, Volume 62, Issue 5, pp 845–859 | Cite as

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

  • Jing Zhang
  • Eunjin Oh
  • Karla E. Merz
  • Arianne Aslamy
  • Rajakrishnan Veluthakal
  • Vishal A. Salunkhe
  • Miwon Ahn
  • Ragadeepthi Tunduguru
  • Debbie C. ThurmondEmail author



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.


DOC2B Glucose homeostasis GLUT4 Insulin sensitivity KLC1 Obesity Skeletal muscle Type 2 diabetes 





Double C2 domain β




Green fluorescent protein


High-fat diet


Insulin resistance (as experimentally induced in vitro)


Insulin receptor


Intraperitoneal glucose tolerance test


Intraperitoneal insulin tolerance test


Kinesin light chain 1


Munc13-interacting domain


Multiplicity of infection


National Disease Research Interchange


Plasma membrane




Reverse tetracycline transactivator


Doxycycline-inducible skeletal-muscle-specific Doc2b overexpression


Soluble N-ethylmaleimide-sensitive factor-attachment protein receptor


Syntaxin 4


Syntaxin binding protein


Tetratricopeptide repeats


Tetracycline-responsive element


Target-associated SNARE


Wild type



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.

Contribution statement

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.

Supplementary material

125_2019_4824_MOESM1_ESM.pdf (522 kb)
ESM 1 (PDF 521 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jing Zhang
    • 1
    • 2
  • Eunjin Oh
    • 1
  • Karla E. Merz
    • 1
  • Arianne Aslamy
    • 1
    • 3
  • Rajakrishnan Veluthakal
    • 1
  • Vishal A. Salunkhe
    • 1
  • Miwon Ahn
    • 1
  • Ragadeepthi Tunduguru
    • 1
    • 4
  • Debbie C. Thurmond
    • 1
    Email author
  1. 1.Department of Molecular and Cellular Endocrinology, Diabetes and Metabolism Research InstituteBeckman Research Institute of City of HopeDuarteUSA
  2. 2.Anwita Biosciences IncSan CarlosUSA
  3. 3.Department of Cellular and Integrative PhysiologyIndiana University School of MedicineIndianapolisUSA
  4. 4.Department of Diabetes Complications and Metabolism, Diabetes and Metabolism Research InstituteBeckman Research Institute of City of HopeDuarteUSA

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