DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle
Skeletal muscle accounts for >80% of insulin-stimulated glucose uptake; dysfunction of this process underlies insulin resistance and type 2 diabetes. Insulin sensitivity is impaired in mice deficient in the double C2 domain β (DOC2B) protein, while whole-body overexpression of DOC2B enhances insulin sensitivity. Whether insulin sensitivity in the skeletal muscle is affected directly by DOC2B or is secondary to an effect on other tissues is unknown; the underlying molecular mechanisms also remain unclear.
Human skeletal muscle samples from non-diabetic or type 2 diabetic donors were evaluated for loss of DOC2B during diabetes development. For in vivo analysis, new doxycycline-inducible skeletal-muscle-specific Doc2b-overexpressing mice fed standard or high-fat diets were evaluated for insulin and glucose tolerance, and insulin-stimulated GLUT4 accumulation at the plasma membrane (PM). For in vitro analyses, a DOC2B-overexpressing L6-GLUT4-myc myoblast/myotube culture system was coupled with an insulin resistance paradigm. Biochemical and molecular biology methods such as site-directed mutagenesis, co-immunoprecipitation and mass spectrometry were used to identify the molecular mechanisms linking insulin stimulation to DOC2B.
We identified loss of DOC2B (55% reduction in RNA and 40% reduction in protein) in the skeletal muscle of human donors with type 2 diabetes. Furthermore, inducible enrichment of DOC2B in skeletal muscle of transgenic mice enhanced whole-body glucose tolerance (AUC decreased by 25% for female mice) and peripheral insulin sensitivity (area over the curve increased by 20% and 26% for female and male mice, respectively) in vivo, underpinned by enhanced insulin-stimulated GLUT4 accumulation at the PM. Moreover, DOC2B enrichment in skeletal muscle protected mice from high-fat-diet-induced peripheral insulin resistance, despite the persistence of obesity. In L6-GLUT4-myc myoblasts, DOC2B enrichment was sufficient to preserve normal insulin-stimulated GLUT4 accumulation at the PM in cells exposed to diabetogenic stimuli. We further identified that DOC2B is phosphorylated on insulin stimulation, enhancing its interaction with a microtubule motor protein, kinesin light chain 1 (KLC1). Mutation of Y301 in DOC2B blocked the insulin-stimulated phosphorylation of DOC2B and interaction with KLC1, and it blunted the ability of DOC2B to enhance insulin-stimulated GLUT4 accumulation at the PM.
These results suggest that DOC2B collaborates with KLC1 to regulate insulin-stimulated GLUT4 accumulation at the PM and regulates insulin sensitivity. Our observation provides a basis for pursuing DOC2B as a novel drug target in the muscle to prevent/treat type 2 diabetes.
KeywordsDOC2B Glucose homeostasis GLUT4 Insulin sensitivity KLC1 Obesity Skeletal muscle Type 2 diabetes
Double C2 domain β
Green fluorescent protein
Insulin resistance (as experimentally induced in vitro)
Intraperitoneal glucose tolerance test
Intraperitoneal insulin tolerance test
Kinesin light chain 1
Multiplicity of infection
National Disease Research Interchange
Reverse tetracycline transactivator
Doxycycline-inducible skeletal-muscle-specific Doc2b overexpression
Soluble N-ethylmaleimide-sensitive factor-attachment protein receptor
Syntaxin binding protein
We are grateful to E. Olson (Department of Cellular and Molecular Endocrinology, City of Hope, Duarte, CA, USA) for technical support and to N. Linford (Department of Cellular and Molecular Endocrinology, City of Hope, Duarte, CA, USA) for editing assistance. Research reported in this publication also includes work performed in the Multi-omics Mass Spectrometry & Biomarker Discovery Core, Integrative Genomics and Bioinformatics Core, Drug Discovery and Structural Biology Core, the Light Microscopy/Digital Imaging Core and the Comprehensive Metabolic Phenotyping Core at City of Hope (Duarte, CA, USA), all supported by a Cancer Center Support Grant from the National Cancer Institute to City of Hope. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Parts of this work were presented at the Federation of American Societies for Experimental Biology (FASEB) science research conference, Glucose Transport: Gateway for Metabolic Systems Biology, July 2017.
JZ performed the majority of the studies, contributed to discussion and wrote and edited the manuscript. EO designed and generated the TRE-Doc2b+/− mice and contributed to the analysis of serum analytes and to the manuscript revision and discussion. KEM assisted with the design of the myotube viral transduction paradigm, the co-immunoprecipitation experiments and IPGTT studies of the obese mice and contributed to the manuscript revision and discussion. AA, RV and VAS assisted with in vivo studies and contributed to manuscript revision and discussion. RT assisted with L6-mycGLUT4 cell culture and manuscript revision and discussion. MA generated the Doc2b adenovirus and contributed to manuscript revision and discussion. DCT conceived the study, contributed to the discussion and wrote, reviewed and edited the manuscript. All authors read and approved the final version of the manuscript. DCT 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.
This work was supported by grants from: the National Institutes of Health, including from the National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK067912; R01 DK102233; R01 DK112917 to DCT); the American Heart Association (17POST33661194, to JZ; 15PRE21970002 to RT); and the National Cancer Institute (P30CA33572). This work was also supported by the Indiana Clinical and Translational Science Institute (predoctoral fellowship to AA). Additional financial support was provided to DCT through City of Hope: the Ruth and Robert Lanman Endowment, the George Schaeffer award and an Excellence award.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
- 15.Kiraly-Borri CE, Morgan A, Burgoyne RD, Weller U, Wollheim CB, Lang J (1996) Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin. Biochem J 314(1):199–203. https://doi.org/10.1042/bj3140199 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Sollner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE (1993) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75(3):409–418. https://doi.org/10.1016/0092-8674(93)90376-2 CrossRefPubMedGoogle Scholar
- 28.Hornbeck PV, Kornhauser JM, Tkachev S et al (2012) PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res 40(D1):D261–D270. https://doi.org/10.1093/nar/gkr1122 CrossRefPubMedGoogle Scholar
- 34.McCarthy AM, Spisak KO, Brozinick JT, Elmendorf JS (2006) Loss of cortical actin filaments in insulin-resistant skeletal muscle cells impairs GLUT4 vesicle trafficking and glucose transport. Am J Physiol Cell Physiol 291(5):C860–C868. https://doi.org/10.1152/ajpcell.00107.2006 CrossRefPubMedPubMedCentralGoogle Scholar
- 35.Chen G, Raman P, Bhonagiri P, Strawbridge AB, Pattar GR, Elmendorf JS (2004) Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin. J Biol Chem 279(38):39705–39709. https://doi.org/10.1074/jbc.C400171200 CrossRefPubMedPubMedCentralGoogle Scholar
- 37.Zhou M, Sevilla L, Vallega G et al (1998) Insulin-dependent protein trafficking in skeletal muscle cells. Am J Phys 275:E187–E196Google Scholar
- 44.Jiang ZY, Zhou QL, Coleman KA, Chouinard M, Boese Q, Czech MP (2003) Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing. Proc Natl Acad Sci U S A 100(13):7569–7574. https://doi.org/10.1073/pnas.1332633100 CrossRefPubMedPubMedCentralGoogle Scholar
- 48.Strommer L, Permert J, Arnelo U et al (1998) Skeletal muscle insulin resistance after trauma: insulin signaling and glucose transport. Am J Phys 275:E351–E358Google Scholar