Dysregulation of cross-talks between endoplasmic reticulum (ER) stress, inflammation, and metabolic pathways hallmark the pathogenesis of obesity, diabetes, atherosclerosis, hypertension, and acute myocardial infarction (Van Gaal et al., 2006; Rocha and Libby, 2009; Flegal et al., 2010). The 78 kDa glucose-regulated protein (GRP78) is an integral ER stress sensor and holds inositol-requiring enzyme 1 (IRE1), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 (ATF6) in the ER lumen to prevent the activation of corresponding downstream substrates under the physiological conditions (Szegezdi et al., 2006; Cnop et al., 2012). Upon the challenge by stress stimuli, however, unfolded or misfolded proteins are overwhelmingly accumulated in ER compartment, disrupt the complexes of GRP78 with IRE1, PERK, and ATF6, and ultimately induce unfolded protein response (Cnop et al., 2012). Obesity and high-fat diet (HFD) not only induce persistent ER stress, leptin resistance, and insulin resistance via activating protein kinase JNK and inducing the phosphorylation of insulin receptor substrate 1 (Ozcan et al., 2004; Ozcan et al., 2009), but also promote the generation of unfolded or misfolded proteins and the activation of proinflammatory signaling pathways (Hotamisligil, 2010; Pagliassotti et al., 2016). On the other hand, hyperactive ER stress drives the proinflammatory M1 polarization of adipose tissue macrophages and exacerbates inflammation and metabolic dysfunctions in obesity (Bujisic and Martinon, 2017; Shan et al., 2017). Importantly, GRP78 is essential for the differentiation of preadipocytes into adipocytes, indicating a critical role in the control of fat contents in body (Zhu et al., 2013). The activation of ER stress pathways (e.g., PERK and eIF2α) inversely induces the upregulation of GRP78 expression (Ozcan et al., 2004; Shan et al., 2017). Thus, GRP78 may be an important molecular target for the development of new therapies against obesity, type 2 diabetes, atherosclerosis, hypertension, and acute myocardial infarction.

Pentacyclic triterpene celastrol as a major bioactive compound from the plant Tripterygium wilfordii Hook.f. was recently identified as the best antiobesity drug candidate without evident toxicity at the dose of 100 μg kg⁻¹ d⁻¹ (Liu et al., 2015). Several recent studies suggest that celastrol may induce rapid weight loss through several mechanisms as follows: (1) To enhance leptin activity and reduce food intake in obese mice (Greenhill, 2015; Liu et al., 2015); (2) To improve insulin sensitivity via inhibiting NF-κB pathway and control the progression of obesity via enhancing antioxidant capacity and lipid metabolism (Kim et al., 2013; Wang et al., 2014); (3) To activate HSF1–PGC1α transcriptional axis and elicit beneficial metabolic changes against obesity (Ma et al., 2015). However, the primary molecular targets have not been well defined for celastrol to bind covalently or noncovalently. At the cell level, celastrol could effectively suppress proinflammatory M1 macrophage polarization and enhance anti-inflammatory M2 macrophage polarization (Luo et al., 2017). These results strongly promoted effort to development novel celastrol-based antiobesity drugs. Nevertheless, the pharmacological potential of celastrol is largely limited by its strong hydrophobicity and poor bioavailability. We recently attempted to load celastrol into PEG-PCL nanomicelles to yield Nano-celastrol (Zhao et al., 2019). Indeed, Nano-celastrol exhibited highly competitive antiobesity effects and less gastrointestinal retention. These results strongly facilitated the pharmaceutical application of celastrol.

The aim of the present study is to discover the protein targets for celastrol and determine the impact of covalent celastrol–protein conjugation on ER stress in diet-induced obesity. We synthesized celastrol-PEG4-alkyne bearing an alkyne (–C≡C–) group as a molecular probe for the affinity isolation of celastrol-bound proteins. We further determined the in vitro and in vivo effects of celastrol on ER stress, inflammation, and lipid metabolism.

Pharmacological modulation of ER stress is a potential therapeutic strategy to treat various metabolic disorders (Cao and Kaufman, 2013). Chaperone protein GRP78 is essential to regulate ER stress, inflammation, and lipid metabolism since genetic knockout of GRP78 abolishes the growth, differentiation, and maturation of preadipocytes (Zhu et al., 2013). By contrast, several small molecule GRP78 inhibitors were recently developed for restoring ER homeostasis, especially, KP1339/IT-139 was evaluated for anticancer activity in a phase I clinical trial (Schoenhacker-Alte et al., 2017; Chen et al., 2018; Gurusinghe et al., 2018). Other purported GRP78 inhibitors such as Verrucocidin also ameliorate ER stress through different mechanisms other than direct binding to GRP78 (Thomas et al., 2013). Currently, little is known about the potential of covalent GRP78 inhibition in the regulation of ER stress. Here, we determined whether celastrol could ameliorate ER stress, inflammation, and metabolic dysfunctions in diet-induced obesity via covalent modification of chaperone GRP78.

HFD is well known to induce the deposition of lipids and alter the composition of fatty acids in liver, adipose tissues, and other organs (Cameron-Smith et al., 2003; Oosterveer et al., 2009; Zhao et al., 2019). Based on transcriptomic profiling and biochemical analysis, HFD upregulated several lipogenic genes for producing fatty acids (Acc, Fas, Scd1, Elovl6) and triglycerides (Dgat, Gpat), elongase for synthesizing long chain fatty acids such as n-3 and n-6 polyunsaturated fatty acids, and Srebp1c and Pgc1b for lipid uptake (Oosterveer et al., 2009). Our group and others have recently demonstrated that celastrol effectively reduces adipose hypertrophy in mouse and rats models of HFD-induced obesity (Wang et al., 2014; Zhang et al., 2017; Zhao et al., 2019). In the present study, firstly, H&E staining confirmed that celastrol ameliorated adipose hypertrophy and reduced lipid accumulation in liver and epididymal adipose tissues (Figure 1A, B). Secondly, GC–MS technology was employed to profile fatty acids in liver and adipose tissues from Control, HFD and HFD + Celastrol groups. Based on the quantitative analysis in Figure 1E, F, celastrol ameliorated HFD-induced elevations of various polyunsaturated fatty acids (e.g., C16:1, trans-C18:1, cis-C18:1, C18:2, C18:3, cis-C20:2, cis-C20:3) in liver but showed little activity against HFD-impaired production of long chain polyunsaturated fatty acids eicosoids (e.g., cis-C20:1, cis-C20:3, cis-C22:6) in epididymal fat pads. These results suggest that celastrol may mainly act on the metabolism of fatty acids in liver. Thirdly, qRT-PCR technique was employed to determine the key biomarkers for adipogensis, hormone secretion and energy expenditure. The key findings include: (1) celastrol could effectively antagonize HFD-induced upregulation of adipogenic genes (e.g., Pparg, Cebpa, Fabp4) and adipocyte-secreted hormones (e.g., Lepa, Retn, Cfd) in livers shortly after 7-day treatment; (2) celastrol markedly elevated the expression of lipolytic genes (e.g., Cpt1, Cat, Aco) and thermogenic gene Ucp2 in a dose-dependent manner (Figure 2A, B); (3) celastrol mainly suppressed HFD-induced upregulation of adipogenic genes (e.g., Pparg, Cebpa, Fabp4) and adipocyte-secreted hormones (e.g., Lepa, Retn, Cfd) in epididymal fat pads (Figure 2C). These results suggest that celastrol mainly reprograms the biosynthesis and degradation of fatty acids in liver while inhibits fatty acid biosynthesis and hormone secretion. To facilitate safe control of body weight, we recently prepared Nano-celastrol by entrapping celastrol in PEG-PCL nanomicelles for the treatment of diet-induced obesity (Zhao et al., 2019). In the present study, we further identified 49 DEGs in the livers from mice in Control, HFD, and HFD + Nano-celastrol groups by next-generation RNA- sequencing technology (Figure 4A). Indeed, Figure 4B classified these DEGs into the functional groups of retinol metabolism (i.e., Aldh1a1, Cyp4a10, Cyp1a2, Cyp2a12, Cyp2c44, Cyp3a25, Cyp4a14, Retsat, Ugt2b5), lipid biosynthesis (e.g., Apoc2, C3, Cyp1a2, Cyp4a10, Cyp4a14, Ehhadh, Elovl5, Pck1, Scd1, Thrsp), oxidative stress (e.g., Aldh1a1, Creg1, Cyp1a2, Cyp2a12, Cyp3a25, Cyp4a10, Cyp4a14, Ehhadh, Hsd17b10, Mdh1, Retsat, Scd1), lipid degradation (i.e., Acat1, Cyp4a10, Cyp4a14, Ehhadh), glucose metabolism (i.e., Mup1, Mup11, Mup12, Mup18, Mup20), cholesterol efflux (i.e., Apoa4, Apoc2, Apom), and inflammatory response (e.g., C3, C8a, Serpina1e) although some DEGs might be involved in several other biological processes and pathways. By using online software STRING, functional enrichment analysis (Figure 4C) revealed four protein–protein interaction networks: (1) lipid and glucose metabolism (i.e., Mdh1, Pck1, Aldh1a1, Cyp1a2, Cyp2a12, Cyp2c44, Cyp3a25, Cyp4a10, Cyp4a14, Ugt2b5); (2) apolipoprotein metabolism and inflammatory response (i.e., Apoa4, Apoc2, Apom, Ltih3, Ltih4, Serpina1e, C3, Creg1, C8a); (3) mitochondrial energy homeostasis (i.e., Acat1, Enhadh, Hsd17b10); (4) adipose tissue energy homeostasis (Mt1, Mt2). Thus, RNA-seq profiling basically confirmed the potential of celastrol in the regulation of oxidative stress, lipid accumulation, and inflammatory response in the liver. The genes encoding ER stress signaling proteins were not selected probably due to the cutoff of >2.0-fold.

On the other hand, free fatty acids induce ER stress and inflammatory response in macrophages and adipose tissues via TLR-mediated mechanisms (Han and Kaufman, 2016). ER stress in turn orchestrates unfolded protein response, inflammation, metabolic dysfunctions, and insulin resistance in obesity and diabetes (Ozcan et al., 2004; Hummasti and Hotamisligil, 2010). ER chaperone protein GRP78 is integral to the celluluar sensing of fatty acid-induced ER stress and inflammatory stimuli and the regulation of lipid uptake, adipogenesis, lipolysis, glucose metabolism, cholesterol efflux, inflammatory response, and immune response (Zhu et al., 2013). GRP78 and CHOP modulate macrophage apoptosis and control the progression of bleomycin-induced pulmonary fibrosis (Ayaub et al., 2016). The activation of ER stress sensor IRE1α exacebates obesity-associated inflammation (Shan et al., 2017). However, celastrol could reduce ER stress via reducing the protein level of p-PERK/PERK in hypothalamus of diet-induced obese mice (Liu et al., 2015). In the present study, the effects of celastrol on the ER stress signaling pathway were examined in the in vitro and in vivo systems. Firstly, livers and epididymal fat pads were collected from experimental mice and examined by immunofluorescence staining and Western blotting. As results, Figure 3A, B shows that HFD upregulated GRP78 expression in macrophages whereas celstrol restored the expression level of GRP78. On the other hand, Western blot analysis in Figure 3C, D confirmed that HFD upregulated the expression GRP78, ATF6, and p-eIF2α in livers and epididymal fat pads. Celstrol not only restored the expression level of GRP78 but also reduced the levels of ATF6 and p-eIF2α in livers and epididymal fat pads. Secondly, RAW264.7 macrophages were treated by fatty acid palmitate to induce ER stress. Following celastrol treatment, we subsequently employed Western blotting and fluorescence immunostaining to determine the expression levels of ER stress biomarkers. As result, celastrol not only reduced the levels of GRP78, IRE1α, ATF6, CHOP, and XBP1, but also inhibited palmitate-stimulated eIF2α phosphorylation in a concentration-dependent manner (Figure 5A, B). Based on the immunostaining results (Figure 5C), celastrol decreased the signal intensity of GRP78, ATF6, and IRE1α and preserved the colocalization of GRP78 with ATF6 and IRE1α in the ER compartment. These results indicate that celastrol could effectively suppress the ER stress signaling pathway in the hepatic and adipose tissues.

Celastrol bears a chemically reactive quinone methide moiety and thereby tends to form covalent conjugate with certain proteins such as HIV-Tat, IKKalpha, and beta kinases, Cdc37 and HSF although such covalent attachment appears to be reversible (Salminen et al., 2010; Narayan et al., 2011; Zhang et al., 2018). However, it is critical to identify protein targets with convincing association to ER signaling, anti-inflammatory, and metabolic reprogramming. In the present study, a novel celastrol-PEG4-alkyne bearing an alkyne (–C≡C–) group was firstly synthesized as a small molecule probe through the in-house synthetic procedure (Cheng et al., 2020). Secondly, RAW264.7 macrophages were treated with celastrol-PEG4-alkyne and lysed. The cellular proteins were biotinylated via Click chemistry ‘azide-alkyne cycloaddition’ and separated by affinity enrichment onto streptavidin-coated magnetic beads (Figure 6A). The protein fractions were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and identified by MALDI-MS technology. The major protein band was identified to be GRP78 and subsequently verified by Western blotting using specific antibody (Figure 6B). Thirdly, recombinant mouse GRP78 protein was prepared in E. coli BL21 cells and incubated with celastrol. Following trypsin digestion, peptides were mapped by LC–MS/MS analysis. Peptide mapping revealed a celstrol-bound peptide with the sequence of GSEEDKKEDVGTVVGIDLGTTYSC⁴¹VGVFK (Figure 6C). This experiment suggests that celastrol interacts with GRP78 by covalent attachment to Cys⁴¹. Fourthly, the binding of celastrol to GRP78 was simulated by molecular docking. Interestingly, celastrol binds to the Cys⁴¹-containing region in GRP78 structure with binding energy of −8.1 kcal mol⁻¹ and inhibition constant of 1.55 μM (Figure 6D). Fifthly, a recently identified peptide WDLAWMFRLPVG was synthesized to examine the effects of celstrol on the chaperone activity of GRP78 as described (Arap et al., 2004). Biacore X100 SPR system was employed to evaluate whether celastrol could alter the binding of peptide WDLAWMFRLPVG to GRP78 on the Sensor Chip. Indeed, celastrol attenuated the chaperone activity of GRP78 since the average Kd value of peptide binding was increased from 17.7 nM to 2.23 μM (Figure 7A, B). Sixthly, two new celastrol derivatives were prepared to explore the importance of quinone methide moiety for celastrol to bind to GRP78 (Figure 7C). Celastrol-PEG3-biotin was readily detected in association with GRP78 although the binding could be blocked by excess celastrol. By contrast, analog-PEG3-biotin failed to bind to GRP78 due to the lack of quinone methide moiety (Figure 7D). Seventhly, the present study addressed whether the formation of celastrol–GRP78 conjugate could be colocalized with the intracellular GRP78 (Figure 8). The cells were treated with celastrol-PEG4-alkyne and subsequently labeled with fluorescence dye AFDye555-picolyl azide whereas GRP78 and another ER biomarker calnexin were detected by immunostaining. As result, the fluorescence of celastrol-PEG4-alkyne labeling was well colocalized with GRP78, and, to a large extent, ER biomarker calnexin. Last but not least, the present study determined whether quinone methide moirty was essential for antiobesity effects (Figure 9). Celastrol was reduced by NaBH₄ and protected by O-methylation to yield celastrol analog. Unlike celastrol, such analog became totally inactive to induce weight loss, reduce fat mass and ameliorate glucose tolerance and insulin tolorance in diet-induced obese mice as assessed by previous described procedure (Luo et al., 2017; Zhao et al., 2019). These results suggest that the unique quinone methide moiety is critical for celastrol to ameliorate ER stress, inflammation, and lipid accumulation via covalent inhibition of GRP78.

In summary, the present study demonstrated that celastrol ameliorated adipose hypertrophy and reduced lipid accumulation in liver and adipose tissues of diet-induced obese mice. Celastrol might induce rapid reduction of body weight through suppressing lipid uptake, lipogenesis and increasing lipolysis and thermogenesis. As depicted in Figure 10, the key finding from the present study was that celastrol covalently inhibited the chaperone activity of GRP78 and could disconnect the transduction of ER stress signals to downstream inflammatory response and lipid metabolism. Therefore, the covalent inhibition of GRP78 is a novel antiobesity mechanism for reprograming the signaling networks that control inflammation, lipid accumulation, ER stress in liver and adipose tissues. Celastrol may be an important lead compound for the development of new selective and irreversible GRP78 inhibitors to treat obesity, diabetes, and related cardiovascular diseases.

Figure 10

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All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided.

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Author details

1. Dan Luo

   School of Chinese Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

   Contribution

   Conceptualization, Data curation, Investigation, Methodology, Writing – original draft

   Contributed equally with

   Ni Fan

   Competing interests

   No competing interests declared

2. Ni Fan

   School of Chinese Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing - review and editing

   Contributed equally with

   Dan Luo

   Competing interests

   No competing interests declared

3. Xiuying Zhang

   School of Chinese Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Fung Yin Ngo

   School of Chinese Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

   Contribution

   Data curation

   Competing interests

   No competing interests declared

5. Jia Zhao

   School of Chinese Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

   Contribution

   Data curation

   Competing interests

   No competing interests declared

6. Wei Zhao

   School of Chinese Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

   Contribution

   Data curation

   Competing interests

   No competing interests declared

7. Ming Huang

   Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Ding Li

   Colleage of Chemistry and Pharmacy, College of Chemistry & Pharmacy, Northwest A&F University, Shaanxi, China

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Yu Wang

   Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

   Contribution

   Data curation

   Competing interests

   No competing interests declared

10. Jianhui Rong

    School of Chinese Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

    Contribution

    Conceptualization, Project administration, Supervision, Writing - review and editing

    For correspondence

    jrong@hku.hk

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-3545-2811

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