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

In the paper

“Indeed, mitochondrial dysfunction and/or a reduction in mitochondrial content, as characterised by a fall in oxidative capacity, has been reported in skeletal muscle and adipose tissue from obese and diabetic subjects [8, 9].”

What the sources say

• Not checked yetcites [8] Kelley DE et al (2002) Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes. Diabetes 51:2944–2950 source
• Partly supported

  The Subcellular distribution of mitochondria section reports reduced skeletal-muscle mitochondrial DNA content and electron transport chain activity in obese and type 2 diabetic subjects relative to lean volunteers. The paper does not report corresponding findings in adipose tissue, so it supports only the skeletal-muscle part of the cited sentence. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  cites [9] Ritov VB et al (2005) Deficiency of subsarcolemmal mitochondria in obesity and type 2 diabetes. Diabetes 54:8–14 sourceExperimental — accuracy not yet measured

In the paper

“In skeletal muscle, reduced mitochondrial respiration has also been linked to aberrant control of mitochondrial dynamics [64–67].”

What the sources say

• Partly supported

  The “Mitochondrial dysfunction” section reports reduced mitochondrial ATP production in aging skeletal muscle and describes enlarged, disrupted mitochondria. It does not establish that reduced respiration is linked to aberrant control of mitochondrial dynamics, so the claimed link is unsupported. (LLM, conf=0.78)

  cites [64] Johnson ML, Robinson MM, Nair KS (2013) Skeletal muscle aging and the mitochondrion. Trends Endocrinol Metab 24:247–256 sourceExperimental — accuracy not yet measured
• Not checked yetcites [65] Iqbal S et al (2013) Expression of mitochondrial fission and fusion regulatory proteins in skeletal muscle during chronic use and disuse. Muscle Nerve 48:963–970 source
• Partly supported

  The Abstract links aberrant mitochondrial fission in skeletal muscle cells to mitochondrial dysfunction, including loss of ATP production. But the paper does not report reduced respiration linked to altered dynamics in skeletal muscle: the Introduction’s explicit examples of decreased respiration concern fibroblasts and HeLa cells, and the Discussion notes unchanged muscle respiration rates in cited short-term feeding studies. The claim is supported for mitochondrial dysfunction, but not for respiration specifically. (LLM, conf=0.83)

  cites [66] Jheng HF et al (2012) Mitochondrial fission contributes to mitochondrial dysfunction and insulin resistance in skeletal muscle. Mol Cell Biol 32:309–319 sourceExperimental — accuracy not yet measured
• Not checked yetcites [67] Smith ME et al (2013) Mitochondrial fission mediates ceramide-induced metabolic disruption in skeletal muscle. Biochem J 456:427–439 source

In the paper

“MEF cells lacking IKKα (but not IKKβ) exhibit reduced cellular expression of OPA-1 and display enhanced mitochondrial fission [69].”

What the sources say

• Partly supported

  The Results and discussion report that IKKα-deficient MEFs, but not IKKβ-deficient MEFs, have markedly lower OPA1 levels. They also report a fragmented mitochondrial network in IKKα-deficient MEFs, but do not establish that mitochondrial fission itself is enhanced; fragmentation could also reflect reduced fusion. (LLM, conf=0.84)

  cites [69] Laforge M et al (2016) NF-kappaB pathway controls mitochondrial dynamics. Cell Death Differ 23:89–98 sourceExperimental — accuracy not yet measured

In the paper

“Figure 1a, b shows that myotubes exposed to increasing PA concentrations exhibit a dose-dependent reduction in cellular IkBα abundance that was associated with a concomitant increase in IL-6 gene expression.”

In the paper

“Figure 1e–h shows that compared to myotubes incubated with either 5 mM GLC or 0.4 mM PA alone, those exposed to both carbon fuels simultaneously had a lower OCR and also exhibited a significant reduction in basal respiration, OXPHOS coupling, and respiratory capacity.”

In the paper

“Strikingly, in myotubes that had been incubated with GLC or PA alone, the green-fluorescent mitochondrial dye highlights that ~ 80% of the mitochondrial population is part of an organised elongated/tubular network, which becomes structurally fragmented and spheroid in nature when myotubes are subjected to a sustained period of fuel overloading with GLC and PA (Fig. 3a, b).”

In the paper

“Analysis of ROS production in L6 myotubes revealed that in the absence of PA provision, BI605906 per se has no detectable effect upon cellular O2–. or H2O2, but significantly reduces the increase in both species when myotubes were subjected to chronic nutrient oversupply (Fig. 5a, b).”

In the paper

“Moreover, consistent with this observation, both BI605906 and IκBαS32A/S36A expression attenuated nuclear localisation of the p65 subunit of NFkB and reduced transcription of the IL-6 gene (an NFkB target gene) that is otherwise seen in GLC+PA-overloaded myotubes (Fig. 4c, d).”

In the paper

“In line with the findings presented in Fig. 1e–h, sustained (16 h) exposure of myotubes to nutrient excess (GLC and PA) induced a significant decline in the basal and maximal respiratory rate, which was associated with a reduction in ATP-linked respiration and a modest decline in mitochondrial proton leak (Fig. 6a–j).”

In the paper

“However, cotreatment of fuel-loaded myotubes with BI605906 or expression of the IκBαS32A/S36A super-repressor (Fig. 9d) not only mitigates the reduction in insulin-stimulated Akt phosphorylation (Fig. 9a, b, d, e), but also partially rescues the loss in hormone-stimulated glucose uptake (Fig. 9c, f).”

In the paper

“Whilst this approach does not quantitatively assess mitophagic flux, it indicates that the combined provision of GLC and PA induces a significant increase in mitophagic particles (Fig. 8d, f) whose appearance is restrained upon cotreatment of myotubes with BI605906 (Fig. 8e, f).”

In the paper

“Furthermore, allied to these observations, it is worth stressing that withholding either GLC or PA from the media or inhibiting GLC use as a metabolic fuel in the presence of PA not only averts activation of the NFkB pathway but antagonises mitochondrial fragmentation and the loss in respiratory function (Figs. 1b, 3a, 4a).”

In the paper

“Strikingly, our data indicate that pharmacological or genetic inhibition of the IKK–NFkB axis not only obviates detrimental changes in mitochondrial morphology and function, but also helps ameliorate disturbances in insulin signalling and insulin-dependent glucose uptake.”

In the paper

“However, our analysis indicates that whilst exposing myotubes to two distinct mitochondrial targeted anti-oxidants (Mitotempo and Mito Q) limits fragmentation of the mitochondrial network induced by nutrient oversupply, neither compound could rescue the loss in UCP3, ANT1, or PGC1α expression or prevent the decline in mitochondrial respiratory capacity (Supplementary Fig. S4).”

In the paper

“pharmacological inhibition of the IKK–NFkB axis in GLC/PA-overloaded myotubes not only suppresses the relative changes in Drp1 and MFN2, but reduces fragmentation of the mitochondrial network, in which, significantly, we find was also associated with amelioration in cellular respiratory capacity and insulin action.”

In the paper

“The fact that suppressing NFkB activation in fuel-overloaded myotubes with BI605906 or 2DG antagonises loss of UCP3 and ANT-1 and that, under these circumstances, we not only see a measurable increase in mitochondrial proton leak, but observe improved insulin sensitivity in L6 myotubes (Fig. 9) and in muscle of obese Zucker rats [17] is fully congruent with this view.”

What the sources say

• Not checked yetcites [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 source

In the paper

“The increase in Drp1 (a profission protein) and reduction in MFN2 (a profusion protein) that we see within a mitochondrial-enriched membrane fraction is fully consistent with this view.”

In the paper

“we observed a significant increase in the number of mitophagic particles in myotubes subjected to GLC/PA loading, and in line with the protective effects, we suggest that the inhibition of canonical NFkB signalling confers upon mitochondrial integrity and function; this increase in mitophagy was blunted in myotubes that had been cotreated with BI606906.”

In the paper

“In support of this latter idea, signalling initiated by toll-like receptor-4 (TLR-4) and the tumor necrosis factor-α (TNF-α) receptor results in activation of the NFkB pathway, which has been linked to reduced mitochondrial respiration and suppressed activation of transcriptional regulators that promote mitochondrial biogenesis and the shift towards a muscle oxidative phenotype [23, 24].”

What the sources say

• Not checked yetcites [23] Kwon OS et al (2016) Intramyocellular ceramides and skeletal muscle mitochondrial respiration are partially regulated by Toll-like receptor 4 during hindlimb unloading. Am J Physiol Regul Integr Comp Physiol 311:R879–R887 source
• Not checked yetcites [24] Remels AH et al (2010) TNF-alpha impairs regulation of muscle oxidative phenotype: implications for cachexia? FASEB J 24:5052–5062

In the paper

“This metabolic inflexibility imposes a major substrate burden on the oxidative machinery of muscle and the continued oversupply of carbon fuel eventually surpasses the respiratory drive and cellular demand for ATP synthesis [10].”

What the sources say

• Not checked yetcites [10] Muoio DM (2014) Metabolic inflexibility: when mitochondrial indecision leads to metabolic gridlock. Cell 159:1253–1262 source

In the paper

“As a result, FAs undergo incomplete oxidation and greater partitioning into lipotoxic derivatives [e.g., diacylglycerol (DAG) and ceramides] which have been strongly implicated in the pathogenesis of insulin resistance [11, 12].”

What the sources say

• Not checked yetcites [11] Koves TR et al (2008) Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance. Cell Metab 7:45–56 source
• Not checked yetcites [12] Lipina C, Hundal HS (2011) Sphingolipids: agents provocateurs in the pathogenesis of insulin resistance. Diabetologia 54:1596–1607 source

In the paper

“which most likely forms part of a cellular defence mechanism designed to help limit oxidative damage/stress under these circumstances”

In the paper

“potentially signifying increased mitochondrial uncoupling that would also help offset ROS generation.”

In the paper

“The first utilised BI605906, a potent pharmacological inhibitor that shows high selectivity for IKKβ (IC50 = 380 nM) when tested against over 100 kinases in a panel screen [36]”

What the sources say

• Not checked yetcites [36] Clark K et al (2011) Novel cross-talk within the IKK family controls innate immunity. Biochem J 434:93–104 source

In the paper

“This increase in mitochondrial-associated Drp1 may have resulted from its recruitment from a cytoplasmic pool, which showed a corresponding decline in its abundance.”

In the paper

“These nutrient-induced changes collectively signal a marked loss in mitochondrial integrity/function that impact negatively upon myocellular energy metabolism and, by extension, upon processes involved in fuel utilisation, such as glucose uptake and its regulation by insulin.”

In the paper

“It is plausible that the increased respiratory drive seen in myotubes that have been subject to fuel overloading, but in which activation of the NFkB pathway has been blunted (Fig. 6) is a benefit derived from suppressing mitochondrial ROS production (Fig. 5).”

In the paper

“The relative changes in mitochondrial Drp1 and MFN2 is consistent with a shift in mitochondrial dynamics that favours increased fission and would be in line with our morphological analysis (Fig. 7a).”

In the paper

“we have shown previously that its cellular abundance declines dramatically when the presence of PA in GLC-containing media is raised above 0.4 mM [17, 31].”

What the sources say

• Not checked yetcites [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 source
• Not checked yetcites [31] Macrae K et al (2013) Defining the role of DAG, mitochondrial function and lipid deposition in palmitate-induced proinflammatory signalling and its counter-modulation by palmitoleate. J Lipid Res 54:2366–2378 source

In the paper

“This observation is most likely explained by the fact that neither anti-oxidant was able to mitigate activation of the IKK–NFkB pathway that we believe initiates the loss of key mitochondrial proteins, such as SDHA, UCP3, and ANT-1 in fuel-overloaded myotubes (Supplementary Fig. S4).”

In the paper

“its expression in cultured myotubes and rodent skeletal muscle has been linked to modulation of fatty acid oxidation [56–58], mitochondrial integrity, and to management of ROS/oxidative stress [59–62].”

What the sources say

• Not checked yetcites [56] Costford SR et al (2008) Long-term high-fat feeding induces greater fat storage in mice lacking UCP3. Am J Physiol Endocrinol Metab 295:E1018–E1024 source
• Not checked yetcites [57] Senese R et al (2011) Uncoupling protein 3 expression levels influence insulin sensitivity, fatty acid oxidation, and related signaling pathways. Pflug Arch 461:153–164 source
• Not checked yetcites [58] MacLellan JD et al (2005) Physiological increases in uncoupling protein 3 augment fatty acid oxidation and decrease reactive oxygen species production without uncoupling respiration in muscle cells. Diabetes 54:2343–2350 source
• Not checked yetcites [59] Sellares J et al (2007) Oxidative stress time course in the rat diaphragm after freezing-thawing cycles. Respir Physiol Neurobiol 155:156–166 source
• Not checked yetcites [60] Jiang N et al (2009) Upregulation of uncoupling protein-3 in skeletal muscle during exercise: a potential antioxidant function. Free Radic Biol Med 46:138–145 source
• Not checked yetcites [61] Brand MD et al (2002) Oxidative damage and phospholipid fatty acyl composition in skeletal muscle mitochondria from mice underexpressing or overexpressing uncoupling protein 3. Biochem J 368:597–603 source
• Not checked yetcites [62] Klumpe I et al (2016) Transgenic overexpression of adenine nucleotide translocase 1 protects ischemic hearts against oxidative stress. J Mol Med (Berl) 94:645–653 source

In the paper

“The production of ceramide and certain DAG species may, in turn, feedback to further suppress the activity of respiratory chain complexes [40, 41], which, associated with the reduced expression of succinate dehydrogenase (complex II) that is induced in response to chronic fuel excess, is likely to impair electron transfer within the respiratory chain.”

What the sources say

• Not checked yetcites [40] Di PM, Cocco T, Lorusso M (2000) Ceramide interaction with the respiratory chain of heart mitochondria. Biochemistry 39:6660–6668 source
• Not checked yetcites [41] Perreault L et al (2018) Intracellular localization of diacylglycerols and sphingolipids influences insulin sensitivity and mitochondrial function in human skeletal muscle. JCI Insight 3(3):e96805. https://doi.org/10.1172/jci.insight.96805 source

In the paper

“However, one potential mechanism by which NFkB may modulate myocellular energy metabolism is via its well-established role as a regulator of gene expression [47].”

What the sources say

• Not checked yetcites [47] Gilmore TD, Garbati MR (2011) Inhibition of NF-kappaB signaling as a strategy in disease therapy. Curr Top Microbiol Immunol 349:245–263

In the paper

“recent work in human glioblastoma cells has highlighted that activated NFkB can bind to two response elements within the gene promoter of the ADP/ATP translocase, ANT-1, resulting in its reduced expression and concomitant decline in mitochondrial ATP production [48].”

What the sources say

• Not checked yetcites [48] Zhang C et al (2017) Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions. Sci Rep 7:44708 source

In the paper

“Increased exposure of myotubes to PA and fatty acid derivatives such as ceramide promote a low mitochondrial fusion to fission ratio resulting in fragmented, discontinuous mitochondria that not only exhibit diminished capacity for respiration and ATP synthesis, but also is thought to be causally linked to impaired insulin sensitivity and metabolic function [66–68].”

What the sources say

• Not checked yetcites [66] Jheng HF et al (2012) Mitochondrial fission contributes to mitochondrial dysfunction and insulin resistance in skeletal muscle. Mol Cell Biol 32:309–319 source
• Not checked yetcites [67] Smith ME et al (2013) Mitochondrial fission mediates ceramide-induced metabolic disruption in skeletal muscle. Biochem J 456:427–439 source
• Not checked yetcites [68] Sebastian D et al (2012) Mitofusin 2 (Mfn2) links mitochondrial and endoplasmic reticulum function with insulin signaling and is essential for normal glucose homeostasis. Proc Natl Acad Sci USA 109:5523–5528 source

In the paper

“very recent work has shown that sustained inhibition of NFkB can adversely affect muscle development and mitochondrial function during early life its repression has no detrimental effect in adult muscle [71].”

What the sources say

• Not checked yetcites [71] Valentine JM et al (2018) NFkappaB regulates muscle development and mitochondrial function. J Gerontol A Biol Sci Med Sci. https://doi.org/10.1093/gerona/gly262 source

In the paper

“In conclusion, our findings indicate that muscle cells subjected to sustained oversupply of metabolic fuel exhibit an increase in proinflammatory NFkB signalling that is mechanistically linked to diminished mitochondrial function as evidenced by our ability to antagonise the marked decline in respiratory capacity, expression of key mitochondrial maker proteins and the increased fission and mitoph”

In the paper

“studies in cultured C2C12 myotubes, in which partial silencing of ANT-1 results in reduced cellular sensitivity to fatty acid-induced uncoupling and a significant reduction in insulin-stimulated glucose uptake [63].”

What the sources say

• Not checked yetcites [63] Sparks LM et al (2016) ANT1-mediated fatty acid-induced uncoupling as a target for improving myocellular insulin sensitivity. Diabetologia 59:1030–1039 source

In the paper

“One potential explanation for this apparent paradox is that whilst our mitophagy reporter assay is highly sensitive and the loss in mitochondrial proteins may be accounted for by a reduction in the transcription and/or translation of their respective genes and products, analysis of mitochondrial DNA may also capture that present within mitochondria during the early stages of the mitophagic process”

In the paper

“the activation of the classical NFkB pathway in C2C12 myotubes can result in suppressed IKKα expression and that this has implications for expression of OXPHOS genes [70].”

What the sources say

• Not checked yetcites [70] Remels AH et al (2014) Activation of alternative NF-kappaB signaling during recovery of disuse-induced loss of muscle oxidative phenotype. Am J Physiol Endocrinol Metab 306:E615–E626 source

In the paper

“in addition to catalysing the exchange of cytoplasmic ADP for mitochondrial ATP, ANT-1 can mediate fatty acid-induced uncoupling via its ability to transfer fatty acid anions across the inner mitochondrial membrane [63].”

What the sources say

• Not checked yetcites [63] Sparks LM et al (2016) ANT1-mediated fatty acid-induced uncoupling as a target for improving myocellular insulin sensitivity. Diabetologia 59:1030–1039 source