Myokine secretion and metabolic pathways under resting and contractile conditions in primary human myotubes: A matter of oxygen level?

Abstract

Diabetologia (2024) 67 (Suppl 1):S1-S593 1 3 postprandial glycaemic control in fertile women with type 1 diabetes (T1DM) during the absorptive and postabsorptive states. This is especially relevant in T1DM since the amount of prandial insulin in circulation is often different when exercising before lunch (low insulin on board) vs. after lunch (high insulin on board), which considerably affects glucose excursions. We aimed to determine which timing of exercise (exercise before or after lunchtime) is more effective to opti-mise postprandial glycaemic control (18-h, 4-h, and nocturnal post-prandial periods) in women of reproductive age with T1DM. Materials and methods: The MERIT1D study is randomized crosso-ver trial. A total of 13 women at the early-middle follicular phase of the menstrual cycle underwent 2 experimental conditions: pre-meal exercise and post-meal exercise. These tests were conducted in 3 non-consecutive days within an 8-day period, with a wash-out period of 48 h between visits. On the 1 st day (screening visit), women underwent a resting energy expenditure test followed by an incremental walk test using a gas analyser, which allowed us to standardize meal energy content and exercise intensity in the experimental days. In the pre-meal and post-meal exercise days, the meal intake test consisted of a mixed milkshake equivalent to 35% of women's resting energy expenditure, and the exercise stimulus of an individualized 35min submaximal walking test whose intensity was equivalent to the 1 st ventilatory threshold. Postprandial glucose excursions were continuously monitored using last-generation continuous glucose monitoring systems (CGM, Dexcom G7). Participants were instructed to follow the same tailored diet, and physical activity and sleep patterns, in the study days. Results: Regarding the 18-h postprandial period, women spent more time in euglycemia and less time in hyperglycaemia when they exercised after lunchtime compared to before lunchtime. However, these trends were non-significant (evidence of statistical significance, p=0.1). Time in hypoglycaemia was similar between both stimuli (p>0.05). In addition, women showed lower glucose variability (standard deviation of mean CGM glucose levels) in the post-meal vs. pre-meal exercise day (p<0.05). Regarding the 4-h and nocturnal postprandial periods, similar non-significant trends were observed (p>0.05). Conclusion: Overall, post-meal exercise appeared to be slightly more effective to optimise postprandial glucose excursions in women with T1DM compared to pre-meal exercise, at least in the 18-h postprandial period. Ongoing analyses will shed more light on these results. Background and aims: Physical activity is a powerful tool to prevent and reverse type 2 diabetes. It has been shown that myokines released by contracting skeletal muscle affect glucose homeostasis. Oxygen availability in the skeletal muscle microenvironment, which is lower during (high-intensity) exercise, might be a key regulator involved in myokine secretion and glucose uptake in skeletal muscle cells. Here, we investigated the effects of oxygen level on myokine secretion and metabolic pathways under resting and contractile conditions in primary human myotubes. Materials and methods: Ex vivo primary human satellite cells were differentiated towards functional myotubes and exposed to different oxygen levels for 3h (1% vs 3% vs 21% O 2) under resting or contractile (Electrical Pulse Stimulation (EPS), 30V, 1Hz, 2ms) conditions (n=7 independent experiments, 6 biological replicates/condition). We determined lactate secretion, gene expression (quantitative RT-PCR) and secretion (high-sensitivity ELISA) of several myokines (IL-6, GDF-15, IL-15, SPARC and myonectin) as well as AMPK activation using Western Blotting. Data were analyzed using the non-parametric Kruskal-Wallis test, with Bonferroni's post-hoc test in case of significance. Results: Both EPS and low oxygen availability induced a significant increase in lactate secretion compared to resting, non-contractile conditions and standard laboratory conditions (21% O 2), respectively (both p<0.001). Highest lactate secretion was found when cells were exposed to 1% O 2 during EPS (p<0.001). Exposure to low physiological oxygen levels evoked pronounced effects on myokine expression and secretion. More specifically, low oxygen exposure increased IL-6 gene expression (1% vs. 21% O 2 , p<0.001; 1% vs. 3% O 2 (p<0.001) and secretion (1% vs. 21% O 2 , p=0.002), increased SPARC secretion (3% vs. 21% O 2 , p<0.001), and reduced myonectin gene expression (1% vs. 21% O 2 , p=0.005) and the secretion of GDF-15 (1% vs. 21% O 2 , p=0.014). Strikingly, myotube contraction during low oxygen exposure increased gene expression of IL-6 (EPS vs. no EPS at 3% O 2 , p=0.002; EPS vs. no EPS at 1% O 2 , p=0.086), GDF-15 (EPS vs. no EPS at 3% O 2 , p<0.001; EPS vs. no EPS at 1% O 2 , p=0.006) and IL-15 (EPS vs. no EPS at 3% O 2 , p=0.011), and further increased IL-6 secretion (EPS vs. no EPS at 1% O 2 , p=0.019) compared to non-contractile conditions. In contrast, EPS under standard laboratory conditions (21% O 2) had no significant effects on myokine gene expression and secretion (P>0.05), except for a modest increase in IL-6 secretion (EPS vs. no EPS at 21% O 2 , p=0.025). Effects of low oxygen exposure with(out) EPS on AMPK activation and glucose uptake are currently being examined. Conclusion: The present findings demonstrate that both contraction and a reduced oxygen availability in the skeletal muscle microenvi-ronment induce a shift towards anaerobic (glycolytic) metabolism and affect myokine expression/secretion.