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

Abstract

Sustained nutrient (fuel) excess, as occurs during obesity and diabetes, has been linked to increased inflammation, impaired mitochondrial homeostasis, lipotoxicity, and insulin resistance in skeletal muscle. Precisely how mitochondrial dysfunction is initiated and whether it contributes to insulin resistance in this tissue remains a poorly resolved issue. Herein, we examine the contribution that an increase in proinflammatory NFkB signalling makes towards regulation of mitochondrial bioenergetics, morphology, and dynamics and its impact upon insulin action in skeletal muscle cells subject to chronic fuel (glucose and palmitate) overloading. 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 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. Strikingly, pharmacological or genetic repression of NFkB activity ameliorated disturbances in mitochondrial respiratory function/morphology, attenuated loss of SDHA, ANT-1, UCP3, and MFN2 and mitigated the increase in ROS and the associated reduction in myotube insulin sensitivity. 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.

Keywords: Glucose; Insulin; Metabolism; Mitochondria; Muscle; NFkB; Palmitate; Respiration.

Fig. 1

Cellular fuel overloading induces NFkB inflammatory signalling, ROS (superoxide and H₂O₂) production and mitochondrial dysfunction. L6 myotubes were incubated with glucose (GLC, 5 mM) in the absence and presence of palmitate (PA) at doses indicated for 16 h prior to analysis of (a) cellular IkBα abundance by immunoblotting and b IL6 and β-actin mRNA abundance by qPCR. For analysis of superoxide (c) and H₂O₂ (d) L6 myotubes were treated as in (a) with PA (0.4 mM) for 16 h in absence or presence GLC (5 mM) and/or 2-deoxyglucose (2DG, 5 mM) as indicated followed by quantification of ROS using 5 μM of either Mitosox or MitoPYI as detailed in methods. For analysis of real-time cellular respiration in L6 myotubes we used a Seahorse XF24 analyser. L6 myotubes were incubated for 16 h with either GLC (5 mM) or PA (0.4 mM) alone, PA (0.4 mM)/GLC (5 mM) together or with a mixture of PA (0.4 mM)/GLC (5 mM)/2DG (5 mM). Oligomycin (1 μM), FCCP (1 μM) and a rotenone (1 μM)/antimycin-A (2 μM) mix were added at times indicated by dotted lines on the Seahorse trace. The trace shown in (e) is a representative readout of oxygen consumption rate (OCR) from a single experiment with measurements (mean ± SD) of triplicate values. Analysis of absolute basal mitochondrial OCR normalised to protein/well (f), coupling efficiency of oxidative phosphorylation (g, determined as the oligomycin sensitivity fraction of the basal respiratory rate) and (h) the respiratory capacity ratio (calculated as a factor of the FCCP-stimulated respiration/oligomycin resistance). The data shown in (f–h) is the combined analyses of three separate experiments. All data are presented as mean ± SEM. Asterisks indicate a significant change (P < 0.05) to the GLC alone condition or between the indicated bars

Fig. 2

Effect of glucose/palmitate overloading and carnitine supplementation on mitochondrial function in L6 myotubes. L6 myotubes were incubated with glucose (GLC, 5 mM), palmitate (PA, 0.4 mM or 0.7 mM) or with GLC (5 mM) and PA (0.4 mM) together for 16 h as shown in (a) or in some experiments (b) when treated with GLC and PA together such treatments were done in the presence of either carnitine (2 mM) or 2-deoxyglucose (2DG, 5 mM) prior to analysis of real-time cellular respiration using a Seahorse XF24 analyser. All data are presented as mean ± SEM. Asterisks indicate a significant change (P < 0.05) between the indicated bars. NS signifies no significant change

Fig. 3

Effect of glucose/palmitate overloading on mitochondrial morphology in L6 myotubes. L6 myotubes were incubated for 16 h with either GLC (5 mM) or PA (0.4 mM) alone, PA (0.4 mM)/GLC (5 mM) together or with a mixture of PA (0.4 mM)/GLC (5 mM)/2DG (5 mM) prior to staining with Mitotracker green (Mitospy) and confocal microscopy. a Confocal images depict mitochondrial morphology in L6 myotubes (the scale bar represents 5 μm). Nuclei (N) are labelled and white boxed areas are magnified to show differences in morphology. The arrow heads depict fragmented mitochondria. b Mitochondrial length was quantified using the imaging software and presented as elongated/tubular if greater than 1 μm and fragmented if < 1 μm in length. Data are presented as mean + SEM from a minimum of three separate experiments. Asterisks indicate a significant change (P < 0.05) between the black filled bars, whereas the hash (#) indicates a significant difference (P < 0.05) between the grey filled bars

Fig. 4

Effects of suppressing NFkB on inflammatory signalling induced by nutrient overload in L6 myotubes. a L6 myotubes were incubated for 16 h with either GLC (5 mM) or PA (0.4 mM) alone, PA (0.4 mM)/GLC (5 mM) together or with a mixture of PA (0.4 mM)/GLC (5 mM)/2DG (5 mM) in the absence or presence of BI605906 (10 μM), an IKKβ inhibitor prior to analysis of cellular IkBα abundance by immunoblotting. b L6 myoblasts were infected with an adenoviral vector expressing HA-tagged non-phosphorylatable IkBα (S32A/S36A) or one expressing an empty vector (EV). Cells were allowed to differentiate prior to treatment of cells with GLC, PA and 2DG and analysis of IkBα abundance as described in (a). L6 myotubes were treated with GLC (5 mM), PA (0.4 mM), BI605906 (10 μM) and adenoviral vectors as indicated prior to (c) subcellular fractionation and analyses of cytosolic and nuclear NFkB p65 abundance by immunoblotting or d analysis of IL-6 gene expression. All data are presented as mean ± SEM from a minimum of three separate experiments. Asterisks indicate a significant change (P < 0.05) between the indicated bars

Fig. 5

Effects of NFkB antagonism on ROS production. a, b L6 myotubes were incubated for 16 h with either GLC (5 mM) or PA (0.4 mM) alone, PA (0.4 mM)/GLC (5 mM) together in the absence or presence of BI605906 (10 μM) or subject to these treatments having been infected with an adenoviral vector expressing HA-tagged non-phosphorylatable IkBα (S32A/S36A) or one expressing an empty vector (EV) c, d prior to analysis of superoxide and hydrogen peroxide. All data are presented as mean ± SEM from four separate experiments. Asterisks indicate a significant change (P < 0.05) between the indicated bars

Fig. 6

Effect of suppressing NFkB activation in response to cellular fuel overloading on mitochondrial respiration. a–e L6 myotubes were incubated for 16 h with either GLC (5 mM) or PA (0.4 mM) alone, PA (0.4 mM)/GLC (5 mM) together in the absence or presence of BI605906 (10 μM) or f–j subject to these treatments having been infected with an adenoviral vector expressing HA-tagged non-phosphorylatable IkBα (S32A/S36A) or one expressing an empty vector (EV) prior to analysis of real-time cellular respiration in L6 myotubes using a Seahorse XF24 analyser. Oligomycin (1 μM), FCCP (1 μM) and a rotenone (1 μM)/antimycin-A (2 μM) mix were added at times indicated by dotted lines. a, f show representative readouts of oxygen consumption rate (OCR) from a single experiment with measurements (mean ± SD) of triplicate values. b, g Depict basal mitochondrial oxygen consumption rate (OCR), c, h maximal respiration (OCR after FCCP stimulation), d, i ATP-linked respiration (oligomycin-sensitive OCR) and e–j proton leak (oligomycin resistance rate). Data are presented as mean ± SEM from five independent experiments. Asterisks indicate a significant change (P < 0.05) between the indicated bars

Fig. 7

Effect of suppressing NFkB activation in response to cellular fuel overloading on mitochondrial morphology, mitochondrial proteins and gene expression. L6 myotubes were incubated with GLC (5 mM), PA (0.4 mM), 2DG (5 mM) and BI605906 (10 μM) for 16 h in the combinations indicated in the various experimental data panels prior to a analysis and quantification of mitochondrial morphology using Mitotracker green (Mitospy) by confocal microscopy (the scale bar represents 5 μm), b mitochondrial DNA copy number by qPCR, c citrate synthase (CS) activity and (d, e), analysis of mitochondria protein and mRNA abundance (UCP3, ANT1, PGC1α, SDHA, and COX4.1) which was normalised to GAPDH. All graphical bar data are presented as mean ± SEM from four separate experiments. Asterisks indicate a significant change (P < 0.05) to the GLC alone condition

Fig. 8

Effects of cellular fuel overloading on mitophagy and upon proteins linked to mitochondrial dynamics. Wild type L6 myotubes or those stably expressing a retroviral vector encoding a GFP–mCherry–Fis1 mitophagy reporter were incubated with GLC (5 mM), PA (0.4 mM) and BI605906 (10 μM) for 16 h as indicated prior to: a subcellular fractionation and isolation of a cytosolic and mitochondrial-enriched membrane fraction for immunoblotting with antibodies to proteins shown. b–e Fixing and confocal imaging to visualise and f quantifying (using the Volocity software) mitophagy in L6 myotubes. Data (mean ± SEM) in (f) are from five separate experiments. Asterisks indicate a significant change (P < 0.05) between the indicated bars. Boxed sections in panels b, c, d and e have been expanded to highlight mitophagic particles, some of which are depicted by the white arrow heads

Fig. 9

Effects of suppressing NFkB signalling in response to cellular fuel overloading on insulin-stimulated PKB phosphorylation and glucose uptake in L6 myotubes a–c L6 myotubes were incubated for 16 h with GLC (5 mM) or PA (0.4 mM) alone or with PA (0.4 mM)/GLC (5 mM) together in the absence or presence of BI605906 (10 μM). Alternatively, d–f muscle cells were subject to incubation with GLC and PA as indicated having been infected with an adenoviral vector expressing HA-tagged non-phosphorylatable IkBα (S32A/S36A) or one expressing an empty vector (EV) prior to acute stimulation with insulin (20 nM) for 15 min (Akt signalling experiments) or insulin (100 nM) for 20 min for glucose uptake studies. Following insulin stimulation, cells were either lysed for immunoblotting using antibodies to proteins indicated (a, d) or used for assay of glucose uptake. The data are mean ± SEM. from 5 separate experiments. Asterisks indicate a significant change (P < 0.05) between the indicated bars