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Glucose Uptake and Insulin Response in Tissue-engineered Human Skeletal Muscle

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Background:

Tissue-engineered muscles (“myobundles”) offer a promising platform for developing a human in vitro model of healthy and diseased muscle for drug development and testing. Compared to traditional monolayer cultures, myobundles better model the three-dimensional structure of native skeletal muscle and are amenable to diverse functional measures to monitor the muscle health and drug response. Characterizing the metabolic function of human myobundles is of particular interest to enable their utilization in mechanistic studies of human metabolic diseases, identification of related drug targets, and systematic studies of drug safety and efficacy.

Methods:

To this end, we studied glucose uptake and insulin responsiveness in human tissue-engineered skeletal muscle myobundles in the basal state and in response to drug treatments.

Results:

In the human skeletal muscle myobundle system, insulin stimulates a 50% increase in 2-deoxyglucose (2-DG) uptake with a compiled EC50 of 0.27 ± 0.03 nM. Treatment of myobundles with 400 µM metformin increased basal 2-DG uptake 1.7-fold and caused a significant drop in twitch and tetanus contractile force along with decreased fatigue resistance. Treatment with the histone deacetylase inhibitor 4-phenylbutyrate (4-PBA) increased the magnitude of insulin response from a 1.2-fold increase in glucose uptake in the untreated state to a 1.4-fold increase after 4-PBA treatment. 4-PBA treated myobundles also exhibited increased fatigue resistance and increased twitch half-relaxation time.

Conclusion:

Although tissue-engineered human myobundles exhibit a modest increase in glucose uptake in response to insulin, they recapitulate key features of in vivo insulin sensitivity and exhibit relevant drug-mediated perturbations in contractile function and glucose metabolism.

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Abbreviations

2-DG:

2-deoxyglucose

4-PBA:

4-phenylbutyrate, histone deacetylase inhibitor

ACA:

6-aminocaproic acid

AMPK:

5′AMP-activated protein kinase

C2C12:

A line of mouse myoblasts

DMEM:

Dulbecco’s Modified Eagle Medium

ECL:

Enhanced chemiluminescent

EGF:

Epidermal growth factor

GLUT1:

Glucose transporter responsible for constitutive glucose uptake

GLUT3:

Glucose transporter most often expressed in neurons

GLUT4:

Glucose transporter responsible for insulin-mediated glucose uptake

HDAC:

Histone deacetylase

HRP:

Horseradish peroxidase

hGM:

Human growth media

IRS-1/PI3K/Akt:

Insulin receptor substrate (IRS)-1 is a signaling molecule that after activation interacts with phosphoinositide 3-kinase (PI3K) and serine/threonine protein kinases to regulate GLUT4

LAA:

Low amino acid

MHC:

Myosin heavy chain protein

MYH1:

Fast twitch type IIX MHC

MYH3:

Immature embryonic form of MHC

MYH7:

Slow twitch type I MHC

MYH8:

Perinatal isoform of MHC

qRT-PCR:

Quantitative reverse transcription polymerase chain reaction

TBST:

Tris-buffered saline and 0.1% Tween 20 solution

PGC-1α:

Peroxisome proliferator-activated receptor-γ coactivator-1α is a transcriptional coactivator involved in cellular energy metabolism

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Acknowledgements

This work was supported by the NIH grants UH2TR000505 and UG3TR002412 from NCATS and NIAMS, the NIH Common Fund for the Microphysiological Systems Initiative to GAT, as well as an NSF Graduate Research Fellowship to MEK.

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  1. Department of Biomedical Engineering, Duke University, Durham, NC, 27708, USA

    Megan E. Kondash, Anandita Ananthakumar, Alastair Khodabukus, Nenad Bursac & George A. Truskey

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  1. Megan E. Kondash
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  2. Anandita Ananthakumar
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  3. Alastair Khodabukus
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Correspondence to George A. Truskey.

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Primary human myogenic cells were isolated from surgical waste samples obtained according to a Duke IRB-approved protocol (IRB No. Pro00063964).

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13770_2020_242_MOESM1_ESM.docx

Supplementary Table S1. Components of the base LAA media compared to DMEM. Parentheses represent components that are not in the custom base media but are supplemented at the indicated concentration. (DOCX 20 kb)

Supplementary Table S2. Primer sequences used for qRT-PCR. (DOCX 18 kb)

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Kondash, M.E., Ananthakumar, A., Khodabukus, A. et al. Glucose Uptake and Insulin Response in Tissue-engineered Human Skeletal Muscle. Tissue Eng Regen Med 17, 801–813 (2020). https://doi.org/10.1007/s13770-020-00242-y

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