perturbed in IR-Mut myotubes with reduced insulin-stimulated expression of metabolic and early growth response genes. Thus, iPS-derived myotubes from individuals with genetically determined insulin resistance demonstrate many of the defects observed in vivo in insulin-resistant skeletal muscle and provide a new model to analyze the molecular impact of muscle insulin resistance.

Insulin resistance is a central feature of type 2 diabetes (T2D), obesity, and metabolic syndrome (1). Insulin action in skeletal muscle plays a particularly important role as mediator of whole-body glucose disposal, and insulin resistance in skeletal muscle has been identified as an early defect in humans in T2D and a major risk factor for development of T2D in genetically susceptible individuals (2–5). However, our understanding of molecular mechanisms underlying human insulin resistance in skeletal muscle and other tissues remains incomplete, because access to muscle and other key insulin-sensitive tissues is limited, especially in preclinical stages of disease.

Induced pluripotent stem cells (iPS cells) represent a unique tool for studying the pathophysiology of human disease (6–8), given their capability to differentiate into multiple cell types from all three embryonic germ layers (8). Furthermore, iPS cells can be derived from human donors at any stage of disease, thus providing unique patient-specific disease models to help elucidate pathways and molecular mechanisms involved in disease pathogenesis.

In previous studies (9), we generated an in vitro model of human insulin resistance by creating iPS cells lines from skin fibroblasts derived from individuals with Donohue syndrome. Donohue syndrome (also called leprechaunism) is caused by homozygous or compound heterozygous mutations in the insulin receptor, resulting in a complex clinical phenotype including marked insulin resistance, failure to thrive, and early death (10–12). iPS cells derived from these patients exhibit severe insulin resistance with defects in insulin signaling, cell proliferation, and gene transcription (9, 13). In the present study, we have taken advantage of our ability to differentiate these iPS cells into functional myotubes in vitro (14) to study the impact of genetic insulin resistance on insulin signaling and glucose homeostasis in this critical insulin target tissue. We demonstrate that iPS-derived myotubes from patients with Donohue syndrome not only exhibit significant defects in insulin signaling but also have defects in insulin-stimulated glucose metabolism and gene expression, thus providing a novel in vitro model of human skeletal muscle insulin resistance.

Results

Generation and Differentiation of iPS Cells Lines into Skeletal Myotubes.

To study the impact of genetic insulin resistance on human myocyte differentiation and function ex vivo, we created iPS cells from four healthy control subjects (C1 to C4) and four patients with Donohue syndrome due to insulin receptor mutations (IR-M1 to IR-M4) (Table S1) and differentiated them in vitro. C1 to C3 and IR-M1 to IR-M3 have been previously reported (9). Two additional iPS cell lines were generated from a healthy 2-y-old, female (C4) and 2-mo-old female with Donohue syndrome (IR-M4) by introduction of octamer-binding transcription factor 4 (OCT4), Kruppel-like factor 4 (KLF4), SRY (sex-determining region Y)-box 2 (SOX2), and myc proto-oncogene (cMYC) genes in skin fibroblasts, as previously described (9). Pluripotency of the iPS cells was confirmed by expression of OCT4, nanog homeobox (NANOG), and stage-specific embryonic antigen 4 (SSEA4) and in vivo teratoma formation (Fig. S1 A and B). Control and insulin receptor-mutant (IR-Mut) iPS cells were then treated with the GSK-3β inhibitor 6-bromoindirubin-3′-oxime (BIO), forskolin and basic fibroblast growth factor (bFGF), a combination that we have previously shown can promote myogenic differentiation (14) (Fig. 1A). Both control and IR-Mut lines differentiated into embryoid bodies (EBs), myoblasts, and myotubes at 7, 12, and 36 d, respectively. No differences in cell viability or morphology were observed between the IR-Mut and control cell lines during the differentiation process (Fig. 1B).

Table S1.

Summary of iPS cells

Cell lines Age at biopsy Gender Genetic mutation Functional domain
C1 (BJ) Neonatal M — —
C2 (GM05400) 6 y M — —
C3 (GM00409) 7 y M — —
C4 (GM00969) 2 y F — —
IR-M1 (Minn1) 1 mo F Arg897→ Stop (one allele) unknown mutation (second allele) exon 14 β-Subunit (one allele) decreased expression (second allele)
IR-M2 (GM10277) 15 y F Ala2→Gly (both alleles) exon 1 α-Subunit signal sequence (L1 domain)
IR-M3 (GM20034) 3 mo M Leu233 →Pro (both alleles) exon 3 α-Subunit (L2 domain)
IR-M4 (GM20327) 2 mo F Glu124→Stop (both alleles) exon 2 α-Subunit signal sequence (L1 domain)
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Summary of iPS cell lines features, including donor age and sex, mutation analysis, and functional domain of INSR mutations.

Fig. S1.

Fig. S1.

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Pluripotency analysis for iPS cells C4 and IR-M4. (A) Teratoma formation analysis of samples C4 and IR-M4, demonstrating formation of all three germ layers. (Scale bar: 100 µm.) (B) Immunocytochemistry for pluripotency factors OCT4, NANOG, and SSEA4. Pictures show merged images for OCT4 (red), SSEA4 (red), NANOG (green), and DAPI (blue).

Fig. 1.

Fig. 1.

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Differentiation of iPS cells into mature myotubes. (A) Schematic representation of myogenic differentiation protocol. (B) Bright-field pictures of differentiating iPS cells at the EB stage (day 5), myoblast stage (day 12), and myotube stage (day 36). (Scale bar: 100 µm.)

Despite normal morphology, IR-Mut cells had higher expression of the early muscle differentiation genes paired box protein7 (PAX7), myogenic factor 5 (MYF5), and myogenic differentiation 1 (MYOD1) (4.3-, 2.4-, and 1.4-fold increases compared with control, respectively) during the EB stage (day 5) compared with controls (Fig. 2A). PAX7 remained significantly increased (2.2-fold) in IR-Mut cells after 12 d of differentiation (myoblast stage) (Fig. 2B). By day 36, when cells were fully differentiated myotubes, PAX7 and MYF5 were almost undetectable in both controls and mutant cell lines consistent with progression through differentiation. At this stage, however, IR-Mut lines had an 80% reduction in myogenin (MYOG) expression, whereas myosin heavy chain 2 (MYH2) expression varied substantially between cell lines, but showed no significant difference in IR-Mut vs. controls. Consistent with normal differentiation, no differences were observed in the extent of multinucleation between controls and IR-Mut cells (Fig. 2C). Thus, although IR-Mut cells had normal myogenic differentiation morphologically, there was altered expression and timing of gene involved in myogenesis with increases in early differentiation genes and decreases in some late differentiation genes.

Fig. 2.

Fig. 2.

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Expression of myogenic markers during myogenesis of iPS cells. (A and B) Quantitative RT-PCR (qRT-PCR) analysis of PAX7, MYF5, and MYOD1 genes after (A) 5 d (EB stage) and (B) 12 d (myoblast stage) of differentiation. Each point represents an individual sample; group means are indicated by solid line. *P < 0.05, ***P < 0.001, IR-Mut vs. controls (n = 3 independent experiments). (C) qRT-PCR analysis of MYOGENIN, MyHC after 36 d of differentiation (myotube stage). The dotted line represents the average of controls; the dashed line represents the mean of IR-Mut. All values represent mean ± SD. *P < 0.05, IR-Mut vs. controls (n = 3 independent experiments).

Insulin Signaling Is Defective in Insulin-Resistant iPS Cell-Derived Myotubes.

Insulin signaling in iPS cell-derived myotubes was assessed by measuring insulin receptor (INSR), insulin receptor substrate-1 (IRS-1), v-akt murine thymoma viral oncogene homolog 2 (AKT), and extracellular-signal–regulated kinases (ERK1/2) phosphorylation following acute insulin stimulation. In control myotubes, Western blotting using an antibody that recognizes the major phosphorylation site in the INSR and the highly homologous region in the IGF1 receptor (IGF1R) showed a 5.5-fold increase in phosphorylation levels following stimulation with 100 nM insulin (Fig. 3A and Fig. S2A). By contrast, insulin-stimulated phosphorylation of INSR/IGF1R was reduced by 80% in IR-Mut myotubes (Fig. 3 A and B, and Fig. S2A). Likewise, insulin-stimulated phosphorylation of IRS-1 was nearly abolished in IR-Mut, and phosphorylation of AKT was reduced by 72% on average in the IR-Mut myotubes (Fig. 3 A and D–F, and Fig. S2 C–E). In contrast, insulin-stimulated phosphorylation of ERK1/2 was normal in IR-M1, IR-M2, and IR-M3, but reduced by about 50% in only IR-M4 myotubes, possibly reflecting a greater dependence of ERK stimulation on IGF1R compared with INSR. No changes were observed in the basal state of ERK1/2 (Fig. 3 A and G, and Fig. S2G).

Fig. 3.

Fig. 3.

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Insulin signaling in iPS-derived myotubes. (A) Western blot analysis of insulin signaling in control and IR-Mut iPS-myotubes was conducted as described in Experimental Procedures. Specific antibodies are indicated adjacent to the respective image. Images are representative of three independent experiments. (B–G) Quantitation of insulin-induced phosphorylation INSR/IGF1R, IRS1, AKT, ERK1/2, and total protein levels. *P < 0.05, **P < 0.01, ***P < 0.001, controls vs. IR-Mut (n = 3 independent experiments).

Fig. S2.

Fig. S2.

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Insulin signaling in iPS-derived myotubes (related to Fig. 3). (A–H) Quantification of insulin-induced phosphorylation and total protein levels of INSR/IGF1R, IRS1, AKT, and ERK1/2 in individual samples. All values represent means ± SD of band densitometry (n = 3 independent experiments); *P < 0.05, **P < 0.01, IR-Mut vs. controls.

Similar to our findings in primary fibroblasts and iPS cells (9), expression of INSR protein was reduced by 90% in myotubes from IR-Mut patients (Fig. 3 A and C, and Fig. S2B). In addition, in the IR-Mut cells, Western blot analysis showed a prominent band of apparent lower molecular weight, suggesting increased degradation of INSR in these cells. Levels of IRS-1, AKT, and ERK1/2 protein were largely unchanged, except for a modest reduction of IRS-1 protein in IR-M2 cells (Fig. 3 A and E, and Fig. S2D). Thus, impairment in insulin signaling in iPS cell-derived myotubes from IR-Mut patients results from both reduced INSR protein expression and decreased signaling capacity of the mutant INSR.

Insulin Action Is Impaired in Insulin-Resistant Myotubes.

Insulin exerts a variety of metabolic effects on skeletal muscle, including stimulation of glucose transport, glycolysis, and glycogen synthesis. These effects were mimicked in all control iPS-derived myotubes with insulin robustly stimulating 2-deoxyglucose uptake, glycogen synthase activity, and glycogen accumulation by >1.5- to >2-fold (Fig. 4 A–C and Table S2). This is similar in magnitude to the best insulin-responsive murine skeletal muscle cell models (15–17), demonstrating that the differentiated myotubes obtained from iPS cells are metabolically active and insulin responsive. In contrast, myotubes from Donohue syndrome patients showed an almost complete failure of insulin to induce glucose uptake, glycogen synthase activity, and glycogen accumulation (Fig. 4 A–C), with no significant differences in basal glucose uptake, glycogen synthase activity, and glycogen accumulation (Table S2).

Fig. 4.

Fig. 4.

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Insulin-stimulated 2-deoxy-d-glucose uptake, glycogen synthase activity, and glycogen accumulation in iPS-derived myotubes. (A) Insulin-stimulated 2-deoxy-d-glucose uptake in differentiated iPS cells. Myotubes were stimulated for 30 min, and uptake of [2-3H]DG was measured as described in Experimental Procedures. (B) Insulin-stimulated glycogen synthase activity in differentiated iPS cells. Myotubes were stimulated for 10 min with 100 nM insulin and glycogen synthase activity was measured as described in Experimental Procedures. (C) Insulin-induced glycogen accumulation in differentiated iPS cells. Myotubes were stimulated for 14 h with 100 nM insulin, and glycogen accumulation was measured. All values represent mean ± SD expressed as fold increase over basal. *P < 0.05, **P < 0.01 vs. unstimulated (n = 3 independent experiments).

Table S2.

Basal and insulin-stimulated glucose levels during insulin-stimulated glucose uptake (related to Fig. 4)

Cell lines pmol of glucose/μg of protein, basal ± SD pmol of glucose/μg of protein, insulin stimulation ± SD
C1 (BJ) 548 ± 36 940 ± 41
C2 (GM05400) 191 ± 46 313 ± 44
C3 (GM00409) 216 ± 40 490 ± 38
C4 (GM00969) 250 ± 55 487 ± 30
IR-MI (Minn1) 612 ± 11 584 ± 117
IR-M2 (GM10277) 241 ± 124 291 ± 61
IR-M3 (GM20034) 261 ± 45 294 ± 55
IR-M4 (GM20327) 280 ± 44 327 ± 67
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Both insulin and diabetes also exert potent effects on transcription of metabolic and growth-regulatory genes in skeletal muscle, including ras-related associated with diabetes (RAD1), hexokinase 2 (HK2), and glucose transporter type 4 (GLUT4) (18–21). Basal expression of these genes was significantly reduced in IR-Mut myotubes compared with controls (Fig. 5A and Fig. S3A). iPS cell-derived control myotubes did respond to insulin with threefold to fourfold increases in expression of the early growth response genes early growth response 1 (EGR1), fos proto-oncogene (cFOS), and jun proto-oncogene (c-JUN) (Fig. 5B and Fig. S3B). By contrast, insulin was unable to stimulate increased expression of these genes in IR-M1, -M3, and -M4 myotubes, and insulin responsiveness was reduced in IR-M2 (Fig. 5B and Fig. S3B).

Fig. 5.

Fig. 5.

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Expression of insulin-regulated genes. (A) qRT-PCR analysis of RAD1, HK2, and GLUT4 genes after 36 d of differentiation. Each dot represents an individual sample. *P < 0.05, **P < 0.01, IR-Mut vs. controls (n = 3 independent experiments). (B) qRT-PCR analysis of insulin-stimulated EGR1 and cFOS mRNA expression, relative to unstimulated cells (dashed line). All values represent mean of fold increase over basal ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. unstimulated; #P < 0.05, IR-Mut vs. controls (n = 3 independent experiments).

Fig. S3.

Fig. S3.

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Expression of insulin-regulated genes (related to Fig. 5). (A) qRT-PCR analysis of RAD1, GLUT4, and HK2 genes after 36 d of differentiation (myotube stage) in individual samples. The dotted line represents the average of controls, whereas the dashed line represents the average of IR-Mut. All values represent mean ± SD of three independent experiments. #P < 0.05, ##P < 0.01, IR-Mut vs. controls. (B) qRT-PCR analysis for insulin-stimulated cJUN mRNA expression, relative to unstimulated cells (dashed line). All values represent mean of fold increase over basal ± SD. *P < 0.05 vs. unstimulated (n = 3 independent experiments).

Discussion

Insulin resistance is a central feature of a variety of physiological and pathological disorders. Insulin resistance is present in humans with obesity, metabolic syndrome, and T2D, and in the latter can be detected years before the clinical presentation of disease (2, 22), suggesting a primary role in the pathophysiology of T2D. Severe forms of insulin resistance are observed in patients with genetic mutations in the insulin receptor or anti-insulin receptor antibodies (1, 23–25), and these have been highly informative in our understanding of insulin signaling pathways and the impact of insulin resistance on systemic metabolism. The molecular mechanisms underlying insulin resistance in these disorders may include both acquired and genetic components; however, dissecting the relative contributions of each is difficult due to the lack of robust and reliable models for the study of disease pathogenesis in humans in vitro. These challenges are compounded by the relative difficulty of obtaining relevant tissue samples, such as skeletal muscle or liver, particularly during the preclinical phases of disease or in childhood, and the inability to perform detailed mechanistic investigations on tissue samples.

The ability to generate iPS cells from human tissue samples represents a cutting-edge technology that has the potential to generate an unlimited source of cells that can be differentiated ex vivo into insulin-responsive tissues, including liver, muscle, fat, and beta cells, and used to probe mechanisms underlying insulin resistance and disease pathogenesis (14, 26–28). In this study, we have used our recently developed method (14) to drive iPS cell differentiation toward the skeletal muscle lineage using a combination of three soluble factors (bFGF, forskolin, and BIO). With this approach, we have produced functional, metabolically active myocytes from four individuals with insulin receptor mutations and four healthy individuals of similar age to determine the effects of genetic insulin resistance on muscle insulin action in human cells in vitro.

During in vitro differentiation, both control and IR-Mut iPS cells were able to form morphologically similar myotubes over 36 d in culture. However, IR-Mut myotubes displayed distinct patterns of differentiation-related genes, with increased PAX7, MYF5, and MYOD1 at early time points, and reduced expression of myogenin at late stages of differentiation, suggesting that insulin resistance impacts early steps of myogenic specification. Both insulin and IGF1 contribute to normal muscle growth, and insulin resistance and diabetes mellitus can be associated with varying degrees of skeletal muscle atrophy and impaired satellite cell proliferation and differentiation (29). Thus, it is possible that alterations in differentiation pattern observed in the mutant lines contribute to the low muscle mass observed in children with Donohue syndrome (10–12).

Myotubes differentiated from iPS cells from healthy control individuals display metabolic activity parallel to that observed in human skeletal muscle in vivo and rodent cell line models in vitro, including insulin-stimulated glucose uptake (80–150% increase over basal), glycogen accumulation (80–100% increase), and induction of glycogen synthase activity (50–90% increase). These responses are comparable with the most robust myotube models currently in use, such as mouse C2C12 cells, rat L6 cells, or primary human myoblasts derived from muscle biopsies (15, 16). By contrast and consistent with their insulin-resistant state, all IR-Mut cells fail to increase glucose uptake, glycogen synthase activity, or glycogen stores in response to insulin stimulation, reflecting the signaling defect linked to insulin receptor mutations in these patients.

As with the primary fibroblasts, iPS cells, and mesenchymal precursor cells derived from these patients (9), mutant myotubes have not only a severe reduction in INSR protein, but also reduced insulin-stimulated phosphorylation of the receptor and reduced phosphorylation of downstream effectors, such as IRS-1 and AKT. Interestingly, activation of ERK1/2 is unchanged, consistent with similar observations in muscles of insulin-resistant subject and a greater role for the IGF1R signals in ERK activation and sustained IGF1 signaling in IR-Mut cells (30–33). As a result of these upstream alterations in insulin signaling, we also observed impaired insulin stimulation of early growth-related genes, such as EGR1, cFOS, and cJUN, as well as reduced expression of insulin-dependent metabolic genes such as RAD1, HK2, and GLUT4 (18, 19). RAD1 encodes a protein that belongs to a class of small GTP binding proteins related to Ras, which was first identified as being overexpressed in skeletal muscle of patients with T2D (18). SNPs in RAD1 locus in skeletal muscle have been linked to T2D (34). Collectively, these data indicate that multiple pathways downstream of insulin receptor signaling are perturbed in IR-Mut myocytes and likely contribute to the metabolic defects observed in these patients in vivo.

Although it has been impossible to study primary myoblasts from Donohue syndrome patients, analysis of primary myoblasts isolated from skeletal muscle biopsies of patients with less severe forms of insulin resistance, such as T2D, also indicates defects in insulin signaling and glucose transport after passage in ex vivo culture (17). In the latter case, however, it still remains unclear whether this is a genetically programmed defect or the result of epigenetic changes that persist in these primary cells. In future studies, myotubes derived from iPS cells should provide a robust cellular model for detailed analysis of cell-autonomous metabolism and the role of genetics in signaling defects in more common forms of insulin resistance such as T2D.

The muscle derived from differentiation of iPS cells present some similarities and some differences from the iPS cells studied in the undifferentiated state, indicating the important role of cell context in studies of insulin resistance. For example, in this study, we find that all IR-Mut–derived myotubes exhibit a dramatic reduction in INSR protein levels. By contrast, at the iPS cell stage, cells from IR-M2 have a decrease in insulin-stimulated insulin receptor phosphorylation but show normal levels of INSR expression (9). Whether this is due to different rates of turnover rate of the insulin receptor in the pluripotent vs. differentiated cells, differences in epigenetic regulation of transcription, or a cell context effect on insulin receptor expression remains to be determined.

In summary, our study shows for the first time (to our knowledge) the ability to use human iPS cells to generate a model of skeletal muscle insulin resistance. Because these cells were initially isolated from skin fibroblasts, this approach provides a less invasive approach to generate human myocytes for future studies of insulin resistance and diabetes risk and an approach to dissect the genetic vs. acquired features of insulin resistance in other states such as T2D.

Experimental Procedures

Cell Lines.

C1 fibroblasts (BJ) were from ATCC; C2, C3, C4, IR-M2 and IR-M3, and IR-M4 fibroblasts were from Coriell. IR-M1 and IR-M3 fibroblasts were previously characterized (11, 12). iPS cells were generated from fibroblasts by retroviral transduction of KLF4, SOX2, OCT4, and c-MYC reprogramming factors and tested for pluripotency as previously described (9).

iPS-Derived Skeletal Muscle Differentiation.

Pluripotent iPS cells were maintained on Matrigel-coated dishes (BD) in mTeSR1 medium (STEMCELL Technologies). iPS cells were differentiated to myotubes as previously described (14). Briefly, iPS cells were induced to form EBs by incubating the plates with dispase (STEMCELL Technologies) for 5–8 min at 37 °C. The resulting clumps were lifted and incubated with the differentiation medium STEM Diff Apel medium (STEMCELL Technologies) supplemented with 10 ng/mL bFGF (Life Technologies), 0.5 μM BIO (Santa Cruz Biotechnologies), and 20 M forskolin (Santa Cruz Biotechnologies) for 7 d (bFGF, BIO, and forskolin added at days 1, 3, and 5). After 7 d of culture, EBs were transferred to Matrigel-coated plates for additional 29 d. Two to 3 d after plating the EBs, the medium was switched to DMEM supplemented with 2% (vol/vol) horse serum for the remaining duration of differentiation (Fig. 1A).

Western Blot Analysis of Insulin Signaling.

iPS-derived myotubes were serum-starved overnight in DMEM containing 0.25% BSA before stimulation with insulin (100 nM for 5 min) and lysis in RIPA buffer. Lysates were prepared for Western blotting as described (9).

Quantitative RT-PCR Analysis.

cDNA was synthesized from 1 µg of total RNA (High Capacity cDNA Reverse Transcription Kit; Life Technologies) and amplified (iTaq Universal SYBR Green Supermix, Bio-Rad; ABI 7900HT Real-Time PCR; Life Technologies). GAPDH and 36B4 were used as housekeeping genes. Relative gene expression was calculated by the 2ΔΔCT method (14).

2-Deoxyglucose Uptake in iPS Cell-Derived Myotubes.

[2-3H]Deoxy-d-glucose ([2-3H]DG) uptake was measured as previously reported (16). Briefly, differentiated myotubes were incubated in DMEM with 0.25% BSA for 14 h at 37 °C after which they were incubated in glucose-free KRB buffer for 30 min. The cells were then stimulated with 100 nM insulin for 30 min, supplemented during the final 10 min with [2-3H]DG. Cells were solubilized in 0.1% SDS buffer, and [2-3H]DG uptake was quantified by liquid scintillation counting.

Glycogen Accumulation and Glycogen Synthase Activity in iPS Cell-Derived Myotubes.

For glycogen accumulation, differentiated myotubes were incubated for 14 h in DMEM supplemented with 0.25% BSA at 37 °C and 100 nM insulin; cells were harvested and glycogen was measured using the Sigma-Aldrich glycogen assay kit following the manufacturer’s instructions. Glycogen synthase activity (35) was assessed in differentiated myotubes incubated in DMEM supplemented with 0.25% BSA for 14 h at 37 °C, and then washed with on ice with PBS and collected in lysis buffer (50 mM Hepes, pH 7.4, 100 mM KF, 10 mM EDTA, 0.5% Triton 100, and protease inhibitors). After centrifugation at 2,000 × g, the supernatant was transferred to new tubes and incubated 1:1 with reaction mix containing 16 mg/mL glycogen, 200 mM UDP-glucose, [14C]UDP glucose, with or without 200 mM glucose-6-phosphate (G6P). Samples were incubated at 37 °C for 15 min, and the reaction was stopped by placing samples on ice. Samples were then spotted on GF/A filters, air-dried, and washed in 70% (vol/vol) ethanol. After further drying overnight, glycogen synthase activity was measured by liquid scintillation counting, dividing –G6P/+G6P activities.

Acknowledgments

We acknowledge the early support provided by C. Cowan and L. Daheron of HSCI iPS Core Facility for assistance with generation of human iPS cells. This work was supported by NIH R01DK031036 (to C.R.K.), funding from the Novo-Nordisk Foundation (to C.R.K. and M.E.P.), and pilot funding from the Harvard Stem Cell Institute. A.M.B. was supported by T32 DK007260, the American Diabetes Association [mentor-based fellowship award (to M.E.P.)], and the Harold Whitworth Pierce Charitable Trust Postdoctoral Fellowship.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1525665113/-/DCSupplemental.

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