Strigolactone GR24 upregulates target genes of the cytoprotective transcription factor Nrf2 in skeletal muscle

GR24 is a synthetic strigolactone analog, demonstrated to regulate the development of plants and arbuscular mycorrhizal fungi. GR24 possesses anti-cancer and anti-apoptotic properties, enhances insulin sensitivity and mitochondrial biogenesis in skeletal myotubes, inhibits adipogenesis, decreases inflammation in adipocytes and macrophages and downregulates the expression of hepatic gluconeogenic enzymes. Transcription factor Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) is a master regulator of antioxidant response, regulating a multitude of genes involved in cellular stress responses and anti-inflammatory pathways, thus maintaining cellular redox homeostasis. Nrf2 activation reduces the deleterious effects of mitochondrial toxins and has multiple roles in promoting mitochondrial function and dynamics. We studied the role of GR24 on gene expression in rat L6 skeletal muscle cells which were differentiated into myotubes. The myotubes were treated with GR24 and analyzed by microarray gene expression profiling. GR24 upregulated the cytoprotective transcription factor Nrf2 and its target genes, activating antioxidant defences, suggesting that GR24 may protect skeletal muscle from the toxic effects of oxidative stress.


Introduction
Strigolactones are carotenoid-derived phytohormones with endogenous roles in regulating plant growth and exogenous roles in establishing symbiosis of host plant with arbuscular mycorrhizal fungi 1 .Strigolactones induce beneficial effects in mycorrhiza, such as mitochondrial biogenesis and ATP production [2][3][4] .The anti-cancer properties [5][6][7][8] and anti-inflammatory potential 9 of strigolactones have recently been investigated in mammalian cells.The positive effects of strigolactone analog GR24 in enhancing insulin sensitivity, mitochondrial function and inhibiting adipogenesis and inflammation in insulin-sensitive cells have also been demonstrated 10,11 .
Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) activates gene transcription by binding to the antioxidant response element (ARE) in the promoters of its target genes.It regulates multiple biological functions ranging from cellular redox metabolism, detoxification, heme, lipid and glucose metabolism, NADPH generation, autophagy, apoptosis, xenobiotic stress reponse to inflammation by interacting with its target genes, regulating an extensive antioxidant protein network.Nrf2 is associated with disease pathologies like cancer 12 , hepatotoxicity 13 , cardiovascular disease 14 and neurodegenerative diseases 15 .
Mitochondria are the major sites of reactive oxygen species (ROS) production and also the targets of their toxic effects.Mitochondrial dysfunction has been associated with the development of insulin resistance, and diabetes is known to induce oxidative stress through the overproduction of ROS and ROS-induced DNA damage 16 .Nrf2 activation defends against mitochondrial toxins and ROS and affects mitochondrial function by regulating mitochondrial biogenesis, mitochondrial fatty acid oxidation, respiration, ATP production and mitochondrial dynamics 17 .With its versatile protective mechanism against oxidative stress, pancreatic β-cell apoptosis and insulin resistance, Nrf2 has become a promising therapeutic target for the treatment of type 2 diabetes 18 .
We have demonstrated that GR24 ameliorates insulin sensitivity, stimulates mitochondrial biogenesis and ATP production and upregulates genes regulating mitochondrial function in L6 myotubes 10 .Very recently, the efficacy of GR24 in promoting cytoprotective responses via Nrf2 activation was reported in hepatic and macrophage cell lines 19 .This work reports a transcriptomic study revealing the potential beneficial effects of GR24 in upregulating Nrf2 and its target genes involved in detoxification, carbohydrate and lipid metabolism, heme metabolism, NADPH regeneration and oxidative stress in L6 myotubes, thus contributing to metabolic homeostasis.

Methods
Results from the methods described in this study have been published previously 10 , although the analyses described here are published for the first time. Cell

Microarray sample preparation
L6 myotubes were treated with 60 µM GR24 at 7 days of differentiation for 24 h in three independent experiments, resulting in three replicate microarrays in each treatment group.Total RNAs were extracted with RNeasy Mini kit (Qiagen, Hilden, Germany) and RNA quality was assessed with the Agilent Bioanalyzer 2100 (Agilent Technologies, Espoo, Finland).Total RNA (200 ng) was converted to cDNA with Agilent AffinityScript RNase Block, labelled according to manufacturer's instructions and purified using RNeasy mini spin columns (Qiagen).RNA Spike-In Kit (Exiqon, Vedbaek, Denmark) was used to monitor the success of labelling.Samples were then mixed with blocking agent, fragmentation and hybridization buffer, and were hybridized to Agilent SurePrint G3 Rat GE 8×60 K Microarrays for 17 h at 65°C before washing and scanning with Agilent Scanner G2505C using manufacturer's protocols.Agilent Feature Extraction software was used to extract data from raw microarray image files 10 .

Microarray data processing
The data was processed with limma package (version 3.28.21) of the Bioconductor software 20 .Microarray data are MIAME compliant.Differentially expressed transcripts were analysed by Bayes moderated t-statistics followed by the Benjamini-Hochberg correction method to control false discovery rate (FDR) with significance threshold set at p < 0.05 20,21 .

Results and discussion
The top 5 GR24-upregulated genes (glutathione S-transferase alpha 1, metallothionein 1M, heme oxygenase 1, glutathione S-transferase alpha 2, and sequestosome 1) were found to be known targets of Nrf2 (Table 1).As the prototypical Nrf2 target gene NAD(P)H quinone dehydrogenase 1 (Nqo1) was also found high on the list, it was compelling to look into the extensive list of Nrf2 target genes.We found 56 known Nrf2 target genes 22 , including Nrf2 itself, to be upregulated by GR24 treatment  1).Prior to our study, the effects of strigolactones treatment on the mammalian cell transcriptome have only been investigated in human osteosarcoma cells, where strigolactone analogs mainly upregulated the heat shock stress proteins and downregulated the cell cycle 5 .
Activation of Nrf2 signaling is known to have beneficial effects in cancer, metabolic syndrome, obesity, nephropathy, retinopathy, neuropathy, and β-cell protection.Nrf2 regulates the transcription of genes involved in antioxidant, detoxification and metabolic processes 23 .In our study, GR24 enhanced the expression of the Nrf2-dependent antioxidant genes Nqo1 and heme oxygenase 1, which are known to block inflammatory pathways 24 .GR24 has recently been shown to alleviate inflammation and upregulate these two NRF2 target genes in hepatic and macrophage cells 19 .
Glutathione and associated enzymes form an important antioxidant defence system.Treatment with GR24 was found to increase root glutathione content in plants 25 , but there are no previous reports on the effects of strigolactones on the glutathione system in mammalian cells.Our results show that GR24 treatment upregulated glutamate-cysteine ligase, the rate-limiting enzyme in glutathione synthesis (Table 1).Many components of the glutathione system are upregulated by Nrf2, and the elevations in the expression of glutaredoxin, glutathione peroxidase 4 and several glutathione S-transferases in GR24-treated cells were evident in our results (Table 1).

Conclusions
GR24 upregulated the cytoprotective transcription factor Nrf2 and its target genes, which are involved in detoxification, antioxidant systems, carbohydrate and lipid metabolism, heme and iron metabolism, NADPH regeneration and regulation of transcription in L6 myotubes.Future research aimed to elucidate the effects of GR24 in the oxidative stress mechanisms in skeletal muscle at the protein level may provide supporting evidence about the therapeutic potential of this compound.
In current research note, a microarray study was carried out to analyze the effect of GR24, a synthetic strigolactone analog, on the gene expression profile of rat L6 myotubes.60 µM of GR24 was used to treat L6 myotubes at 7 day of differentiation for 24 h in three independent experiments.Microarray data was processed and differentially expressed transcripts were analyzed.Upregulated genes were partly presented in Table 1.Accordingly, GR24 upregulated the cytoprotective transcription factor Nrf2 and its several target genes involved in antioxidant defense and phase II detoxification processes.The fold values especially for and , , , , , were considerably and GSTa1 a2 NQO1 GGt1 Hmox1 Mtm1m Sqstm1 significantly high (above 6 fold).Authors previously showed that GR24 (60 µM) ameliorates insulin sensitivity, stimulates mitochondrial biogenesis and ATP production and upregulates genes regulating mitochondrial function in L6 myotubes (Modi et al., 2017 ).Also, in this previous report it was shown that 9838 transcripts were upregulated and 6315 transcripts were downregulated in L6 myotubes as a result of 60 µM GR24 treatment.Up and down regulated transcripts involved in insulin signaling and mitochondrial function in L6 myaotubes have been presented in this previous paper (Modi et al., 2017 ).In the present research note, authors represented up regulated transcripts involved in oxidative stress and cytoprotective signaling when myotubes were treated with 60 µM GR24.Since the work can be classified as original and the results are remarkable, data deserves to be published in F1000Research as research note.However, the data is limited to reach the conclusion suggesting that GR24 may protect skeletal muscle from the toxic effects of oxidative stress and thus contributing to metabolic homeostasis.In the scope of report, there is no any biological assay or disease modelling evaluating the beneficial effects of GR24 on myotubes.Furthermore, gene expression profiling does not always reflect the changes on the protein level.Regarding these facts, title also should not contain the assumption suggesting that GR24 may protect against oxidative stress in skeletal muscle.
In my opinion, both conclusion and title need revision and if possible it is better to represent additional data and interpretation in terms of down regulated genes in specified signaling pathways.Also could you please add an explanation about why you chose 60 µM concentration?References 1. Modi S, Yaluri N, Kokkola T, Laakso M: Plant-derived compounds strigolactone GR24 and pinosylvin activate SIRT1 and enhance glucose uptake in rat skeletal muscle cells. .

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Partly I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
These are very valid questions.The suggested qPCR analyses, protein blots and measures of oxidative stress would be essential next steps to verify the changes in gene expression.For that reason, we published our preliminary analyses as a Research Note, as additional analyses would have been required in order to produce a complete full-length article.When drafting the title, we avoided making strong claims on the effects on oxidative stress.After reading the suggestions by the referee, we have reconsidered the title, and will submit a new version of the article with a title "Strigolactone GR24 upregulates target genes of the cytoprotective transcription factor Nrf2 in skeletal muscle".
No competing interests were disclosed.

Competing Interests:
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th 1 1
Are the conclusions drawn adequately supported by the results?PartlyNo competing interests were disclosed.Competing Interests:

Table 1 . Selected upregulated Nrf2 target genes in L6 myotubes after 24 h treatment with 60 µM GR24. Gene symbol Description Accession logFC Fold change Adjusted P value Detoxication: Phase I drug oxidation, reduction and hydrolysis
(Table