Proinflammatory NFkB signalling promotes mitochondrial dysfunction in skeletal muscle in response to cellular fuel overloading

Sustained nutrient excess of L6 myotubes is associated with a marked reduction in mitochondrial respiratory capacity (>50%).

“We show sustained nutrient excess of L6 myotubes promotes activation of the IKKβ-NFkB pathway (as judged by a six-fold increase in IL-6 mRNA expression; an NFkB target gene) and that this was associated with a marked reduction in mitochondrial respiratory capacity (>50%), a three-fold increase in mitochondrial fragmentation and 2.5-fold increase in mitophagy.”

Under conditions of sustained nutrient excess, there is a reduction in the mRNA and protein abundance of key mitochondrial components SDHA, ANT-1, UCP3, and MFN2 in myotubes.

“Under these circumstances, we also noted a reduction in the mRNA and protein abundance of PGC1α and that of key mitochondrial components (SDHA, ANT-1, UCP3, and MFN2) as well as an increase in cellular ROS and impaired insulin action in myotubes.”

Sustained nutrient excess of L6 myotubes is associated with a three-fold increase in mitochondrial fragmentation.

“We show sustained nutrient excess of L6 myotubes promotes activation of the IKKβ-NFkB pathway (as judged by a six-fold increase in IL-6 mRNA expression; an NFkB target gene) and that this was associated with a marked reduction in mitochondrial respiratory capacity (>50%), a three-fold increase in mitochondrial fragmentation and 2.5-fold increase in mitophagy.”

Sustained nutrient excess of L6 myotubes promotes activation of the IKKβ-NFkB pathway, as judged by a six-fold increase in IL-6 mRNA expression (an NFkB target gene).

“We show sustained nutrient excess of L6 myotubes promotes activation of the IKKβ-NFkB pathway (as judged by a six-fold increase in IL-6 mRNA expression; an NFkB target gene)”

Under conditions of sustained nutrient excess, there is a reduction in the mRNA and protein abundance of PGC1α in myotubes.

“Under these circumstances, we also noted a reduction in the mRNA and protein abundance of PGC1α and that of key mitochondrial components (SDHA, ANT-1, UCP3, and MFN2) as well as an increase in cellular ROS and impaired insulin action in myotubes.”

Under conditions of sustained nutrient excess, there is an increase in cellular ROS in myotubes.

“Under these circumstances, we also noted a reduction in the mRNA and protein abundance of PGC1α and that of key mitochondrial components (SDHA, ANT-1, UCP3, and MFN2) as well as an increase in cellular ROS and impaired insulin action in myotubes.”

Sustained nutrient excess of L6 myotubes is associated with a 2.5-fold increase in mitophagy.

“We show sustained nutrient excess of L6 myotubes promotes activation of the IKKβ-NFkB pathway (as judged by a six-fold increase in IL-6 mRNA expression; an NFkB target gene) and that this was associated with a marked reduction in mitochondrial respiratory capacity (>50%), a three-fold increase in mitochondrial fragmentation and 2.5-fold increase in mitophagy.”

Under conditions of sustained nutrient excess, there is impaired insulin action in myotubes.

“Under these circumstances, we also noted a reduction in the mRNA and protein abundance of PGC1α and that of key mitochondrial components (SDHA, ANT-1, UCP3, and MFN2) as well as an increase in cellular ROS and impaired insulin action in myotubes.”

Treating myotubes with either Mitotempo or MitoQ effectively suppresses increases in both ROS initiated by fuel oversupply.

“Furthermore, we contest that the increase in ROS, we see in response to GLC/PA overloading is principally mitochondrial generated given that treating myotubes with either Mitotempo or MitoQ, two mitochondrial targeted anti-oxidants, which, respectively, scavenge O2–. and quench H2O2, effectively suppress increases in both ROS initiated by fuel oversupply (Supplementary Fig. S1D and S2D).”

The combined presence of GLC (5 mM) and PA (0.4 mM) induces a significant increase in superoxide (O2-.) and H2O2 in myotubes.

“Whilst incubation of myotubes with 5 mM GLC or 0.4 mM PA alone had no significant effect on ROS production in myotubes, the combined presence of both nutrients induced a significant increase in O2–. and H2O2 (Fig. 1c, d).”

Incubation of myotubes with 5 mM GLC or 0.4 mM PA alone has no significant effect on ROS production.

“Whilst incubation of myotubes with 5 mM GLC or 0.4 mM PA alone had no significant effect on ROS production in myotubes, the combined presence of both nutrients induced a significant increase in O2–. and H2O2 (Fig. 1c, d).”

Increases in O2-. and H2O2 are regulated in a PA and GLC concentration-dependent manner.

“The notion that this increase in ROS is driven by nutrient overload is strengthened by our demonstration that increases in O2–. and H2O2 are regulated in a PA and GLC concentration-dependent manner (Supplementary Fig. S1 and S2) and that increases in ROS can be restrained in myotubes exposed to 2-deoxyglucose (2DG); a glycolytic inhibitor that severely restricts GLC use as a metabolic fuel (Fig. 1”

Increases in ROS can be restrained in myotubes exposed to 2-deoxyglucose (2DG), a glycolytic inhibitor that severely restricts GLC use as a metabolic fuel.

“The notion that this increase in ROS is driven by nutrient overload is strengthened by our demonstration that increases in O2–. and H2O2 are regulated in a PA and GLC concentration-dependent manner (Supplementary Fig. S1 and S2) and that increases in ROS can be restrained in myotubes exposed to 2-deoxyglucose (2DG); a glycolytic inhibitor that severely restricts GLC use as a metabolic fuel (Fig. 1”

The threshold concentration at which palmitate induces a significant change in IkBα and IL-6 gene expression is 0.4 mM.

“Since the threshold concentration at which PA induced a significant change in IkBα and IL-6 gene expression was 0.4 mM, all subsequent experiments involving nutrient overloading of myotubes were conducted in the presence of 5 mM GLC and 0.4 mM PA unless otherwise indicated.”

Nutrient-induced disturbances in mitochondrial respiration were ameliorated if activation of the IKKβ-NFkB signalling axis by nutrient excess was repressed by BI605906 in a dose-dependent manner or by expression of the IκBαS32A/S36A mutant.

“Strikingly, these nutrient-induced disturbances in mitochondrial respiration were ameliorated if activation of the IKKβ-NFkB signalling axis by nutrient excess was repressed by BI605906 in a dose-dependent manner (Fig. 6a–e and Supplementary Fig. S3) or expression of the IκBαS32A/S36A mutant (Fig. 6f–j).”

• AssumptionRests on an assumption: Mutation of Ser32 and Ser36 to alanine renders IκBα resistant to IKK phosphorylation, protecting it from proteasomal degradation and retaining its capacity to hold NFκB in an inhibited state.

Repressing NFkB signalling with BI605906 helps attenuate the fragmentation of the elongated/tubular mitochondrial network that is otherwise seen when myotubes are chronically exposed to nutrient excess.

“Figure 7a shows that repressing NFkB signalling with BI605906 helps attenuate the fragmentation of the elongated/tubular mitochondrial network that is otherwise seen when myotubes are chronically exposed to nutrient excess.”

Exogenous supplementation of carnitine (2 mM) to culture media did not enhance or ameliorate the reduction in mitochondrial respiratory capacity seen in PA/GLC-treated myotubes.

“In line with this view, exogenous supplementation of carnitine (2 mM) to culture media did not enhance or ameliorate the reduction in mitochondrial respiratory capacity that we see in PA/GLC treated myotubes (Fig. 2b).”

Repressing NFkB activation by expression of IκBαS32A/S36A blocked the increase in ROS triggered by nutrient overload.

“Similarly, repressing NFkB activation by expression of IκBαS32A/S36A blocked the increase in ROS triggered by nutrient overload (Fig. 5c, d).”

• AssumptionRests on an assumption: Mutation of Ser32 and Ser36 to alanine renders IκBα resistant to IKK phosphorylation, protecting it from proteasomal degradation and retaining its capacity to hold NFκB in an inhibited state.

Irrespective of nutrient overload, 2DG or BI605906 treatment, no significant differences in mitochondrial DNA or citrate synthase activity were observed.

“Figure 7b, c shows that irrespective of whether myotubes were subject to nutrient overload or not, or whether they were treated with 2DG or BI605906, we observed no significant differences in mitochondrial DNA or citrate synthase activity (a representative nuclear-encoded mitochondrial enzyme) in response to the various experimental manipulations.”

• AssumptionRests on an assumption: Mitochondrial DNA copy number and citrate synthase activity are proxies of mitochondrial mass.

The nutrient-induced increase in ROS is accompanied by an increase in the expression of anti-oxidant enzymes SOD2, catalase and GPX1.

“It is noteworthy that this nutrient-induced increase in ROS is accompanied by an attendant increase in the expression of anti-oxidant enzymes [superoxide dismutase 2 (SOD2), catalase and glutathione peroxidase (GPX1)], which most likely forms part of a cellular defence mechanism designed to help limit oxidative damage/stress under these circumstances (Supplementary Fig. S2E).”

The respiratory changes induced by simultaneous GLC and PA exposure are averted by addition of 2DG to inhibit glucose use as a metabolic substrate.

“Strikingly, these respiratory changes are averted in myotubes incubated in media containing GLC and PA, but to which 2DG was also added to inhibit glucose use as a metabolic substrate.”

The morphological change in the mitochondrial network caused by substrate overloading was mitigated by 2DG.

“In line with the finding that glycolytic inhibition with 2DG helps preserve mitochondrial respiratory function (Fig. 1e–h), the morphological change in the mitochondrial network caused by substrate overloading was mitigated by 2DG (Fig. 3).”

The reduction in IkBα instigated by a 16 h period of nutrient (GLC + PA) overloading in myotubes was halted by BI605906 or by cellular expression of the IkBα mutant.

“The data in Fig. 4a, b show that the reduction in IkBα instigated by a 16 h period of nutrient (GLC + PA) overloading in myotubes was halted by BI605906 or by cellular expression of the IkBα mutant.”

• AssumptionRests on an assumption: Mutation of Ser32 and Ser36 to alanine renders IκBα resistant to IKK phosphorylation, protecting it from proteasomal degradation and retaining its capacity to hold NFκB in an inhibited state.

The reduced respiratory rate seen under nutrient overload was associated with a significant reduction in the abundance of UCP3, ANT1, SDHA, and PGC1α proteins.

“Whilst mitochondrial mass in L6 myotubes was unaltered following a 16 h period of nutrient overload, the reduced respiratory rate seen under these circumstances was associated with a significant reduction in the abundance of proteins with key roles in mitochondrial bioenergetics. These include uncoupling protein 3 (UCP3), mitochondrial ADP–ATP translocase (ANT1) and succinate dehydrogenase (SDHA)”

No change in the protein abundance of Cox 4.1 was seen in myotubes exposed to both GLC and 0.4 mM PA.

“Whilst we saw no change in the protein abundance of Cox 4.1 (a subunit of Cytochrome C-oxidase that functions within the mitochondrial respiratory chain) in myotubes exposed to both GLC and 0.4 mM PA”

BI605906 did not suppress the increased expression of SOD2, catalase or GPX1 induced by cellular over-nutrition.

“Intriguingly, however, whilst the elevated ROS generation induced by cellular over-nutrition was restrained by BI605906 (Fig. 5a, b), the inhibitor did not to suppress the increased expression of SOD2, catalase or GPX1 (Supplementary Fig. S2E)”

The improved respiratory drive under these circumstances is associated with a significant increase in mitochondrial proton leak.

“Notably, the improved respiratory drive that we see under these circumstances is associated with a significant increase in mitochondrial proton leak (Fig. 6e, j)”

Associated with the loss in Akt-directed insulin signalling was a substantial loss in insulin-stimulated glucose uptake.

“Associated with this loss in Akt-directed insulin, signalling was a substantial loss in insulin-stimulated glucose uptake (Fig. 9c).”

The observed changes in mitochondrial-associated Drp1 and MFN2 abundance were repressed in fuel-loaded myotubes treated with BI605906.

“In contrast, the observed changes in mitochondrial-associated Drp1 and MFN2 abundance were repressed in fuel-loaded myotubes that had been treated with BI605906 and would fit with the reduced mitochondrial fragmentation that we see (Fig. 7a).”

Insulin induces a robust increase in AktSer473 phosphorylation/activation that is blunted significantly (by ~58%) in myotubes subject to GLC/PA oversupply.

“Figure 9a shows that insulin induces a robust increase in AktSer473 phosphorylation/activation that is blunted significantly (by ~ 58%) in myotubes that have been subject to GLC/PA oversupply (Fig. 9b).”

Targeting NOXII with the cell permeable inhibitor VAS2870 proved ineffective in countering the decline in respiratory capacity caused by nutrient excess.

“We are mindful that ROS production can also occur at extra-mitochondrial sites such as via NADPH oxidase (NOXII) in the cytosol, but targeting NOXII with a cell permeable inhibitor, VAS2870, also proved ineffective in countering the decline in respiratory capacity caused by nutrient excess (Supplementary Fig. S4).”

No notable differences were observed for mitofusin 1 (MFN1) or optic atrophy-1 protein (OPA-1) in the mitochondrial and cytosolic fractions examined.

“No notable differences were observed for mitofusin 1 (MFN1) or optic atrophy-1 protein (OPA-1) in the mitochondrial and cytosolic fractions that we examined (Fig. 8a).”

The observations of nutrient-induced mitochondrial changes are not restricted to L6 myotubes but can also be demonstrated in LHCN-M2 myotubes, a myogenic cell line from skeletal muscle of a healthy male human subject.

“It is also important to highlight that these observations are not just restricted to L6 myotubes, but can also be demonstrated in LHCN-M2 myotubes; a myogenic cell line obtained from skeletal muscle of a healthy male human subject (Supplementary Fig. S5).”

Some recovery in the expression of SDHA was seen with mitochondrial targeted anti-oxidants.

“However, we did see some recovery in the expression of SDHA.”

Sustained exposure of myotubes to 5 mM GLC and 0.4 mM PA induced a very modest increase in the mitochondrial abundance of Drp1.

“Figure 8a shows that sustained exposure of myotubes to 5 mM GLC and 0.4 mM PA induced a very modest increase in the mitochondrial abundance of Drp1, a GTPase that facilitates mitochondrial fission.”

A loss in mitochondrial mitofusin 2 (MFN2) was noted after sustained exposure to GLC and PA.

“By contrast, we noted a loss in mitochondrial mitofusin 2 (MFN2), a GTPase resident on the outer mitochondrial membrane involved in mitochondrial clustering and fusion (Fig. 8a).”

In our in vitro myotube cultures, nutrient oversupply with GLC and PA initially increases gene expression of PGC1α, SDHA, ANT-1, and UCP3 during the first 4 h, then rapidly declines over the succeeding 20 h.

“whilst we observe an initial increase in the gene expression of PGC1α, SDHA, ANT-1, and UCP3 (during the first 4 h of nutrient oversupply), we find that this rapidly declines over the succeeding 20 h myotube incubation period with GLC and PA (Supplementary Fig. S7).”

Heightened ROS production is a feature of mitochondrial substrate overload.

“Our findings reveal that heightened ROS production is indeed a feature of mitochondrial substrate overload (Fig. 5)”

The decline in mitochondrial protein gene expression correlates with an increase in NFkB activation as judged by loss of IkBα and increased IL-6 expression from 4 h onwards in myotubes.

“This decline correlates with an associated increase in NFkB activation as judged by the loss in IkBα and increased expression of IL-6 that is observed from 4 h onwards in myotubes (Supplementary Fig. S7).”

In myotubes incubated with GLC/PA, uncoupled respiration in the presence of FCCP was markedly lower than in myotubes treated with either nutrient alone.

“uncoupled respiration in the presence of FCCP was markedly lower in myotubes incubated with GLC/PA than those treated with either nutrient alone (Fig. 1e)”

In fuel-overloaded myotubes, heightened ROS production occurs despite an attendant increase in expression of SOD2, GPX1 and catalase.

“this occurs despite an attendant increase in the expression of SOD2, GPX1 and catalase (Supplementary Fig. S2)”

Treatment of myotubes with either Mitotempo or MitoQ could not rescue the decline in mitochondrial respiratory capacity induced by fuel overloading.

“treatment of myotubes with either Mitotempo or MitoQ could not rescue the decline in mitochondrial respiratory capacity induced by fuel overloading.”

Targeted inhibition of the IKK-NFkB axis helps mitigate the loss of mitochondrial proteins and the associated decline in respiratory function.

“the targeted inhibition of the IKK–NFkB axis helps mitigate the loss of these mitochondrial proteins and the associated decline in respiratory function.”

Activation of canonical NFkB signalling initiated in response to fuel overloading promotes mitochondrial fragmentation.

“our studies indicate that the activation of canonical NFkB signalling initiated in response to fuel overloading promotes mitochondrial fragmentation”

The observed increase in mitophagy and associated loss of key mitochondrial proteins (SDHA, ANT-1, UCP3) in fuel-overloaded myotubes occur despite no significant change in mitochondrial DNA or citrate synthase activity.

“the observed increase in mitophagy and the associated loss of key mitochondrial proteins (SDHA, ANT-1, and UCP3) that we see in fuel-overloadeded myotubes occur despite their being no significant change in mitochondrial DNA or citrate synthase activity.”

GLC/PA overloading of L6 myotubes in our studies induced a profound morphological change in mitochondria from an elongated/tubular network to one that is highly fragmented, indicative of a dynamic shift towards greater fission.

“GLC/PA overloading of L6 myotubes in our studies induced a profound morphological change in mitochondria from an elongated/tubular network to one that is highly fragmented, indicative of a dynamic shift towards greater fission.”

Sustained oversupply of metabolic fuel to skeletal muscle cells induces heightened NFkB signalling.

“Our findings indicate that sustained oversupply of metabolic fuel to skeletal muscle cells induces heightened NFkB signalling and that this serves as a critical driver for disturbances in mitochondrial function and morphology, redox status, and insulin signalling.”

• AssumptionRests on an assumption: The L6 myotube model and the 16 h GLC/PA overloading protocol recapitulate the metabolic and inflammatory state of skeletal muscle in obesity and Type II diabetes.

The actions of proinflammatory stimuli impair control of numerous signalling pathways regulating skeletal muscle insulin signalling, glucose uptake, mitochondrial fuel oxidation and respiration, impacting negatively upon skeletal muscle energy homeostasis.

“The actions of these stimuli serve to not only further induce tissue expression and secretion of IL-6 and TNFα (via the NFκB pathway, [3, 4]), but impair control of numerous signalling pathways regulating skeletal muscle insulin signalling, glucose uptake, mitochondrial fuel oxidation and respiration that impact negatively upon skeletal muscle energy homeostasis [2, 5–7].”

• cites [2] Funaki M (2009) Saturated fatty acids and insulin resistance. J Med Invest 56:88–92 sourceNot checked yet

  Not checked in the free run.

• cites [5] Boden G (2006) Fatty acid-induced inflammation and insulin resistance in skeletal muscle and liver. Curr Diab Rep 6:177–181 sourceNot checked yet

  Not checked in the free run.

• cites [6] Powell DJ et al (2004) Intracellular ceramide synthesis and protein kinase Czeta activation play an essential role in palmitate-induced insulin resistance in rat L6 skeletal muscle cells. Biochem J 382:619–629 sourceNot checked yet

  Not checked in the free run.

• cites [7] Barma P et al (2009) Lipid induced overexpression of NF-kappaB in skeletal muscle cells is linked to insulin resistance. Biochim Biophys Acta 1792:190–200 sourceNot checked yet

  Not checked in the free run.

The actions of proinflammatory stimuli further induce tissue expression and secretion of IL-6 and TNFα via the NFκB pathway.

“The actions of these stimuli serve to not only further induce tissue expression and secretion of IL-6 and TNFα (via the NFκB pathway, [3, 4]), but impair control of numerous signalling pathways regulating skeletal muscle insulin signalling, glucose uptake, mitochondrial fuel oxidation and respiration that impact negatively upon skeletal muscle energy homeostasis [2, 5–7].”

• cites [3] Jove M et al (2005) Palmitate-induced interleukin 6 production is mediated by protein kinase C and nuclear-factor kappaB activation and leads to glucose transporter 4 down-regulation in skeletal muscle cells. Endocrinology 146:3087–3095 sourceNot checked yet

  Not checked in the free run.

• cites [4] Weigert C et al (2004) Palmitate, but not unsaturated fatty acids, induces the expression of interleukin-6 in human myotubes through proteasome-dependent activation of nuclear factor-kappaB. J Biol Chem 279:23942–23952 sourceNot checked yet

  Not checked in the free run.

Sustained oversupply of metabolic fuel (glucose and fatty acids) to skeletal muscle, as seen during Type II diabetes and obesity, impairs the ability of mitochondria to shift between use of lipid during fasting and use of carbohydrate in the post-prandial state.

“Notably, the sustained oversupply of metabolic fuel (glucose and fatty acids) to skeletal muscle, as seen during Type II diabetes and obesity, impairs the ability of mitochondria to shift between use of lipid during fasting and use of carbohydrate in the post-prandial state.”

• cites [10] Muoio DM (2014) Metabolic inflexibility: when mitochondrial indecision leads to metabolic gridlock. Cell 159:1253–1262 sourceNot checked yet

  Not checked in the free run.

Chronic activation of proinflammatory signalling in skeletal muscle and adipose is a significant contributing factor in the development and progression of metabolic disorders such as insulin resistance, obesity and Type II diabetes.

“There is mounting evidence that chronic activation of proinflammatory signalling in tissues such as skeletal muscle and adipose is a significant contributing factor in the development and progression of metabolic disorders such as insulin resistance, obesity and Type II diabetes [1, 2].”

• cites [1] Feve B, Bastard JP (2009) The role of interleukins in insulin resistance and type 2 diabetes mellitus. Nat Rev Endocrinol 5:305–311 sourceNot checked yet

  Not checked in the free run.

• cites [2] Funaki M (2009) Saturated fatty acids and insulin resistance. J Med Invest 56:88–92 sourceNot checked yet

  Not checked in the free run.

Heightened NFkB signalling serves as a critical driver for disturbances in mitochondrial function and morphology, redox status, and insulin signalling in skeletal muscle cells under fuel oversupply.

“Our findings indicate that sustained oversupply of metabolic fuel to skeletal muscle cells induces heightened NFkB signalling and that this serves as a critical driver for disturbances in mitochondrial function and morphology, redox status, and insulin signalling.”

• AssumptionRests on an assumption: The L6 myotube model and the 16 h GLC/PA overloading protocol recapitulate the metabolic and inflammatory state of skeletal muscle in obesity and Type II diabetes.

Obesity in rodents and chronic oversupply of metabolic fuel to skeletal muscle cells in vitro is associated with an increase in proinflammatory NFkB signalling and insulin resistance.

“We, and others, have also previously demonstrated that obesity in rodents and chronic oversupply of metabolic fuel to skeletal muscle cells in vitro is associated with an increase in proinflammatory NFkB signalling and insulin resistance [17, 25, 26].”

• cites [17] Lipina C et al (2013) Mitochondrial substrate availability and its role in lipid-induced insulin resistance and proinflammatory signalling in skeletal muscle. Diabetes 62:3426–3436 sourceNot checked yet

  Not checked in the free run.

• cites [25] Green CJ et al (2011) Counter-modulation of fatty acid-induced pro-inflammatory nuclear factor kappaB signalling in rat skeletal muscle cells by AMP-activated protein kinase. Biochem J 435:463–474 sourceNot checked yet

  Not checked in the free run.

• cites [26] Coll T et al (2008) Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells. J Biol Chem 283:11107–11116 sourceNot checked yet

  Not checked in the free run.

Mitochondrial fuel overload increases production of reactive oxygen species (ROS).

“Mitochondrial fuel overload also increases production of reactive oxygen species (ROS) [19, 20] that may serve to trigger or prime mechanisms that not only induce, but also sustain the loss in mitochondrial function.”

• cites [19] Bonnard C et al (2008) Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice. J Clin Invest 118:789–800Not checked yet

  Not checked in the free run.

• cites [20] Yuzefovych L, Wilson G, Rachek L (2010) Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress. Am J Physiol Endocrinol Metab 299:E1096–E1105 sourceNot checked yet

  Not checked in the free run.

Combined provision of GLC (5 mM) and PA (0.4 mM) for 16 h did not invoke notable death or loss of terminally differentiated myotubes.

“It is important to stress that combined provision of GLC and PA at these concentrations did not invoke any notable death or loss of terminally differentiated myotubes on culture plates.”

Expression of SOD2, catalase and GPX1 is likely regulated by mechanisms distinct from those involved in promoting generation of O2- and H2O2.

“suggesting that expression of these enzymes was likely to be regulated by mechanisms that are distinct to those involved in promoting generation of O2– and H2O2.”

The increase in ROS observed in response to GLC/PA overloading is principally mitochondrial generated.

“Furthermore, we contest that the increase in ROS, we see in response to GLC/PA overloading is principally mitochondrial generated given that treating myotubes with either Mitotempo or MitoQ, two mitochondrial targeted anti-oxidants, which, respectively, scavenge O2–. and quench H2O2, effectively suppress increases in both ROS initiated by fuel oversupply (Supplementary Fig. S1D and S2D).”

It is unlikely that carnitine is limiting for mitochondrial uptake and oxidation of PA at the concentrations used in these studies.

“Consequently, we believe it unlikely that carnitine would be limiting for mitochondrial uptake and oxidation of PA at the concentrations used in our studies.”

Adenoviral mediated expression of a mutant form of IκBα (S32A/S36A) functions as a super-repressor of NFκB activity.

“the second involved adenoviral mediated expression of a mutant form of IκBα (S32A/S36A) that functions as a super-repressor of NFκB activity [25].”

• cites [25] Green CJ et al (2011) Counter-modulation of fatty acid-induced pro-inflammatory nuclear factor kappaB signalling in rat skeletal muscle cells by AMP-activated protein kinase. Biochem J 435:463–474 sourceNot checked yet

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The decline in UCP3, ANT1, SDHA, and PGC1α proteins may have been driven by a decrease in gene expression based on the reduced mRNA abundance that respectively encode the four proteins.

“Our analysis reveals that the decline in these proteins may have been driven by a decrease in gene expression based on the reduced mRNA abundance that, respectively, encode the four proteins (Fig. 7e).”

Heightened NFkB signalling associated with sustained nutrient oversupply, as occurs during obesity, plays an important role in lipid-induced insulin resistance and metabolic dysfunction in skeletal muscle both in vitro and in vivo.

“We have previously shown that heightened NFkB signalling associated with sustained nutrient oversupply, as occurs during obesity, plays an important role in lipid-induced insulin resistance and metabolic dysfunction in skeletal muscle both in vitro and in vivo [17].”

• cites [17] Lipina C et al (2013) Mitochondrial substrate availability and its role in lipid-induced insulin resistance and proinflammatory signalling in skeletal muscle. Diabetes 62:3426–3436 sourceNot checked yet

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The increased expression of SOD2, GPX1 and catalase most likely represents a cellular stress response designed to limit oxidative stress.

“that most likely represent a cellular stress response designed to limit oxidative stress.”

The observed perturbations in mitochondrial homeostasis in this study may in part be a consequence of cellular lipotoxicity initiated by accumulation of molecules such as DAG and ceramide associated with sustained oversupply of fatty acids.

“It is, therefore, plausible that the observed perturbations in mitochondrial homeostasis seen in this study may, in part, be a consequence of cellular lipotoxicity initiated by the accumulation of molecules such as DAG and ceramide that would be associated with sustained oversupply of fatty acids.”

The combined carbon load of GLC and PA appears to overwhelm the respiratory capacity of myotubes, especially if it is not matched by any increase in energy demand.

“These findings imply that whilst myotubes can efficiently metabolise GLC (5 mM) or PA (0.4 mM) when presented independently at the concentrations indicated, their combined carbon load appears to overwhelm the respiratory capacity of myotubes, especially if it is not matched by any increase in energy demand.”

Increased fission is likely secondary to increased production of ROS that stems from mitochondrial dysfunction induced by activation of the NFkB pathway.

“we postulate that increased fission is likely to be secondary to the increased production of ROS that stems from mitochondrial dysfunction induced by activation of the NFkB pathway.”

Therapeutic strategies that restrain NFkB activation during energy excess, such as obesity and age-onset Type II diabetes, may help counter disturbances in mitochondrial homeostasis and impart beneficial effects upon skeletal muscle insulin sensitivity.

“our observations would imply that therapeutic strategies that help restrain NFkB activation as seen during circumstances of energy excess, such as obesity and age-onset Type II diabetes, may help counter disturbances in mitochondrial homeostasis and impart beneficial effects upon skeletal muscle insulin sensitivity.”