N = 13?7 for ND, 9?5 for T2D. *p<0.05 vs ND doi:10.1371/journal.

N = 13?7 Roc-A chemical information for ND, 9?5 for T2D. *p<0.05 vs ND doi:10.1371/journal.pone.0158209.g[6]. Since skeletal muscle contains a number of different cell types in addition to myotubes that can also produce cytokines and chemokines, the question arises of what is needed to classify a factor as a myokine? A key feature of such a definition is that a myokine be secreted, placing importance on measuring release from muscle, in addition to monitoring changes in gene expression [6]. Furthermore, the site of myokine synthesis and secretion as specific to muscle should be verified by studying muscle cells [6]. For the latter reason rodent skeletal muscle cell lines have been useful in identifying myokines and exploring their regulation. For example, the expression of a number of myokines is modified during myogenesis in C2C12 cells [29]. However, rodent muscle cell models can provide limited insights into human pathophysiology. Fortunately, multiple investigators have shown that hSMCs proliferated and differentiated in vitro display many of the features of mature muscle [16]. Indeed, hSMCs obtained from T2D subjects retain many of the abnormalPLOS ONE | DOI:10.1371/journal.pone.0158209 July 25,9 /Myokine Secretion in Type 2 DiabetesFig 5. Regulation of inflammatory signaling in hSMC. ND (open bars) and T2D (solid bars) cells extracted after 48 hr treatment with LPS, Pioglitazone (Pio), palmitate or oleate. Results expressed relative to untreated control for each individual set of cells, Ave + SEM. (A) IkBa protein, n = 7?2 and 6?5 for ND and T2D, respectively. (B) Total and phospho-p38, n = 6?4 and 5?. (C) Total and phospho-p44/42, n = 7?3 and 7?1. (D) Total and phospho-JNK, n = 9?2 and 3?. * p<0.05 T2D vs ND. p<0.05 T2D response vs ND response doi:10.1371/journal.pone.0158209.gmetabolic properties that their donors express in vivo. These include: Impaired basal and insulin-stimulated glucose uptake [17, 30], reduced glycogen synthesis [18], impaired fatty acid oxidation [19], and altered protein expression [31], Under the criteria described above, IL6 was identified as early as 1994 as a myokine, constitutively secreted by human myotubes [32]. This was followed by the work of Pedersen and colleagues on the dynamic exercise-mediated regulation of IL6 synthesis and secretion [4]. Mirroring what is seen in vivo, production of multiple myokines by human myotubes have been reported to be regulated by differentiation [8], exercise [5] and electrical stimulation [9, 10]. FPS-ZM1 chemical information information regarding whether myokine secretion is altered in T2D is somewhat more limited. Novel information provided by the current report includes: 1) GROa, IL8, IL15 and follistatin are myokines whose secretion is higher in T2D, 2) secretion of IL1?and VEGF is similar between ND and T2D myotubes, and, 3) IL10 secretion by resting myotubes is modest. Furthermore, the results indicate that INFg does not meet the criteria for a myokine, in agreement with the recent report of Brown et al [33]. Our current observation that TNFa secretion, while low, is elevated in T2D myotubes is confirmatory of our earlier results [13] and in agreement with Green et al [34] and Vandanmagsar et al at the level of gene expression [12]. Consistent with these results, induction of an insulin resistant state by TNFa treatment of hSMC from healthy individuals also resulted in increased TNFa production and secretion [11]. The picture regarding IL6 is more mixed. While we found IL6 secretion to be higher in T2D hSMC, as.N = 13?7 for ND, 9?5 for T2D. *p<0.05 vs ND doi:10.1371/journal.pone.0158209.g[6]. Since skeletal muscle contains a number of different cell types in addition to myotubes that can also produce cytokines and chemokines, the question arises of what is needed to classify a factor as a myokine? A key feature of such a definition is that a myokine be secreted, placing importance on measuring release from muscle, in addition to monitoring changes in gene expression [6]. Furthermore, the site of myokine synthesis and secretion as specific to muscle should be verified by studying muscle cells [6]. For the latter reason rodent skeletal muscle cell lines have been useful in identifying myokines and exploring their regulation. For example, the expression of a number of myokines is modified during myogenesis in C2C12 cells [29]. However, rodent muscle cell models can provide limited insights into human pathophysiology. Fortunately, multiple investigators have shown that hSMCs proliferated and differentiated in vitro display many of the features of mature muscle [16]. Indeed, hSMCs obtained from T2D subjects retain many of the abnormalPLOS ONE | DOI:10.1371/journal.pone.0158209 July 25,9 /Myokine Secretion in Type 2 DiabetesFig 5. Regulation of inflammatory signaling in hSMC. ND (open bars) and T2D (solid bars) cells extracted after 48 hr treatment with LPS, Pioglitazone (Pio), palmitate or oleate. Results expressed relative to untreated control for each individual set of cells, Ave + SEM. (A) IkBa protein, n = 7?2 and 6?5 for ND and T2D, respectively. (B) Total and phospho-p38, n = 6?4 and 5?. (C) Total and phospho-p44/42, n = 7?3 and 7?1. (D) Total and phospho-JNK, n = 9?2 and 3?. * p<0.05 T2D vs ND. p<0.05 T2D response vs ND response doi:10.1371/journal.pone.0158209.gmetabolic properties that their donors express in vivo. These include: Impaired basal and insulin-stimulated glucose uptake [17, 30], reduced glycogen synthesis [18], impaired fatty acid oxidation [19], and altered protein expression [31], Under the criteria described above, IL6 was identified as early as 1994 as a myokine, constitutively secreted by human myotubes [32]. This was followed by the work of Pedersen and colleagues on the dynamic exercise-mediated regulation of IL6 synthesis and secretion [4]. Mirroring what is seen in vivo, production of multiple myokines by human myotubes have been reported to be regulated by differentiation [8], exercise [5] and electrical stimulation [9, 10]. Information regarding whether myokine secretion is altered in T2D is somewhat more limited. Novel information provided by the current report includes: 1) GROa, IL8, IL15 and follistatin are myokines whose secretion is higher in T2D, 2) secretion of IL1?and VEGF is similar between ND and T2D myotubes, and, 3) IL10 secretion by resting myotubes is modest. Furthermore, the results indicate that INFg does not meet the criteria for a myokine, in agreement with the recent report of Brown et al [33]. Our current observation that TNFa secretion, while low, is elevated in T2D myotubes is confirmatory of our earlier results [13] and in agreement with Green et al [34] and Vandanmagsar et al at the level of gene expression [12]. Consistent with these results, induction of an insulin resistant state by TNFa treatment of hSMC from healthy individuals also resulted in increased TNFa production and secretion [11]. The picture regarding IL6 is more mixed. While we found IL6 secretion to be higher in T2D hSMC, as.