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Association between insulin resistance and impairment of FGF21 signal transduction in skeletal muscles

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Abstract

Fibroblast growth factor (FGF) 21, was identified as a potent metabolic regulator of glucose and lipid metabolism. We investigated whether the levels and signalings of FGF21 changed in the skeletal muscle of type 2 diabetes mellitus (T2DM) patients, participants with impaired glucose tolerance (IGT), human skeletal muscle myotubes (HSMMs) under insulin-resistant conditions, and mice with diet-induced obesity (DIO). A percutaneous biopsy sample of the vastus lateralis muscle of T2DM patients, IGT subjects, and participants with normal glucose tolerance was obtained and the levels and signalings of FGF21 were assessed. We determined whether the expression and signalings of FGF21 in HSMMs altered according to palmitate concentrations and exposure time. Also, we confirmed whether changes of FGF21 signal transduction resulted in the alteration of FGF21 functions. DIO mice were treated intravenously with recombinant FGF21, and the levels and signalings of FGF21 were assessed in their soleus muscles. We checked whether or not FGF21 played a role in the gene transcription related to lipid oxidation. Levels of FGF21 increased, whereas levels of phosphorylated FGF receptor (p-FGFR), phosphorylated FGFR substrates 2α (p-FRS2α), and phosphorylated extracellular signal-regulated kinases (p-ERK) decreased in the skeletal muscle of both T2DM patients and IGT subjects. In vitro, palmitate increased the levels of FGF21 and significantly reduced the levels of β-klotho, p-FGFR, p-FRS2α, and p-ERK1/2 in HSMMs exposed to palmitate. Palmitate also decreased glucose uptake and glycogen contents of FGF21. Consistently, the levels of FGF21 were significantly higher and the levels of β-klotho and p-FGFR were lower in the DIO mice than in normal lean mice. The levels of FGF21 increased but its signal transduction and actions were impaired in skeletal muscles of T2DM patients, IGT subjects, in insulin-resistant HSMMs, and DIO mice.

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Acknowledgments

This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare (HI12C1006/A121102 to K.W. Lee) and Basic Science Research Program through the National Research Foundation of Korea, Ministry of Science, ICT & Future Planning (NRF-2014R1A1A3050777 to H.J. Kim), Republic of Korea. The biospecimens (liver tissue) for this study were provided by the Ajou Human Bio-Resource Bank (AHBB), a member of the National Biobank of Korea, which is supported by the Ministry of Health and Welfare. Liver tissues derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols.

Author contribution

JYJ designed the study, analyzed data, and wrote the manuscript. SEC designed the study, performed experiments, analyzed data, and wrote the manuscript. ESH, JGJ performed experiments, and analyzed data. THK analyzed data, and wrote the manuscript. SJH, HJK, DJK, and YK contributed to the discussion and reviewed and edited the manuscript. KWL designed the study and wrote the manuscript. All authors contributed to data interpretation and reviewed and approved the final manuscript. KWL is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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Authors and Affiliations

  1. Department of Endocrinology and Metabolism, Ajou University School of Medicine, San 5, Wonchon-dong, Yeongtong-gu, Suwon, 443-721, Republic of Korea

    Ja Young Jeon, Eun Suk Ha, Jong Gab Jung, Seung ** Han, Hae ** Kim, Dae Jung Kim & Kwan-Woo Lee

  2. Department of Physiology, Ajou University School of Medicine, Suwon, Republic of Korea

    Sung-E Choi & Yup Kang

  3. Division of Endocrine and Metabolism, Department of Internal Medicine, Seoul Medical Center, Seoul, Republic of Korea

    Tae Ho Kim

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12020_2015_845_MOESM1_ESM.tif

Supplemental Fig. 1 The original Western blot of figure 1. The numbers 1 and 2 are indications of the identical subject repeated in each blot. FGF, Fibroblast growth factor; p-FGFR, phosphorylated fibroblast growth factor receptor; p-FRS2α, phosphorylated fibroblast growth factor receptor substrates 2α; T2DM, type 2 diabetes mellitus patients; IGT, subjects with impaired glucose tolerance; NGT, participants with normal glucose tolerance.. Supplementary material 1 (TIFF 248 kb)

12020_2015_845_MOESM2_ESM.tif

Supplemental Fig. 2 Validation of β-klotho. (A) β-klotho protein in human skeletal muscle myotubes (HSMMs), (B) knock down of β-klotho using β-klotho siRNA, (C) FGF21 signaling proteins with or without β-klotho siRNA. HSMMs were transfected with siRNA β-klotho, incubated for 24h, differentiated and then treated with palmitate 200 μM for 24 h. Levels of β-klotho were significantly decreased after treatment of siRNA β-klotho and after treatment of palmitate. PA, palmitate; BSA, Bovine serum albumin; FGF, Fibroblast growth factor; p-FRS2α, phosphorylated fibroblast growth factor receptor substrates 2α; p-ERK1/2, phosphorylated extracellular signal-regulated kinases 1 and 2. Supplementary material 2 (TIFF 108 kb)

12020_2015_845_MOESM3_ESM.tif

Supplemental Fig. 3 Expression of β-klotho in each tissue from C57BL/6J mice. (A) the expression of β-klotho in adipose tissue and (B) in a few other tissues. To investigate β-klotho expression in a few tissues of mice, tissues from liver, gastrocnemius muscle, soleus muscles, and epididymal fat of normal diet mice and only epididymal fat of DIO mice were isolated, then the levels of β-klotho, using western blotting were measured. The levels of β-klotho were high in liver, adipose tissue from normal mice, and soleus muscles, but epididymal fat from DIO mice and gastrocnemius muscle weakly expressed β-klotho. DIO, diet-induced obesity model with high fat diet; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.. Supplementary material 3 (TIFF 113 kb)

12020_2015_845_MOESM4_ESM.tif

Supplemental Fig. 4 FGF21 signal transduction in gastrocnemius muscle, and soleus muscles of normal C57BL/6J mice. FGF21 stimulated tyrosine phosphorylation of FRS2α and ERK in soleus muscles, not gastrocnemius muscle in normal mice. Normal mice were injected via the tail with FGF21 (50 mg/kg) then after 15 min, liver, gastrocnemius muscle, and soleus muscles were isolated. The β-klotho protein was strongly detected in soleus muscles than in gastrocnemius muscle. The levels of p-FRS2 and p-ERK increased more in response to FGF21 in soleus muscles than in gastrocnemius muscle. FGF21 signaling was measured by immunoprecipitation (IP, FRS2 antibody)/immunoblot (IB, p-Y). Phosphorylated and total ERK and phosphorylated and total FRS2 were detected by immunoblot using each antibody. FGF21, fibroblast growth factor 21; p-FRS2α, phosphorylated fibroblast growth factor receptor substrates 2α; p-ERK1/2, phosphorylated extracellular signal-regulated protein kinases 1 and 2. Supplementary material 4 (TIFF 127 kb)

12020_2015_845_MOESM5_ESM.tif

Supplemental Fig. 5 Gene expression by FGF21 in soleus muscles of normal mice. FGF21 stimulated expression of peroxisome proliferator-activated receptor α. Normal mice were injected via the tail with FGF21 (50 mg/kg) then after 15 min, soleus muscles were isolated. The mRNA expression was measured by qRT-PCR. ppara, peroxisome proliferator-activated receptor α; pparb/d peroxisome proliferator-activated receptor β/δ; mcad, medicum-chain acul-coenzyme A dehydrogenase. Data represent mean ± SEs. †P < 0.05 vs. normal without FGF21. Supplementary material 5 (TIFF 63 kb)

12020_2015_845_MOESM6_ESM.tif

Supplemental Fig. 6 Expression of β-klotho in each tissue from humans. To compare the levels of β-klotho expressions, samples from the liver, vastus lateralis, and subcutaneous fat of different subjects were obtained, then the levels of β-klotho, using western blotting were measured. Although the levels of β-klotho were relatively weaker than that of adipose tissue, the human muscle did show an expression of β-klotho. Supplementary material 6 (TIFF 106 kb)

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Jeon, J.Y., Choi, SE., Ha, E.S. et al. Association between insulin resistance and impairment of FGF21 signal transduction in skeletal muscles. Endocrine 53, 97–106 (2016). https://doi.org/10.1007/s12020-015-0845-x

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