The effect of acute and chronic thermotherapy on type 2 diabetic skeletal muscle gene expression and inflammatory responses
Bachnak, Louay Z.
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Diabetes is a complex chronic illness associated with a state of high blood glucose level occurring from deficiencies in insulin secretion, action, or both. The disease affects millions of people globally. Type 2 Diabetes is associated with insulin resistance or a defective secretion of insulin from the pancreas. The skeletal muscle system accounts for 80% of glucose uptake and is a vital player in healthy aging and muscle mass maintenance. This muscle system also adapts to various stimuli, which are interdependent to fiber-type distribution, metabolism, and changes in muscle size. PURPOSE: The purpose of this study was to investigate the effects of thermotherapy on skeletal muscle metabolism, with an emphasis on gene expression, inflammation, and cell viability, in Type 2 Diabetic skeletal muscle. METHODS: Human skeletal muscle myoblast (HSMM) and Diabetic Type 2 human skeletal muscle myoblast (D-HSMM) (Lonza Inc, Walkersville, MD) cells were cultured until 70% confluency was reached, and then subjected to heat treatment – acute or chronic. The chronic heat treatment consisted of a 30-minute exposure to 40°C, three times a week for three weeks, while the acute heat treatment consisted of a one-time exposure to 40°C for 30 minutes. Approximately, 105 cells of each cell type (HSMM and DHSMM), along with growth media, were seeded into 24-well plates for a total volume of 2 mL per well. Groups included control cells (CON), chronic treatment (CH), and acute treatment (AC). Following a 48-hour incubation period, the chronic treatment was initiated, while the acute treatment was performed during the last session of the chronic treatment. Following the treatments, cell viability and density were determined. The cDNA was isolated and a real-time polymerase chain reaction (QuantStudio 3 Real-time PCR Instrument, Thermofisher, Waltham, MS) was performed to assess an array of gene expression. To assess inflammation, ELISA was performed on cytokines Interleukin 1β, IL-4, IL-6, IL-10, and tumor necrosis factor (TNF) -α. RESULTS: There was significant evidence supporting a difference between cell viability percentage following the acute thermotherapy (p = 0.0006) on D-HSMM, however, no significant change occurred in chronic thermotherapy. Through a qualitative assessment of gene expression, HSMM yielded the highest elevations under the chronic treatment compared to the control - AcvR2B (215.613-fold), IGF-2 (59.063-fold), and CTNNB1 (47.492-fold). Similarly, in D-HSMM cells, chronic heat treatment yielded an up-regulation of IGF-2 (0.485-fold), TNNT3 (0.399-fold), and PGC1B (0.328-fold), while in the acute treatment, an up-regulation of ACTB (1.648-fold) was observed. Acute and chronic heat treatments of HSMM cells significantly down-regulated IL-1β, IL-6, IL10, and TNF-α. Acute heat treatment of D-HSMM cells resulted in a significant decrease in IL-1β, IL-6, and up-regulation of IL-10 and TNF-α levels. In contrast, chronic treatment yielded a reduction in IL-4, TNF-α, and an up-regulation of IL-6 and IL-10 levels. CONCLUSION: The results suggest an increase in gene expression is related to actin activity (ACTB), sarcolemma repair (SGCA), and cellular responses (IGFBP3, CTNNB1, TNNT3), which could be indicative of an increase in transcriptional regulation. The observed increase in ACTB may indicate an increase in the regulation of energy balance due to the environmental change in both treatments. A significant difference is evident between acute treatment and control regarding cell viability percentage. In D-HSMM, proinflammatory cytokine IL-6 was seen upregulated following the chronic treatment, along with IL-10. This significant upregulation was negatively correlated with a decrease in TNF-α and IL-1β, which may be indicative of improved adverse inflammatory effects associated with the diabetic population.
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Bachnak, Louay Z,