Covalent inhibition of endoplasmic reticulum chaperone GRP78 disconnects the transduction of ER stress signals to inflammation and lipid accumulation in diet-induced obese mice
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Evaluation Summary:
This paper is of interest to a broad audience of cell biologists, pharmacologists and researchers who work in metabolic diseases. The work provides substantial new insights into the mechanism of action for a plant derived pentacyclic triterpene called celastrol elastrol, in effectively reducing the high fat diet induced tissue hypertrophy in mouse liver and adipose. A series of compelling experiments depict the site of covalent inhibition of the ER stress sensor GRP78 as essential for the beneficial effects in-vivo, supporting the main conclusions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Targeting endoplasmic reticulum (ER) stress, inflammation, and metabolic dysfunctions may halt the pathogenesis of obesity and thereby reduce the prevalence of diabetes, cardiovascular disesases, and cancers. The present study was designed to elucidate the mechnaisms by which plant-derived celastrol ameliorated inflammation and lipid accumulation in obesity. The mouse model of diet-induced obesity was induced by feeding high-fat diet for 3 months and subsequently intervented with celastrol for 21 days. Hepatic and adipose tissues were analyzed for lipid accumulation, macrophage activation, and biomarker expression. As result, celastrol effectively reduced body weight, suppressed ER stress, inflammation, and lipogenesis while promoted hepatic lipolysis. RNA-sequencing revealed that celastrol-loaded nanomicelles restored the expression of 49 genes that regulate ER stress, inflammation, and lipid metabolism. On the other hand, celastrol-PEG4-alkyne was synthesized for identifying celastrol-bound proteins in RAW264.7 macrophages. ER chaperone GRP78 (78 kDa glucose-regulated protein) was identified by proteomics approach for celastrol binding to the residue Cys 41 . Upon binding and conjugation, celastrol diminished the chaperone activity of GRP78 by 130-fold and reduced ER stress in palmitate-challenged cells, while celastrol analog lacking quinone methide failed to exhibit antiobesity effects. Thus, covalent GRP78 inhibition may induce the reprograming of ER signaling, inflammation, and metabolism against diet-induced obesity.
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Evaluation Summary:
This paper is of interest to a broad audience of cell biologists, pharmacologists and researchers who work in metabolic diseases. The work provides substantial new insights into the mechanism of action for a plant derived pentacyclic triterpene called celastrol elastrol, in effectively reducing the high fat diet induced tissue hypertrophy in mouse liver and adipose. A series of compelling experiments depict the site of covalent inhibition of the ER stress sensor GRP78 as essential for the beneficial effects in-vivo, supporting the main conclusions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Inflammation has been recently found as a crucial factor underlying the pathogenesis mechanism of obesity and the metabolic syndrome. Plant-derived celastrol was identified as an important therapeutic agent for obesity; however, the drug target of celastrol was totally unknown. Luo Dan et al employed affinity isolation, protein identification and biochemical validation to study the molecular mechanism by which celastrol attenuates inflammation and lipid accumulation in diet-induced obese mice. As result, the authors found that celastrol formed covalent conjugate with endoplasmic reticulum chaperone GRP78 and thereby disconnected the interactions between ER stress, inflammation and lipid metabolism. The findings are very significant to the field of natural product chemistry, pharmacology and metabolic diseases.
Reviewer #1 (Public Review):
Inflammation has been recently found as a crucial factor underlying the pathogenesis mechanism of obesity and the metabolic syndrome. Plant-derived celastrol was identified as an important therapeutic agent for obesity; however, the drug target of celastrol was totally unknown. Luo Dan et al employed affinity isolation, protein identification and biochemical validation to study the molecular mechanism by which celastrol attenuates inflammation and lipid accumulation in diet-induced obese mice. As result, the authors found that celastrol formed covalent conjugate with endoplasmic reticulum chaperone GRP78 and thereby disconnected the interactions between ER stress, inflammation and lipid metabolism. The findings are very significant to the field of natural product chemistry, pharmacology and metabolic diseases.
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Reviewer #2 (Public Review):
Luo et. al., have proposed a mechanism of action for a plant derived pentacyclic triterpene called celastrol. This drug has been previously shown to reduce obesity in mouse models by increasing leptin sensitivity and decreasing ER stress (PMC4768733) but a detailed mechanism of action has not been fully elucidated. The authors have identified GRP78, an ER resident chaperone, as a molecular target of celastrol. The evidence that authors provide in favor of their hypothesis shows that celastrol treatment is effective in reducing the deleterious effects of high fat diet on tissue hypertrophy in mouse liver and adipose. They further show that celastrol mitigates the high fat diet induced changes in lipid composition in mouse liver but not in adipose tissue. They also provide data to show that the expression of …
Reviewer #2 (Public Review):
Luo et. al., have proposed a mechanism of action for a plant derived pentacyclic triterpene called celastrol. This drug has been previously shown to reduce obesity in mouse models by increasing leptin sensitivity and decreasing ER stress (PMC4768733) but a detailed mechanism of action has not been fully elucidated. The authors have identified GRP78, an ER resident chaperone, as a molecular target of celastrol. The evidence that authors provide in favor of their hypothesis shows that celastrol treatment is effective in reducing the deleterious effects of high fat diet on tissue hypertrophy in mouse liver and adipose. They further show that celastrol mitigates the high fat diet induced changes in lipid composition in mouse liver but not in adipose tissue. They also provide data to show that the expression of lipolytic and lipogenic genes are regulated by celastrol in both mouse liver and adipose tissue. These results support and add to the previous literature that celastrol decreases lipid accumulation and affects the regulation of important lipogenic and lipolytic genes in mouse liver and adipose tissue. But these results lack important information about the status of ER stress response in their particular experimental set up and how celastrol is affecting this stress pathway.
Authors solely rely on GRP78 immunofluorescence staining to show that its expression is reduced only in resident macrophage cells after celastrol treatment in mouse liver and adipose tissue. Another approach would have been to isolate macrophages from mouse liver which then can be used for protein or mRNA analysis and further broaden the findings.
In figure 4, authors have done a transcriptome profiling after nano-celastrol treatment. This provides valuable high throughput data which shows major cellular pathways that are affected by celstrol in-vivo. But it is also interesting to see that the proposed target molecule for celastrol i.e GRP78 as well as other canonical markers of ER stress are not among the list of differentially expressed genes. This raises the question that the effects of celastrol might not be limited to the modulation of ER stress.
The authors have done several experiments which greatly increase the confidence in their findings that celastrol binds to GRP78. Their discovery that quinone methide is essential for the beneficial effects of celastrol in-vivo is remarkable and of great clinical significance. But their data does not rule out the possibility that celastrol has molecular targets other than GRP78 and that the physiological effects of celastrol might not be limited to inhibition of GRP78 only.
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Reviewer #3 (Public Review):
The article focuses on the anti-obesity effects of celastrol on HFD-induced mouse model. The impact of the article provided evidences that celastrol contains anti-obese effects in animal model. By using RAW264.7 marcrophages cell model and biotinylation method, they revealed that celastrol covalently bonded to the residue Cys41 and inhibit the ER stress sensor GRP78. The findings provide insights that covalent inhibition on GRP78 may serve as target against diet induced obesity. A synthesized celastrol analogue lacking quinone function group in ring A provided further evidence that the triterpene celastrol was covalently bound to GRP78 due to its containing the specific quinone substructure.
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