Strigolactone GR24 upregulates Nrf2 target genes and may protect against oxidative stress in skeletal muscle

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 fungi¹. Strigolactones induce beneficial effects in mycorrhiza, such as mitochondrial biogenesis and ATP production^2–4. The anti-cancer properties^5–8 and anti-inflammatory potential⁹ 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 cancer¹², hepatotoxicity¹³, cardiovascular disease¹⁴ and neurodegenerative diseases¹⁵.

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 damage¹⁶. 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 dynamics¹⁷. 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 diabetes¹⁸.

We have demonstrated that GR24 ameliorates insulin sensitivity, stimulates mitochondrial biogenesis and ATP production and upregulates genes regulating mitochondrial function in L6 myotubes¹⁰. Very recently, the efficacy of GR24 in promoting cytoprotective responses via Nrf2 activation was reported in hepatic and macrophage cell lines¹⁹. 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 previously¹⁰, although the analyses described here are published for the first time.

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 genes²², including Nrf2 itself, to be upregulated by GR24 treatment (Table 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 cycle⁵.

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 processes²³. In our study, GR24 enhanced the expression of the Nrf2-dependent antioxidant genes Nqo1 and heme oxygenase 1, which are known to block inflammatory pathways²⁴. GR24 has recently been shown to alleviate inflammation and upregulate these two NRF2 target genes in hepatic and macrophage cells¹⁹.

Glutathione and associated enzymes form an important antioxidant defence system. Treatment with GR24 was found to increase root glutathione content in plants²⁵, 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. The ability of GR24 to enhance key cellular defence mechanisms through the activation of Nrf2 signaling may beneficially protect cells from oxidative stress and inflammation, and may alleviate the complications associated with metabolic syndrome.

Data availability

Microarray raw data have been deposited in the NCBI Gene Expression Omnibus (GEO), accession number GSE90833: https://identifiers.org/geo/GSE90833.