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Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype
Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype Michele Longo, Rosa Spinelli, Vittoria D’Esposito, Federica Zatterale, Francesca Fiory, Cecilia Nigro, Gregory A. Raciti, Claudia Miele, Pietro Formisano, Francesco Beguinot, Bruno Di Jeso PII: DOI: Reference:
S0167-4889(16)30044-1 doi: 10.1016/j.bbamcr.2016.02.019 BBAMCR 17815
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BBA - Molecular Cell Research
Received date: Revised date: Accepted date:
30 July 2015 2 February 2016 26 February 2016
Please cite this article as: Michele Longo, Rosa Spinelli, Vittoria D’Esposito, Federica Zatterale, Francesca Fiory, Cecilia Nigro, Gregory A. Raciti, Claudia Miele, Pietro Formisano, Francesco Beguinot, Bruno Di Jeso, Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype, BBA - Molecular Cell Research (2016), doi: 10.1016/j.bbamcr.2016.02.019
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PATHOLOGIC ENDOPLASMIC RETICULUM STRESS INDUCED BY GLUCOTOXIC INSULTS INHIBITS ADIPOCYTE DIFFERENTIATION AND INDUCES AN INFLAMMATORY PHENOTYPE. Michele Longo, Rosa Spinelli, Vittoria D’Esposito°, Federica Zatterale, Francesca Fiory, Cecilia Nigro°, Gregory A. Raciti°, Claudia Miele°, Pietro Formisano, Francesco Beguinot , and Bruno Di Jeso .
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Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Strada Monteroni, 73100 Lecce, Italy. Dipartimento di Scienze Mediche Traslazionali, Università “Federico II”, IEOS/CNR, via S. Pansini 5 80131 Napoli, Italy.
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To whom correspondence should be addressed: Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Strada Monteroni, 73100 Lecce, Italy. Tel. 0039 0832 298 912 Email [email protected] These authors equally contributed to the work.
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ABSTRACT Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is “physiologically” activated during adipocyte differentiation, the “pathologic” part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype. Keywords Adipocyte differentiation, ER stress, inflammation. 1
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Abbreviations BiP, Binding protein; CCL2, chemokine (C-C motif) ligand 2; CCL3, chemokine (C-C motif) ligand 3; CCL5, chemokine (C-C motif) ligand 5; C/EBP, CCAAT/enhancer binding protein; CHOP, C/EBP homologous protein; Ct, Comparative threshold cycle; eIF2, Eukaryotic initiation factor 2 alpha; ER, Endoplasmic reticulum; GM-CSF, Granulocyte-macrophage colony-stimulating factor; HBP, Hexosamine biosynthetic pathway; HFD, High Fat Diet; IL, interleukin; MCP-1, Monocyte chemoattractant protein 1; MIP-1, Macrophage inflammatory protein1-alpha; MTT, (3-(4,5-dimethyl- thiazol-2yl)-2,5-diphenol tetrazolium bromide); PERK, Protein kinase RNA-like ER kinase; PPAR, Peroxisome proliferator-activated receptor; PBA, 4-Phenyl butyric acid; RANTES, Regulated upon activation normally T expressed, and secreted; REU, relative expression units; Tg, thapsigargin; TH1, T helper 1; TH2, T helper 2; TUDCA, taurine-conjugated ursodeoxycholic acid; TSH, thyroid stimulating hormone; UPR, Unfolded protein response.
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1. INTRODUCTION Obesity has become a major worldwide health problem, because of its dramatically increased incidence, high prevalence and associated mortality 1. Obesity is characterized by increased fat mass caused by increased adipocyte number (hyperplasia) and/or size (hypertrophy) 2. Hypertrophic obesity is mostly associated with abdominal obesity, which in turn is associated with increased insulin resistance, risk of developing type 2 diabetes, and cardiovascular disease 3-5. Increasing evidence indicates that obesity is causally linked to a chronic low-grade inflammatory state, called “metainflammation”, which contributes to the development of obesity-linked disorders 6-9.
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The ER is a multifunctional organelle. The ER acts as gatekeeper to the secretory pathway by folding, modifying, and exporting nascent secretory and transmembrane proteins, but it is also a stress-sensing organelle. It senses stress caused by disequilibrium between ER load and folding capacity, ER stress, and responds by activating the unfolded protein response (UPR) 10. In recent years, the perspective of the UPR as an exclusive stress response has been challenged. Thus the UPR can be activated by stimuli that represent physiological oscillations of environmental and/or cellular conditions, such as diurnal induction of the UPR in dependency of the circadian clock 11, and, in thyrocytes, stimulation with TSH 12 that increases the synthesis of the highly expressed thyroglobulin 13-17. Perhaps the most dramatic examples of physiologic UPR upregulation are those taking place during cellular differentiation, such as that of B cells into plasma cells 18 . However, also the opposite effect has been observed, in that UPR activation inhibits differentiation. In transgenic mice models expressing mutant collagen X, the UPR was activated in hypertrophic chondrocytes and terminal differentiation is interrupted, producing a chondrodysplasia phenotype 19. Interestingly, in a transgenic mouse model using the collagen X promoter to drive expression of the cog mutant of thyroglobulin in hypertrophic chondrocytes, the same phenotype was observed 20, confirming the causal role of ER stress in inhibiting chondrocyte differentiation. Data reported on adipogenic differentiation reproduce this apparent contradiction. Some studies suggest that ER stress and UPR activation might be important for adipogenesis 21-23. However, it has been also reported that mice heterozygous for BiP gain less fat mass compared with wild-type mice 24. A reduction of BiP decreases the folding capacity and favors UPR activation. Thus, the reduced fat mass in these mice is an effect of inhibited adipogenesis that likely follows an activation of UPR. In addition, highfat diet (HFD)-fed mice with a heterozygous mutation at the phosphorylation site in Eif2 (Ser51Ala, S/A) became significantly more obese than HFD-fed wild-type mice, suggesting that the PERK-Eif2 branch of the UPR inhibits adipogenesis 25. Thus, eIF2α phosphorylation inhibits adipocyte differentiation in response to ER stress in vitro and in vivo, by increasing CHOP production 26. In this study we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We sought to substantiate the hypothesis that while ER stress and UPR activation that occurs during adipocyte differentiation is a physiologic response 3
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instrumental to differentiation, as shown by others 21-23, the ER stress and UPR secondary to glucotoxic insults is pathologic, causing an inhibition of differentiation. Moreover, we sought to determine the effect of ER stress and UPR activation during differentiation on the inflammatory phenotype of preadipocyte/adipocyte population, which may be relevant to the onset and development of the low grade inflammation of the adipose tissue.
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2. MATERIALS AND METHODS. 2.1. Materials. Media, sera and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA). Chemicals were from Sigma-Aldrich (St Louis, MO, USA). Glucosamine was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Insulin, TRIzol, and SuperScript III were from Invitrogen (Carlsbad, CA,USA). Cytokine assay kit and Lowry protein assay were from BioRad (Hercules, CA, USA). ECLWestern Blot detection reagents were from GE Healthcare (Pittsburgh, PA, USA). Antibodies were anti-tubulin (monoclonal; Sigma), anti-binding protein (BiP) and anti-Chop (Santa Cruz). 2.2. Cell culture, adipocyte differentiation, and primary adipocyte isolation. 3T3-L1 mouse embryo fibroblasts were obtained from the American Type Culture Collection (Manassas, VA, USA). They were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, penicillin (200 IU/ml), streptomycin (100 g/ml), in a humidified CO2 incubator. Two days after confluency, cell differentiation into adipocytes was induced with DMEM containing 1 g/ml insulin, 0.5 mmol/l 3-isobutyl-1methylxanthine and 1 mol/l dexamethasone for 2 days, followed by DMEM supplemented with 10% FBS and 1 g/ml insulin only, for another 5 days. Oil red O working solution was added to formalin-fixed cells and incubated for 10 min at room temperature. Images were taken using an Olympus microscope system (Olympus, Center Valley, PA, USA). For quantification, absorbance was measured at 500 nm using a spectrophotometer (Beckman, CA, USA). Human adipose tissue samples were obtained from abdominal adipose tissue biopsies of healthy subjects (n=5; age 25-63 years; BMI 20.2-25.3) undergoing biliary surgery. All subjects were otherwise free of metabolic or endocrine diseases. Informed consent was obtained from every patient before the surgical procedure. This procedure was approved by the ethical committee of the University of Naples. Adipose tissue was digested with collagenase and mature adipocytes were isolated as previously reported 27. Isolated adipocytes were filtered through a 250-m nylon mesh and washed four times with fresh DMEM -F12 (1:1) 10% FBS medium. The cells were incubated at 37 °C with 20mM GlcN for 6h. Next, adipocytes were washed with PBS 1X and RNA extracted as described previously. 2.3. RNA isolation and real Time RT-PCR Total RNA was extracted with the TRIzol reagent, according to the manufacturer’s protocol. Reverse transcription of 1 µg of total RNA was performed using SuperScript III, following the manufacturer’s instructions. Quantitative real time PCR was performed in triplicate by using iQ SYBR Green Supermix on iCycler real time detection system (BioRad). Relative quantification of gene expression was calculated by the ΔΔCt method. Each Ct value was first normalized to the respective Cyclophilin Ct value of a sample to account for variability in the concentration of RNA and in the conversion efficiency of the RT reaction. The primers used are listed in Supplementary Materials and Methods. For the selective amplification of Xbp1-s, the forward primer was designed across the Xbp1s deletion. Thus, the Xbp1-s forward primer corresponded to a non-contiguous sequence in the Xbp1-t cDNA. 2.4. Cytokine and growth factor assay
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3T3-L1 adipocyte conditioned media were screened for the concentration of several cytokines/chemokines using a custom-blended Bio-Plex ProTM (Bio-Rad) assay according to the manufacturer’s protocol. 2.5. Western blotting and cell viability assay. Cell lysates were obtained in cell lysis buffer (50 mmol/l Tris HCl [pH 7.4], 150 mmol/l NaCl, 1% [vol./vol.] Triton X-100, 0.1% [wt/vol.] SDS, 1% [wt/vol.] sodium deoxycholate and protease inhibitors) and total protein concentration in each sample was measured using the Lowry protein assay kit (Biorad). Chemiluminescence detection was performed using ECLWestern Blot detection reagents. Membranes were exposed to imaging film and developed using a Kodak X-OMAT processor (Kodak, Rochester, NY, USA). Cell viability was assessed by the MTT assay as previously reported 28. 2.6. Statistical analysis All data are presented as means ± SEM. The difference between groups was evaluated using Student’s t test, and, when more than two groups were present, ANOVA and post hoc tests (Bonferroni/Dunn) were also performed. p<0.05 was considered significant.
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3. RESULTS 3.1. ER stress and UPR are “physiologically” activated during adipogenesis, but, when overactivated, inhibits adipocyte differentiation. During differentiation of 3T3-L1 cells, the mRNAs of Cebp, Ppar2, Fabp4/Ap2, Adipoq and Glut4 genes increased (Fig. 1A). At day 7, Oil red O staining showed a robust lipid accumulation in these cells (Fig. 1C). Next, we investigated the activation of the UPR during differentiation. The mRNA of UPR markers BiP, total and spliced Xbp1 accumulated during adipocyte differentiation (Fig. 1B). Chop mRNA, on the other hand, decreased at day 2 and subsequently increased between days 4 and 7, as has been reported by others 26 (Fig. 1B). Notably, a similar activation of the UPR during adipocyte differentiation was present also in a murine multipotent mesenchymal cell line, W20-17 cells (Suppl. Fig.1). Next, we sought to investigate the effect of an exogenous induction of ER stress during adipocyte differentiation, such as that elicited by the classical ER stress inductor thapsigargin (Tg). As shown in Fig. 2A, a low dose (0.1 nM) of Tg, which is not cytotoxic (Table 1), led to a higher UPR activation respect to the “physiologic” activation that occurs during unperturbed adipocyte differentiation. This UPR overactivation was accompanied by an inhibition of adipogenesis, assessed by expression of adipocyte marker genes (Fig. 2B). Next, the effect of a pathologically relevant inducer of ER stress, glucosamine (GlcN), was evaluated. GlcN activates the hexosamine biosynthetic pathway (HBP). Increased HBP flux has been implicated in many of the adverse effects of hyperglycemia. Thus, we have previously shown that GlcN causes ER stress and activates the UPR in skeletal muscle (L6) and pancreatic beta cells (INS-1E) 29-30. Culturing 3T3-L1 cells in differentiation media in the presence of 7.5 mM GlcN activated UPR above the physiological levels (Fig. 2C). Notably, GlcN increased BiP and Chop mRNAs, and Xbp1 splicing, respect to their levels in the absence of perturbation, already at day 2 of differentiation. At the protein level, GlcN increased eIF2 phosphorylation, and BiP and Chop levels, respect to control adipocytes (Fig. 2E). In parallel with the UPR overactivation, GlcN treatment inhibited adipogenesis as shown by the reduced expression of mature adipocyte marker genes (Fig. 2D) and Oil red O staining (Fig. 2F). We sought to confirm these findings in primary adipocytes.
FABP4/AP2 ADIPOQ Culturing 3T3-L1 cells in differentiation media in the presence of high glucose (40 mM) gave analogous results (activation of ER stress above the physiological levels and inhibition of differentiation, Fig. 6A-B). GlcN enhanced UPR activation above the physiological level and concomitantly inhibited adipogenesis also in W20-17 cells (Supplemental Fig. S1). As a control for the osmotic effects of GlcN treatments, 3T3-L1 cells were exposed to 7.5 and 15 mmol/l xylose, which showed no effect neither on UPR and adipocyte-related genes nor on Oil red O staining (not shown). 3.2. Overactivation of UPR by GlcN is accompanied by increased pro-inflammatory
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cytokine release. Obesity is causally linked to a chronic low-grade inflammatory state 6-9. We sought to evaluate if the “pathologic” ER stress elicited by GlcN during adipocyte differentiation may result in a pro-inflammatory phenotype. Thus, we begun to evaluate the release of cytokines/chemokines in the culture medium of 3T3-L1 cells differentiated in the presence or in the absence of GlcN. Both, 3T3-L1 differentiated in the absence or presence of GlcN released in the culture medium detectable amounts of the pro-inflammatory cytokines/chemokines IL-12, IL-8, MCP-1 (CCL2), MIP-1 (CCL3), RANTES (CCL5), and TNF (Table 2). However, 3T3L1 differentiated in the presence of GlcN released higher amounts of IL-12, MCP-1, MIP-1, TNF, and RANTES (Table 2). To understand if these variations in release of cytokines/chemokines were concomitant with variations of mRNA expression, we measured the mRNA levels of them. Thus, we found an increase of mRNA levels of Tnf, Mip-1,, Mcp1, Il12, and Rantes (Fig. 4). Thus, the variations in cytokine release were accompanied by variations in the respective mRNA levels. The effect of GlcN on cytokine release was reproduced by exposing 3T3-L1 cells to high glucose (40 mM) during differentiation (Fig. 6C).
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3.3. Low concentrations of PBA inhibiting only the “pathologic” part of the UPR, do not inhibit adipocyte differentiation, but rescue the inhibitory effect of GlcN on adipogenesis, and the shift towards the inflammatory phenotype. In order to verify the causal role of GlcN-induced ER stress on adipocyte differentiation and cytokine production and release, we used the chemical chaperone PBA, which has been shown to alleviate ER stress in a number of systems 31-32. It has been recently shown that PBA inhibits adipocyte differentiation by inhibiting ER stress 33. However, this has been shown at high PBA concentrations (10-20 mM), that completely suppressed the “physiologic” ER stress required for a proper adipocyte differentiation. To reveal the link between “pathologic” ER stress and its cellular effects (differentiation/cytokine production/release), we reasoned that only this part of stress should be inhibited, trying to keep as low as possible, or even abolish, the inhibition of the “physiologic” part. We tried to achieve this goal by lowering the concentrations of PBA. We added PBA together with GlcN resulting in a co-treatment of seven days. In the paper of Basseri et al. 33, even 1 mM PBA had an inhibitory effect on differentiation, already at day 5 of treatment (although the effect was not significant). Moreover, although 1 mM PBA did not affect the physiologic up-regulation of BiP, this was checked after only one day of treatment. Thus, we decided to lower further the PBA concentration to 250 M, and it still inhibited, albeit partially, the physiologic upregulation of BiP and Ppar2 (Supplemental Table 1). Interestingly, 50 M PBA leaved unaltered BiP and Chop physiologic upregulation (Fig. 5A) and it was able to rescue the inhibitory effect of GlcN on adipocyte differentiation, quantified by expression of mature adipocyte marker genes (Fig. 5B). Paralleling the effect on adipocyte differentiation, 50 M PBA alone did not affect cytokine/chemokine mRNA levels in the differentiated adipocyte, but when given with GlcN removed the GlcN upregulation of the same mRNA levels (Fig. 5C). These data were confirmed in W20-17 cells (Supplemental Fig. S2). Moreover, 50 M PBA was also able to revert (albeith not completely) the pathologic activation of UPR by high glucose and the inhibitory effect of high glucose on 3T3-L1 8
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adipocyte differentiation (Fig. 6A-B). Next, to strengthen our results, we sought to inhibit ER stress with a complimentary method in addition to PBA. We used TUDCA which has been already shown to reproduce the effects of PBA 32. As shown in Supplemental Table 2, 5 M TUDCA was able to revert the effect of GlcN, although at the concentration tested was less effective than PBA.
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3.4. The shift towards an inflammatory phenotype elicited by GlcN is sensitive to a NFkB inhibitor. ER stress has been reported to activate NF-kB 34-36, and the cytokine/chemokine upregulated by GlcN are all well known targets of NF-kB 37. Thus, we investigated if 4Methyl-N-(3-phenylpropyl)benzene-1,2-diamine (JSH-23), that selectively blocks nuclear translocation of NF-kB p65 and its transcriptional activity, has an effect on glucotoxicinduced inflammatory phenotype. Pre-adipocytes were differentiated in the absence or in the presence of GlcN and in the last day of differentiation we added 7.5 M JSH-23, to minimize possible side effects. As shown in Fig. 7, JSH-23 alone did not affect Mcp1, Mip1, Rantes, and Tnf mRNA levels of the differentiated adipocyte, but when given to GlcN-treated cells, rescued the GlcN-mediated up-regulation of the same mRNAs (Fig. 7). Notably, also the release in the culture medium of IL-12, MCP-1, MIP-1, RANTES, TNF was decreased by JSH-23 (Table 2). Thus, the GlcN-induced increase of cytokine/chemokine mRNA levels and protein release is sensitive to a, bona fide, NF-kB inhibition.
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The aim of this study was to clarify the effect of ER stress and UPR activation on adipocyte differentiation. There are several reports suggesting that UPR activation is necessary for adipocyte differentiation 21-23 and on the contrary, others indicate a detrimental effect of UPR activation on adipocyte differentiation. Mice with decreased folding capacity and increased UPR gain less weight than wild type counterparts 24, and, conversely, HFD-fed mice with a defective PERK branch with a heterozygous mutation at the phosphorylation site in Eif2 (Ser51Ala, S/A) or Chop -/- are significantly more obese upon HFD than wild type mice 25-26.
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To clarify these conflicting evidences we have measured UPR activation and adipocyte differentiation in normal condition, in the presence of a strong UPR activator, Tg, and in the presence of a pathologically relevant inducer of ER stress, GlcN. Hyperglycemia increases HBP flux and generates GlcN, which is involved in hyperglycaemic-induced damage 38. GlcN activates UPR in several cell types, such as HepG2 liver cells 39, L6 skeletal muscle cells 29, and INS-1 pancreatic -cells 30, among others, impairing their function. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W2017 cells. Indeed, W20-17 cells express Nanog, a key mediator of embryonic stem cell (ESC) maintenance, present in various mulitpotential post-natal mesenchymal stem cells 40, 41 at levels comparable to murine bone marrow stromal cells 42. Their use avoids the high variability in adipocyte differentiation yield, a major drawback of the cell type mixture indicated as stromal vascular fraction (SVF) 43. Both Tg and GlcN induce ER stress above that occurring in the absence of perturbation and inhibit adipocyte differentiation (Fig. 2 and Supplemental S1). This suggests that ER stress and UPR activation has a dual effect on adipocyte differentiation. A low ER stress and UPR activation, like that occurring without perturbation during normal differentiation, is instrumental in adipocyte differentiation, as shown by others 21-23, probably to adapt the ER folding capacity to increased protein synthesis during differentiation. Instead, an ER stress quantitatively and/or qualitatively above this level, secondary to metabolic insults, has detrimental effects on differentiation. By using low PBA concentrations, we were able to inhibit only the GlcN-induced part of the UPR. Under these conditions PBA did not inhibit adipocyte differentiation, but reversed the inhibitory effect of GlcN (Fig. 5 and Supplemental S2). Thus, we were able to dissect the effects of the “physiologic” and “pathologic” part of ER stress. Downstream ER stress the inhibition of differentiation is very likely exerted by the PERK-Eif2a-CHOP axis, as shown by Han et al. 26. These results seem to be at variance with the finding of Basseri et al. 33 that showed that a treatment of 3T3-L1 with PBA, decreasing UPR activation, inhibited adipogenesis. As noted above, in that study, high concentrations of PBA were used, that suppressed the “physiologic” ER stress and adipocyte differentiation. In human obesity the increased adipose mass leads to an increased size of adipose 10
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cells (cell hypertrophy), mostly associated with abdominal adipose, although there is also recruitment of new adipocytes, prevalently present in peripheral obesity 3-5, 44. The development of insulin resistance and its complications are mostly associated with hypertrophic abdominal obesity and deposition of NEFA in other tissues, as liver and muscle 3-5, 44. Thus, ensuring adipose tissue expansion by new adipocyte recruitment is a safe-guard mechanism against the metabolic consequences of obesity. This mechanism is suppressed by a glucotoxic insult, which through a “pathologic” UPR activation, inhibits adipocyte differentiation and exacerbates hypertrophic obesity and its deleterious consequences.
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Our conclusion that physiologic and pathologic UPR activations have opposite effects on differentiation may extend beyond the adipocyte system and explain conflicting data present in literature. Indeed, the UPR can be activated by oscillation of cellular and/or environmental conditions in the physiological range, such those occurring during differentiation of highly secretory phenotypes 18. In other instances, however, UPR activation has been reported to inhibit differentiation. This is the case of hypertrophic chondrocytes expressing mutant collagen X, 19 or even a mutant thyroglobulin 20, and thyrocytes challenged with Tg and tunicamycin 45, 46. In such cases, the difference may be, indeed, physiologic versus pathologic UPR activation.
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Obesity (especially hypertrophic obesity) is characterized by a low grade inflammation present in several tissue but mostly in the adipose tissue. Several inflammatory cytokines 6,7 and immune cells (macrophages, limphocytes) 8-9, 47 are increased in obese tissues. Moreover, a shift in the macrophage phenotype is observed: recently recruited macrophages in obesity have a pro-inflammatory phenotype (M1-type or “classically” activated) compared with the “alternatively” activated phenotype (M2-type) of the resident adipose tissue macrophages from lean mice 48. While it is clear that these different cell types (adipocytes, macrophages, limphocytes) may cross-talk, amplifying inflammation, the question of how the inflammatory response occurs in obesity in the first place is not entirely clear. One mechanism may be that the progressive hypertrophy of adipocytes tends to lead to cell death, releasing cytokines and fatty acids. Macrophages will be recruited as scavengers 49 and trigger inflammation. It has been reported that ER stress markers are upregulated in adipose tissue and liver of obese subjects and are reduced after weight loss 50. Thus, ER stress and the inflammatory state of the adipose and other tissues, may be causally linked. Here we show that a glucotoxic insult during adipocyte differentiation is capable of inducing ER stress and this stimulates proinflammatory cytokine/chemokine production (Fig 4 and Supplemental S1, Table 1). Thus, the preadipocyte/adipocyte may sense in the first place the insult originating from nutrient excess and may trigger the inflammation of the adipose tissue. Secondarily, inflammatory cytokines/chemokines attract and activate immune cells in the adipose tissue, and these will produce more cytokines/chemokines with the build-up of feed-forward mechanisms. Indeed, we have shown the causal role of ER stress in shifting the phenotype of preadipocyte/adipocyte population to an inflammatory type, by its reversion elicited by
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PBA (Fig. 5 and Supplemental S2). The ER stressed preadipocyte/adipocyte releases increased amount of a primary cytokine like TNF, of chemokines like MCP-1, MIP-1, RANTES, and of the limphokine IL-12, which constitute an array of cytokines/chemokines able to trigger inflammation. TNF is one of the most important promoter of endothelial activation 51, MCP-1 promotes recruitment of inflammatory LY6ChiCCR2+ monocytes 52 which differentiate into M1-type macrophages 53. MIP1 and RANTES, also contribute to recruit monocytes through CCR1 and CCR5 receptors 54. The M1/M2 macrophages imbalance in obesity is also secondary to an imbalance between TH1 and TH2/CD4+Foxp3+ limphocytes towards the TH1 population 47. Thus, M1 macrophages are generated by TH1 cytokines, including IFN-gamma, while M2 macrophages are generated by TH2 and CD4+Foxp3+ cytokines, IL-4 and IL-13 55. However, the changes in the adipose tissue that initiate TH1 cell influx/expansion are not fully understood, but may include IL12, which participates in the generation of TH1 cells 56 and/or the TH1 chemokine RANTES 57. Thus, our results, by showing Il12 and Rantes production by ER stressed preadipocytes/adipocytes, suggest the possibility of their participation in the influx/expansion of TH1 cells in the adipose tissue.
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However, the effects of a “pathologic” ER stress on adipocyte differentiation and cytokine secretion, may also positively cross-talk. The increased TNF production by adipocytes may exacerbate the inhibition of preadipocyte differentiation 58.
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5. CONCLUSIONS In conclusion, we have shown how a quantitative and/or qualitative overactivation of ER stress and UPR, secondary to a glucotoxic insult dictates the outcome on adipocyte (and perhaps other cell types) differentiation. The inhibition of differentiation by the “pathologic” ER stress will drive the adipocyte population towards an hypertrophic phenotype. Moreover, we have shown that a “pathologic” ER stress stimulates the preadipo/adipocyte population to release several cytokines/chemokines able to trigger inflammation by acting on inflammatory cells such as macrophages and limphocytes. Thus, the metabolic cells may represent the primary target of the insults that lead to the inflammatory state characteristic of the adipose tissue in obesity. ACKNOWLEDGEMENTS This work has been supported, in part, by the European Foundation for the Study of Diabetes (EFSD), by the Ministero dell’Università e della Ricerca Scientifica (grants PRIN and FIRB-MERIT, and PON 01_02460) and by the Società Italiana di Diabetologia (SID-FO.DI.RI). This work was also supported by the P.O.R. Campania FSE 2007-2013, Project CREMe.
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Figure 1. During differentiation 3T3-L1 cells experience ER stress and UPR activation. Confluent 3T3-L1 cells were cultured in differentiation media for 7 days. (A) mRNA levels of adipocyte-related genes and (B) of UPR genes. Total RNA was isolated from 3T3-L1 cells at the indicated times after the induction of differentiation and mRNA levels were measured by quantitative real-time RT-PCR and quantified as relative expression units (REU) vs. cells at 0 time. Xbp1-s and Xbp1-t indicate spliced and total Xbp1, respectively. Data are means ± SEM of three independent experiments with duplicates. p<0.01, p<0.001. C) On days 0 and 7, cells were fixed and stained with Oil red O. Representative images of Oil red O staining are shown.
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Figure 2. Tg and GlcN induce ER stress above that occurring during normal differentiation and inhibit adipocyte differentiation of 3T3-L1 cells. (A, B) Confluent 3T3-L1 cells were cultured in differentiation media in the presence of 0.1 nM Tg. On day 7, total RNA was isolated, and mRNA levels of UPR (A) and adipocytes-related (B) genes were measured by quantitative real-time RT-PCR and quantified as REU vs. control cells. Data are means ± SEM of three independent experiments with duplicates. p<0.001. (C-F) Confluent 3T3-L1 cells were cultured in differentiation media in the presence of 7.5 mM GlcN. At the indicated times after initiation of differentiation, mRNA levels of UPR genes (C) and adipocyte-related genes (D) were measured by quantitative real-time RTPCR and quantified as REU vs. control cells at the same time. Data are means ± SEM of three independent experiments with duplicates. p<0.05, p<0.01, p<0.001 indicates significance of the value of GlcN-treated cells vs. control cells at the same time. At day 7, cell lysates were collected and analyzed by western blot (E), or cells were fixed and stained with Oil red O (F). Representative images of Oil red O staining and quantification of Oil red O staining of three independent experiments are shown. p<0.001. Figure 3. GlcN induces ER stress and inhibit adipocyte differentiation of human primary adipocytes. Human primary adipocytes were isolated as outlined in Materials and Methods and incubated in control conditions or in the presence of 20 mM GlcN for 6 hrs. mRNA levels of UPR genes (A) and adipocyte-related genes (B) were measured by quantitative realtime RT-PCR and quantified as REU vs. control cells. Data are means ± SEM of three independent experiments with duplicates. p<0.05, p<0.01. Figure 4. 3T3-L1 cells differentiated in the presence of GlcN are shifted towards a proinflammatory phenotype. Confluent 3T3-L1 cells were cultured in differentiation media in the presence of 7.5 mM GlcN. At day 7, total RNA was isolated, and mRNA levels of cytokines/chemokines were measured by quantitative real-time RT-PCR and quantified as REU vs. control cells. Data are means ± SEM of three independent experiments with duplicates. p<0.05,
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Figure 5. A low PBA concentration inhibits only the “pathologic” part of ER stress, and revert the inhibitory effect of GlcN on differentiation. Confluent 3T3-L1 cells were cultured in differentiation media in the presence of 7.5 mM GlcN and/or 50 M PBA. At day 7, total RNA was isolated, and mRNA levels of UPR (A), adipocyte-related (B) and cytokine/chemokine (C) genes were measured by quantitative real-time RT-PCR and quantified as relative REU vs. control cells. Data are means ± SEM of three independent experiments with duplicates. p<0.05, p<0.01, p<0.001.
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Figure 6. High glucose induces ER stress, inhibits adipocyte differentiation, and increases Mip-1 and Rantes expression in 3T3-L1 cells. Confluent 3T3-L1 cells were cultured in differentiation media in control condition, in the presence of 40 mM glucose, and in the presence of 40 mM glucose and 50 M PBA. At day 7, total RNA was isolated, and mRNA levels of UPR (A), adipocyte-related (B) and cytokine/chemokine (C) genes were measured by quantitative real-time RT-PCR and quantified as relative REU vs. control cells. Data are means ± SEM of three independent experiments with duplicates. p<0.05, p<0.01.
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Figure 7. The p65Rel translocation inhibitor JSH-23 opposes the GlcN-induced shift towards a pro-inflammatory phenotype. Confluent 3T3-L1 cells were cultured in differentiation media in the presence of 7.5 mM GlcN and in the last day of 7.5 M JSH-23. At day 7, total RNA was isolated, and mRNA levels of cytokine/chemokine genes were measured by quantitative real-time RT-PCR and quantified as REU vs. control cells. Data are means ± SEM of three independent p<0.05, p<0.01, experiments with duplicates. p<0.001.
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Table 1: 3T3-L1 cell viability upon Tg treatments. % cell viability respect to control Control 100 1.0 nM Tg 88.6 4.6* 0.1 nM Tg 95.2 6.6 3T3-L1 cells were differentiated, as reported in Materials and Methods, in vehicle or in presence of 1.0 or 0.1 nM Th. At the end of the differentiation period, cell viability was assessed by the MTT assay as previously reported 25. The percentage of survival was calculated as the absorbance ratio of treated to untreated cells. The data presented are the mean ± SEM from four replicate wells, replicated three times. *p0.05 versus control.
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Table 2: Effect of GlcN and GlcN+JSH on 3T3-L1-released cytokines. Cytokine 3T3-L1 CM 3T3-L1 GlcN 3T3-L1 GlcN+JSH (pg/ml) CM CM (pg/ml) (pg/ml) IL-2 ND ND IL-8 5023.3425.6 6493567.6 5492.7486.8 IL-12 4.20.52 10.140.75, 18.31.12 IL-13 ND ND ND GM-CSF ND ND ND MCP-1 (CCL2) 16113115.4 13527121.12 21605103.7 MIP-1 81.17 18.11.45, 272.35 (CCL3) RANTES 205.311.12 426.315.72 358.210.56, (CCL5) TNF- 4.12.32 12.671.89, 28.93.11 Data represent the mean ± SD of at least four independent triplicate experiments. 3T3L1 cells were differentiated under standard conditions (control, 3T3-L1 CM), in the presence of GlcN (3T3-L1 GlcN CM), or in the presence of GlcN and, in the last day, of JSH (3T3-L1 GlcN+JSH CM). At day 7, conditioned media (CM) were collected for 24 h in Dulbecco's modified Eagle's medium without serum, with 0.5% BSA (manufacturer’s instructions), and tested by using a custom premixed assay from Biorad (Bio-Plex ProTM) as described in the Materials and Methods. p<0.05, p<0.01, p<0.001, for 3T3-L1 GlcN adipocytes vs. 3T3-L1 control adipocytes; p<0.05, p<0.01, for GlcN+JSH adipocytes vs. GlcN adipocytes; p<0.05, p<0.01 for GlcN+JSH adipocytes vs. control adipocytes.
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ACCEPTED MANUSCRIPT Highlights Glucosamine induces ER stress above the level occurring during differentiation and inhibits adipocyte differentiation
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Glucosamine increases the release of proinfammatory cytokines
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Low concentration of PBA (a chemical chaperone) inhibits only the “pathologic” part of ER stress
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PBA reverses the inhibitory effect of glucosamine on adipocyte differentiation
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PBA reverses the stimulatory effect of glucosamine on cytokine release
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S0167-4889(16)30044-1 doi: 10.1016/j.bbamcr.2016.02.019 BBAMCR 17815
To appear in:
BBA - Molecular Cell Research
Received date: Revised date: Accepted date:
30 July 2015 2 February 2016 26 February 2016
Please cite this article as: Michele Longo, Rosa Spinelli, Vittoria D’Esposito, Federica Zatterale, Francesca Fiory, Cecilia Nigro, Gregory A. Raciti, Claudia Miele, Pietro Formisano, Francesco Beguinot, Bruno Di Jeso, Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype, BBA - Molecular Cell Research (2016), doi: 10.1016/j.bbamcr.2016.02.019
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PATHOLOGIC ENDOPLASMIC RETICULUM STRESS INDUCED BY GLUCOTOXIC INSULTS INHIBITS ADIPOCYTE DIFFERENTIATION AND INDUCES AN INFLAMMATORY PHENOTYPE. Michele Longo, Rosa Spinelli, Vittoria D’Esposito°, Federica Zatterale, Francesca Fiory, Cecilia Nigro°, Gregory A. Raciti°, Claudia Miele°, Pietro Formisano, Francesco Beguinot , and Bruno Di Jeso .
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Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Strada Monteroni, 73100 Lecce, Italy. Dipartimento di Scienze Mediche Traslazionali, Università “Federico II”, IEOS/CNR, via S. Pansini 5 80131 Napoli, Italy.
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To whom correspondence should be addressed: Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Strada Monteroni, 73100 Lecce, Italy. Tel. 0039 0832 298 912 Email [email protected] These authors equally contributed to the work.
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ABSTRACT Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is “physiologically” activated during adipocyte differentiation, the “pathologic” part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype. Keywords Adipocyte differentiation, ER stress, inflammation. 1
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Abbreviations BiP, Binding protein; CCL2, chemokine (C-C motif) ligand 2; CCL3, chemokine (C-C motif) ligand 3; CCL5, chemokine (C-C motif) ligand 5; C/EBP, CCAAT/enhancer binding protein; CHOP, C/EBP homologous protein; Ct, Comparative threshold cycle; eIF2, Eukaryotic initiation factor 2 alpha; ER, Endoplasmic reticulum; GM-CSF, Granulocyte-macrophage colony-stimulating factor; HBP, Hexosamine biosynthetic pathway; HFD, High Fat Diet; IL, interleukin; MCP-1, Monocyte chemoattractant protein 1; MIP-1, Macrophage inflammatory protein1-alpha; MTT, (3-(4,5-dimethyl- thiazol-2yl)-2,5-diphenol tetrazolium bromide); PERK, Protein kinase RNA-like ER kinase; PPAR, Peroxisome proliferator-activated receptor; PBA, 4-Phenyl butyric acid; RANTES, Regulated upon activation normally T expressed, and secreted; REU, relative expression units; Tg, thapsigargin; TH1, T helper 1; TH2, T helper 2; TUDCA, taurine-conjugated ursodeoxycholic acid; TSH, thyroid stimulating hormone; UPR, Unfolded protein response.
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1. INTRODUCTION Obesity has become a major worldwide health problem, because of its dramatically increased incidence, high prevalence and associated mortality 1. Obesity is characterized by increased fat mass caused by increased adipocyte number (hyperplasia) and/or size (hypertrophy) 2. Hypertrophic obesity is mostly associated with abdominal obesity, which in turn is associated with increased insulin resistance, risk of developing type 2 diabetes, and cardiovascular disease 3-5. Increasing evidence indicates that obesity is causally linked to a chronic low-grade inflammatory state, called “metainflammation”, which contributes to the development of obesity-linked disorders 6-9.
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The ER is a multifunctional organelle. The ER acts as gatekeeper to the secretory pathway by folding, modifying, and exporting nascent secretory and transmembrane proteins, but it is also a stress-sensing organelle. It senses stress caused by disequilibrium between ER load and folding capacity, ER stress, and responds by activating the unfolded protein response (UPR) 10. In recent years, the perspective of the UPR as an exclusive stress response has been challenged. Thus the UPR can be activated by stimuli that represent physiological oscillations of environmental and/or cellular conditions, such as diurnal induction of the UPR in dependency of the circadian clock 11, and, in thyrocytes, stimulation with TSH 12 that increases the synthesis of the highly expressed thyroglobulin 13-17. Perhaps the most dramatic examples of physiologic UPR upregulation are those taking place during cellular differentiation, such as that of B cells into plasma cells 18 . However, also the opposite effect has been observed, in that UPR activation inhibits differentiation. In transgenic mice models expressing mutant collagen X, the UPR was activated in hypertrophic chondrocytes and terminal differentiation is interrupted, producing a chondrodysplasia phenotype 19. Interestingly, in a transgenic mouse model using the collagen X promoter to drive expression of the cog mutant of thyroglobulin in hypertrophic chondrocytes, the same phenotype was observed 20, confirming the causal role of ER stress in inhibiting chondrocyte differentiation. Data reported on adipogenic differentiation reproduce this apparent contradiction. Some studies suggest that ER stress and UPR activation might be important for adipogenesis 21-23. However, it has been also reported that mice heterozygous for BiP gain less fat mass compared with wild-type mice 24. A reduction of BiP decreases the folding capacity and favors UPR activation. Thus, the reduced fat mass in these mice is an effect of inhibited adipogenesis that likely follows an activation of UPR. In addition, highfat diet (HFD)-fed mice with a heterozygous mutation at the phosphorylation site in Eif2 (Ser51Ala, S/A) became significantly more obese than HFD-fed wild-type mice, suggesting that the PERK-Eif2 branch of the UPR inhibits adipogenesis 25. Thus, eIF2α phosphorylation inhibits adipocyte differentiation in response to ER stress in vitro and in vivo, by increasing CHOP production 26. In this study we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We sought to substantiate the hypothesis that while ER stress and UPR activation that occurs during adipocyte differentiation is a physiologic response 3
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instrumental to differentiation, as shown by others 21-23, the ER stress and UPR secondary to glucotoxic insults is pathologic, causing an inhibition of differentiation. Moreover, we sought to determine the effect of ER stress and UPR activation during differentiation on the inflammatory phenotype of preadipocyte/adipocyte population, which may be relevant to the onset and development of the low grade inflammation of the adipose tissue.
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2. MATERIALS AND METHODS. 2.1. Materials. Media, sera and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA). Chemicals were from Sigma-Aldrich (St Louis, MO, USA). Glucosamine was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Insulin, TRIzol, and SuperScript III were from Invitrogen (Carlsbad, CA,USA). Cytokine assay kit and Lowry protein assay were from BioRad (Hercules, CA, USA). ECLWestern Blot detection reagents were from GE Healthcare (Pittsburgh, PA, USA). Antibodies were anti-tubulin (monoclonal; Sigma), anti-binding protein (BiP) and anti-Chop (Santa Cruz). 2.2. Cell culture, adipocyte differentiation, and primary adipocyte isolation. 3T3-L1 mouse embryo fibroblasts were obtained from the American Type Culture Collection (Manassas, VA, USA). They were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, penicillin (200 IU/ml), streptomycin (100 g/ml), in a humidified CO2 incubator. Two days after confluency, cell differentiation into adipocytes was induced with DMEM containing 1 g/ml insulin, 0.5 mmol/l 3-isobutyl-1methylxanthine and 1 mol/l dexamethasone for 2 days, followed by DMEM supplemented with 10% FBS and 1 g/ml insulin only, for another 5 days. Oil red O working solution was added to formalin-fixed cells and incubated for 10 min at room temperature. Images were taken using an Olympus microscope system (Olympus, Center Valley, PA, USA). For quantification, absorbance was measured at 500 nm using a spectrophotometer (Beckman, CA, USA). Human adipose tissue samples were obtained from abdominal adipose tissue biopsies of healthy subjects (n=5; age 25-63 years; BMI 20.2-25.3) undergoing biliary surgery. All subjects were otherwise free of metabolic or endocrine diseases. Informed consent was obtained from every patient before the surgical procedure. This procedure was approved by the ethical committee of the University of Naples. Adipose tissue was digested with collagenase and mature adipocytes were isolated as previously reported 27. Isolated adipocytes were filtered through a 250-m nylon mesh and washed four times with fresh DMEM -F12 (1:1) 10% FBS medium. The cells were incubated at 37 °C with 20mM GlcN for 6h. Next, adipocytes were washed with PBS 1X and RNA extracted as described previously. 2.3. RNA isolation and real Time RT-PCR Total RNA was extracted with the TRIzol reagent, according to the manufacturer’s protocol. Reverse transcription of 1 µg of total RNA was performed using SuperScript III, following the manufacturer’s instructions. Quantitative real time PCR was performed in triplicate by using iQ SYBR Green Supermix on iCycler real time detection system (BioRad). Relative quantification of gene expression was calculated by the ΔΔCt method. Each Ct value was first normalized to the respective Cyclophilin Ct value of a sample to account for variability in the concentration of RNA and in the conversion efficiency of the RT reaction. The primers used are listed in Supplementary Materials and Methods. For the selective amplification of Xbp1-s, the forward primer was designed across the Xbp1s deletion. Thus, the Xbp1-s forward primer corresponded to a non-contiguous sequence in the Xbp1-t cDNA. 2.4. Cytokine and growth factor assay
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3T3-L1 adipocyte conditioned media were screened for the concentration of several cytokines/chemokines using a custom-blended Bio-Plex ProTM (Bio-Rad) assay according to the manufacturer’s protocol. 2.5. Western blotting and cell viability assay. Cell lysates were obtained in cell lysis buffer (50 mmol/l Tris HCl [pH 7.4], 150 mmol/l NaCl, 1% [vol./vol.] Triton X-100, 0.1% [wt/vol.] SDS, 1% [wt/vol.] sodium deoxycholate and protease inhibitors) and total protein concentration in each sample was measured using the Lowry protein assay kit (Biorad). Chemiluminescence detection was performed using ECLWestern Blot detection reagents. Membranes were exposed to imaging film and developed using a Kodak X-OMAT processor (Kodak, Rochester, NY, USA). Cell viability was assessed by the MTT assay as previously reported 28. 2.6. Statistical analysis All data are presented as means ± SEM. The difference between groups was evaluated using Student’s t test, and, when more than two groups were present, ANOVA and post hoc tests (Bonferroni/Dunn) were also performed. p<0.05 was considered significant.
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3. RESULTS 3.1. ER stress and UPR are “physiologically” activated during adipogenesis, but, when overactivated, inhibits adipocyte differentiation. During differentiation of 3T3-L1 cells, the mRNAs of Cebp, Ppar2, Fabp4/Ap2, Adipoq and Glut4 genes increased (Fig. 1A). At day 7, Oil red O staining showed a robust lipid accumulation in these cells (Fig. 1C). Next, we investigated the activation of the UPR during differentiation. The mRNA of UPR markers BiP, total and spliced Xbp1 accumulated during adipocyte differentiation (Fig. 1B). Chop mRNA, on the other hand, decreased at day 2 and subsequently increased between days 4 and 7, as has been reported by others 26 (Fig. 1B). Notably, a similar activation of the UPR during adipocyte differentiation was present also in a murine multipotent mesenchymal cell line, W20-17 cells (Suppl. Fig.1). Next, we sought to investigate the effect of an exogenous induction of ER stress during adipocyte differentiation, such as that elicited by the classical ER stress inductor thapsigargin (Tg). As shown in Fig. 2A, a low dose (0.1 nM) of Tg, which is not cytotoxic (Table 1), led to a higher UPR activation respect to the “physiologic” activation that occurs during unperturbed adipocyte differentiation. This UPR overactivation was accompanied by an inhibition of adipogenesis, assessed by expression of adipocyte marker genes (Fig. 2B). Next, the effect of a pathologically relevant inducer of ER stress, glucosamine (GlcN), was evaluated. GlcN activates the hexosamine biosynthetic pathway (HBP). Increased HBP flux has been implicated in many of the adverse effects of hyperglycemia. Thus, we have previously shown that GlcN causes ER stress and activates the UPR in skeletal muscle (L6) and pancreatic beta cells (INS-1E) 29-30. Culturing 3T3-L1 cells in differentiation media in the presence of 7.5 mM GlcN activated UPR above the physiological levels (Fig. 2C). Notably, GlcN increased BiP and Chop mRNAs, and Xbp1 splicing, respect to their levels in the absence of perturbation, already at day 2 of differentiation. At the protein level, GlcN increased eIF2 phosphorylation, and BiP and Chop levels, respect to control adipocytes (Fig. 2E). In parallel with the UPR overactivation, GlcN treatment inhibited adipogenesis as shown by the reduced expression of mature adipocyte marker genes (Fig. 2D) and Oil red O staining (Fig. 2F). We sought to confirm these findings in primary adipocytes.
FABP4/AP2 ADIPOQ Culturing 3T3-L1 cells in differentiation media in the presence of high glucose (40 mM) gave analogous results (activation of ER stress above the physiological levels and inhibition of differentiation, Fig. 6A-B). GlcN enhanced UPR activation above the physiological level and concomitantly inhibited adipogenesis also in W20-17 cells (Supplemental Fig. S1). As a control for the osmotic effects of GlcN treatments, 3T3-L1 cells were exposed to 7.5 and 15 mmol/l xylose, which showed no effect neither on UPR and adipocyte-related genes nor on Oil red O staining (not shown). 3.2. Overactivation of UPR by GlcN is accompanied by increased pro-inflammatory
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cytokine release. Obesity is causally linked to a chronic low-grade inflammatory state 6-9. We sought to evaluate if the “pathologic” ER stress elicited by GlcN during adipocyte differentiation may result in a pro-inflammatory phenotype. Thus, we begun to evaluate the release of cytokines/chemokines in the culture medium of 3T3-L1 cells differentiated in the presence or in the absence of GlcN. Both, 3T3-L1 differentiated in the absence or presence of GlcN released in the culture medium detectable amounts of the pro-inflammatory cytokines/chemokines IL-12, IL-8, MCP-1 (CCL2), MIP-1 (CCL3), RANTES (CCL5), and TNF (Table 2). However, 3T3L1 differentiated in the presence of GlcN released higher amounts of IL-12, MCP-1, MIP-1, TNF, and RANTES (Table 2). To understand if these variations in release of cytokines/chemokines were concomitant with variations of mRNA expression, we measured the mRNA levels of them. Thus, we found an increase of mRNA levels of Tnf, Mip-1,, Mcp1, Il12, and Rantes (Fig. 4). Thus, the variations in cytokine release were accompanied by variations in the respective mRNA levels. The effect of GlcN on cytokine release was reproduced by exposing 3T3-L1 cells to high glucose (40 mM) during differentiation (Fig. 6C).
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3.3. Low concentrations of PBA inhibiting only the “pathologic” part of the UPR, do not inhibit adipocyte differentiation, but rescue the inhibitory effect of GlcN on adipogenesis, and the shift towards the inflammatory phenotype. In order to verify the causal role of GlcN-induced ER stress on adipocyte differentiation and cytokine production and release, we used the chemical chaperone PBA, which has been shown to alleviate ER stress in a number of systems 31-32. It has been recently shown that PBA inhibits adipocyte differentiation by inhibiting ER stress 33. However, this has been shown at high PBA concentrations (10-20 mM), that completely suppressed the “physiologic” ER stress required for a proper adipocyte differentiation. To reveal the link between “pathologic” ER stress and its cellular effects (differentiation/cytokine production/release), we reasoned that only this part of stress should be inhibited, trying to keep as low as possible, or even abolish, the inhibition of the “physiologic” part. We tried to achieve this goal by lowering the concentrations of PBA. We added PBA together with GlcN resulting in a co-treatment of seven days. In the paper of Basseri et al. 33, even 1 mM PBA had an inhibitory effect on differentiation, already at day 5 of treatment (although the effect was not significant). Moreover, although 1 mM PBA did not affect the physiologic up-regulation of BiP, this was checked after only one day of treatment. Thus, we decided to lower further the PBA concentration to 250 M, and it still inhibited, albeit partially, the physiologic upregulation of BiP and Ppar2 (Supplemental Table 1). Interestingly, 50 M PBA leaved unaltered BiP and Chop physiologic upregulation (Fig. 5A) and it was able to rescue the inhibitory effect of GlcN on adipocyte differentiation, quantified by expression of mature adipocyte marker genes (Fig. 5B). Paralleling the effect on adipocyte differentiation, 50 M PBA alone did not affect cytokine/chemokine mRNA levels in the differentiated adipocyte, but when given with GlcN removed the GlcN upregulation of the same mRNA levels (Fig. 5C). These data were confirmed in W20-17 cells (Supplemental Fig. S2). Moreover, 50 M PBA was also able to revert (albeith not completely) the pathologic activation of UPR by high glucose and the inhibitory effect of high glucose on 3T3-L1 8
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adipocyte differentiation (Fig. 6A-B). Next, to strengthen our results, we sought to inhibit ER stress with a complimentary method in addition to PBA. We used TUDCA which has been already shown to reproduce the effects of PBA 32. As shown in Supplemental Table 2, 5 M TUDCA was able to revert the effect of GlcN, although at the concentration tested was less effective than PBA.
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3.4. The shift towards an inflammatory phenotype elicited by GlcN is sensitive to a NFkB inhibitor. ER stress has been reported to activate NF-kB 34-36, and the cytokine/chemokine upregulated by GlcN are all well known targets of NF-kB 37. Thus, we investigated if 4Methyl-N-(3-phenylpropyl)benzene-1,2-diamine (JSH-23), that selectively blocks nuclear translocation of NF-kB p65 and its transcriptional activity, has an effect on glucotoxicinduced inflammatory phenotype. Pre-adipocytes were differentiated in the absence or in the presence of GlcN and in the last day of differentiation we added 7.5 M JSH-23, to minimize possible side effects. As shown in Fig. 7, JSH-23 alone did not affect Mcp1, Mip1, Rantes, and Tnf mRNA levels of the differentiated adipocyte, but when given to GlcN-treated cells, rescued the GlcN-mediated up-regulation of the same mRNAs (Fig. 7). Notably, also the release in the culture medium of IL-12, MCP-1, MIP-1, RANTES, TNF was decreased by JSH-23 (Table 2). Thus, the GlcN-induced increase of cytokine/chemokine mRNA levels and protein release is sensitive to a, bona fide, NF-kB inhibition.
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The aim of this study was to clarify the effect of ER stress and UPR activation on adipocyte differentiation. There are several reports suggesting that UPR activation is necessary for adipocyte differentiation 21-23 and on the contrary, others indicate a detrimental effect of UPR activation on adipocyte differentiation. Mice with decreased folding capacity and increased UPR gain less weight than wild type counterparts 24, and, conversely, HFD-fed mice with a defective PERK branch with a heterozygous mutation at the phosphorylation site in Eif2 (Ser51Ala, S/A) or Chop -/- are significantly more obese upon HFD than wild type mice 25-26.
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To clarify these conflicting evidences we have measured UPR activation and adipocyte differentiation in normal condition, in the presence of a strong UPR activator, Tg, and in the presence of a pathologically relevant inducer of ER stress, GlcN. Hyperglycemia increases HBP flux and generates GlcN, which is involved in hyperglycaemic-induced damage 38. GlcN activates UPR in several cell types, such as HepG2 liver cells 39, L6 skeletal muscle cells 29, and INS-1 pancreatic -cells 30, among others, impairing their function. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W2017 cells. Indeed, W20-17 cells express Nanog, a key mediator of embryonic stem cell (ESC) maintenance, present in various mulitpotential post-natal mesenchymal stem cells 40, 41 at levels comparable to murine bone marrow stromal cells 42. Their use avoids the high variability in adipocyte differentiation yield, a major drawback of the cell type mixture indicated as stromal vascular fraction (SVF) 43. Both Tg and GlcN induce ER stress above that occurring in the absence of perturbation and inhibit adipocyte differentiation (Fig. 2 and Supplemental S1). This suggests that ER stress and UPR activation has a dual effect on adipocyte differentiation. A low ER stress and UPR activation, like that occurring without perturbation during normal differentiation, is instrumental in adipocyte differentiation, as shown by others 21-23, probably to adapt the ER folding capacity to increased protein synthesis during differentiation. Instead, an ER stress quantitatively and/or qualitatively above this level, secondary to metabolic insults, has detrimental effects on differentiation. By using low PBA concentrations, we were able to inhibit only the GlcN-induced part of the UPR. Under these conditions PBA did not inhibit adipocyte differentiation, but reversed the inhibitory effect of GlcN (Fig. 5 and Supplemental S2). Thus, we were able to dissect the effects of the “physiologic” and “pathologic” part of ER stress. Downstream ER stress the inhibition of differentiation is very likely exerted by the PERK-Eif2a-CHOP axis, as shown by Han et al. 26. These results seem to be at variance with the finding of Basseri et al. 33 that showed that a treatment of 3T3-L1 with PBA, decreasing UPR activation, inhibited adipogenesis. As noted above, in that study, high concentrations of PBA were used, that suppressed the “physiologic” ER stress and adipocyte differentiation. In human obesity the increased adipose mass leads to an increased size of adipose 10
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cells (cell hypertrophy), mostly associated with abdominal adipose, although there is also recruitment of new adipocytes, prevalently present in peripheral obesity 3-5, 44. The development of insulin resistance and its complications are mostly associated with hypertrophic abdominal obesity and deposition of NEFA in other tissues, as liver and muscle 3-5, 44. Thus, ensuring adipose tissue expansion by new adipocyte recruitment is a safe-guard mechanism against the metabolic consequences of obesity. This mechanism is suppressed by a glucotoxic insult, which through a “pathologic” UPR activation, inhibits adipocyte differentiation and exacerbates hypertrophic obesity and its deleterious consequences.
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Our conclusion that physiologic and pathologic UPR activations have opposite effects on differentiation may extend beyond the adipocyte system and explain conflicting data present in literature. Indeed, the UPR can be activated by oscillation of cellular and/or environmental conditions in the physiological range, such those occurring during differentiation of highly secretory phenotypes 18. In other instances, however, UPR activation has been reported to inhibit differentiation. This is the case of hypertrophic chondrocytes expressing mutant collagen X, 19 or even a mutant thyroglobulin 20, and thyrocytes challenged with Tg and tunicamycin 45, 46. In such cases, the difference may be, indeed, physiologic versus pathologic UPR activation.
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Obesity (especially hypertrophic obesity) is characterized by a low grade inflammation present in several tissue but mostly in the adipose tissue. Several inflammatory cytokines 6,7 and immune cells (macrophages, limphocytes) 8-9, 47 are increased in obese tissues. Moreover, a shift in the macrophage phenotype is observed: recently recruited macrophages in obesity have a pro-inflammatory phenotype (M1-type or “classically” activated) compared with the “alternatively” activated phenotype (M2-type) of the resident adipose tissue macrophages from lean mice 48. While it is clear that these different cell types (adipocytes, macrophages, limphocytes) may cross-talk, amplifying inflammation, the question of how the inflammatory response occurs in obesity in the first place is not entirely clear. One mechanism may be that the progressive hypertrophy of adipocytes tends to lead to cell death, releasing cytokines and fatty acids. Macrophages will be recruited as scavengers 49 and trigger inflammation. It has been reported that ER stress markers are upregulated in adipose tissue and liver of obese subjects and are reduced after weight loss 50. Thus, ER stress and the inflammatory state of the adipose and other tissues, may be causally linked. Here we show that a glucotoxic insult during adipocyte differentiation is capable of inducing ER stress and this stimulates proinflammatory cytokine/chemokine production (Fig 4 and Supplemental S1, Table 1). Thus, the preadipocyte/adipocyte may sense in the first place the insult originating from nutrient excess and may trigger the inflammation of the adipose tissue. Secondarily, inflammatory cytokines/chemokines attract and activate immune cells in the adipose tissue, and these will produce more cytokines/chemokines with the build-up of feed-forward mechanisms. Indeed, we have shown the causal role of ER stress in shifting the phenotype of preadipocyte/adipocyte population to an inflammatory type, by its reversion elicited by
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PBA (Fig. 5 and Supplemental S2). The ER stressed preadipocyte/adipocyte releases increased amount of a primary cytokine like TNF, of chemokines like MCP-1, MIP-1, RANTES, and of the limphokine IL-12, which constitute an array of cytokines/chemokines able to trigger inflammation. TNF is one of the most important promoter of endothelial activation 51, MCP-1 promotes recruitment of inflammatory LY6ChiCCR2+ monocytes 52 which differentiate into M1-type macrophages 53. MIP1 and RANTES, also contribute to recruit monocytes through CCR1 and CCR5 receptors 54. The M1/M2 macrophages imbalance in obesity is also secondary to an imbalance between TH1 and TH2/CD4+Foxp3+ limphocytes towards the TH1 population 47. Thus, M1 macrophages are generated by TH1 cytokines, including IFN-gamma, while M2 macrophages are generated by TH2 and CD4+Foxp3+ cytokines, IL-4 and IL-13 55. However, the changes in the adipose tissue that initiate TH1 cell influx/expansion are not fully understood, but may include IL12, which participates in the generation of TH1 cells 56 and/or the TH1 chemokine RANTES 57. Thus, our results, by showing Il12 and Rantes production by ER stressed preadipocytes/adipocytes, suggest the possibility of their participation in the influx/expansion of TH1 cells in the adipose tissue.
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However, the effects of a “pathologic” ER stress on adipocyte differentiation and cytokine secretion, may also positively cross-talk. The increased TNF production by adipocytes may exacerbate the inhibition of preadipocyte differentiation 58.
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5. CONCLUSIONS In conclusion, we have shown how a quantitative and/or qualitative overactivation of ER stress and UPR, secondary to a glucotoxic insult dictates the outcome on adipocyte (and perhaps other cell types) differentiation. The inhibition of differentiation by the “pathologic” ER stress will drive the adipocyte population towards an hypertrophic phenotype. Moreover, we have shown that a “pathologic” ER stress stimulates the preadipo/adipocyte population to release several cytokines/chemokines able to trigger inflammation by acting on inflammatory cells such as macrophages and limphocytes. Thus, the metabolic cells may represent the primary target of the insults that lead to the inflammatory state characteristic of the adipose tissue in obesity. ACKNOWLEDGEMENTS This work has been supported, in part, by the European Foundation for the Study of Diabetes (EFSD), by the Ministero dell’Università e della Ricerca Scientifica (grants PRIN and FIRB-MERIT, and PON 01_02460) and by the Società Italiana di Diabetologia (SID-FO.DI.RI). This work was also supported by the P.O.R. Campania FSE 2007-2013, Project CREMe.
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Figure 1. During differentiation 3T3-L1 cells experience ER stress and UPR activation. Confluent 3T3-L1 cells were cultured in differentiation media for 7 days. (A) mRNA levels of adipocyte-related genes and (B) of UPR genes. Total RNA was isolated from 3T3-L1 cells at the indicated times after the induction of differentiation and mRNA levels were measured by quantitative real-time RT-PCR and quantified as relative expression units (REU) vs. cells at 0 time. Xbp1-s and Xbp1-t indicate spliced and total Xbp1, respectively. Data are means ± SEM of three independent experiments with duplicates. p<0.01, p<0.001. C) On days 0 and 7, cells were fixed and stained with Oil red O. Representative images of Oil red O staining are shown.
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Figure 2. Tg and GlcN induce ER stress above that occurring during normal differentiation and inhibit adipocyte differentiation of 3T3-L1 cells. (A, B) Confluent 3T3-L1 cells were cultured in differentiation media in the presence of 0.1 nM Tg. On day 7, total RNA was isolated, and mRNA levels of UPR (A) and adipocytes-related (B) genes were measured by quantitative real-time RT-PCR and quantified as REU vs. control cells. Data are means ± SEM of three independent experiments with duplicates. p<0.001. (C-F) Confluent 3T3-L1 cells were cultured in differentiation media in the presence of 7.5 mM GlcN. At the indicated times after initiation of differentiation, mRNA levels of UPR genes (C) and adipocyte-related genes (D) were measured by quantitative real-time RTPCR and quantified as REU vs. control cells at the same time. Data are means ± SEM of three independent experiments with duplicates. p<0.05, p<0.01, p<0.001 indicates significance of the value of GlcN-treated cells vs. control cells at the same time. At day 7, cell lysates were collected and analyzed by western blot (E), or cells were fixed and stained with Oil red O (F). Representative images of Oil red O staining and quantification of Oil red O staining of three independent experiments are shown. p<0.001. Figure 3. GlcN induces ER stress and inhibit adipocyte differentiation of human primary adipocytes. Human primary adipocytes were isolated as outlined in Materials and Methods and incubated in control conditions or in the presence of 20 mM GlcN for 6 hrs. mRNA levels of UPR genes (A) and adipocyte-related genes (B) were measured by quantitative realtime RT-PCR and quantified as REU vs. control cells. Data are means ± SEM of three independent experiments with duplicates. p<0.05, p<0.01. Figure 4. 3T3-L1 cells differentiated in the presence of GlcN are shifted towards a proinflammatory phenotype. Confluent 3T3-L1 cells were cultured in differentiation media in the presence of 7.5 mM GlcN. At day 7, total RNA was isolated, and mRNA levels of cytokines/chemokines were measured by quantitative real-time RT-PCR and quantified as REU vs. control cells. Data are means ± SEM of three independent experiments with duplicates. p<0.05,
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Figure 5. A low PBA concentration inhibits only the “pathologic” part of ER stress, and revert the inhibitory effect of GlcN on differentiation. Confluent 3T3-L1 cells were cultured in differentiation media in the presence of 7.5 mM GlcN and/or 50 M PBA. At day 7, total RNA was isolated, and mRNA levels of UPR (A), adipocyte-related (B) and cytokine/chemokine (C) genes were measured by quantitative real-time RT-PCR and quantified as relative REU vs. control cells. Data are means ± SEM of three independent experiments with duplicates. p<0.05, p<0.01, p<0.001.
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Figure 6. High glucose induces ER stress, inhibits adipocyte differentiation, and increases Mip-1 and Rantes expression in 3T3-L1 cells. Confluent 3T3-L1 cells were cultured in differentiation media in control condition, in the presence of 40 mM glucose, and in the presence of 40 mM glucose and 50 M PBA. At day 7, total RNA was isolated, and mRNA levels of UPR (A), adipocyte-related (B) and cytokine/chemokine (C) genes were measured by quantitative real-time RT-PCR and quantified as relative REU vs. control cells. Data are means ± SEM of three independent experiments with duplicates. p<0.05, p<0.01.
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Figure 7. The p65Rel translocation inhibitor JSH-23 opposes the GlcN-induced shift towards a pro-inflammatory phenotype. Confluent 3T3-L1 cells were cultured in differentiation media in the presence of 7.5 mM GlcN and in the last day of 7.5 M JSH-23. At day 7, total RNA was isolated, and mRNA levels of cytokine/chemokine genes were measured by quantitative real-time RT-PCR and quantified as REU vs. control cells. Data are means ± SEM of three independent p<0.05, p<0.01, experiments with duplicates. p<0.001.
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Table 1: 3T3-L1 cell viability upon Tg treatments. % cell viability respect to control Control 100 1.0 nM Tg 88.6 4.6* 0.1 nM Tg 95.2 6.6 3T3-L1 cells were differentiated, as reported in Materials and Methods, in vehicle or in presence of 1.0 or 0.1 nM Th. At the end of the differentiation period, cell viability was assessed by the MTT assay as previously reported 25. The percentage of survival was calculated as the absorbance ratio of treated to untreated cells. The data presented are the mean ± SEM from four replicate wells, replicated three times. *p0.05 versus control.
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Table 2: Effect of GlcN and GlcN+JSH on 3T3-L1-released cytokines. Cytokine 3T3-L1 CM 3T3-L1 GlcN 3T3-L1 GlcN+JSH (pg/ml) CM CM (pg/ml) (pg/ml) IL-2 ND ND IL-8 5023.3425.6 6493567.6 5492.7486.8 IL-12 4.20.52 10.140.75, 18.31.12 IL-13 ND ND ND GM-CSF ND ND ND MCP-1 (CCL2) 16113115.4 13527121.12 21605103.7 MIP-1 81.17 18.11.45, 272.35 (CCL3) RANTES 205.311.12 426.315.72 358.210.56, (CCL5) TNF- 4.12.32 12.671.89, 28.93.11 Data represent the mean ± SD of at least four independent triplicate experiments. 3T3L1 cells were differentiated under standard conditions (control, 3T3-L1 CM), in the presence of GlcN (3T3-L1 GlcN CM), or in the presence of GlcN and, in the last day, of JSH (3T3-L1 GlcN+JSH CM). At day 7, conditioned media (CM) were collected for 24 h in Dulbecco's modified Eagle's medium without serum, with 0.5% BSA (manufacturer’s instructions), and tested by using a custom premixed assay from Biorad (Bio-Plex ProTM) as described in the Materials and Methods. p<0.05, p<0.01, p<0.001, for 3T3-L1 GlcN adipocytes vs. 3T3-L1 control adipocytes; p<0.05, p<0.01, for GlcN+JSH adipocytes vs. GlcN adipocytes; p<0.05, p<0.01 for GlcN+JSH adipocytes vs. control adipocytes.
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ACCEPTED MANUSCRIPT Highlights Glucosamine induces ER stress above the level occurring during differentiation and inhibits adipocyte differentiation
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Glucosamine increases the release of proinfammatory cytokines
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Low concentration of PBA (a chemical chaperone) inhibits only the “pathologic” part of ER stress
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PBA reverses the inhibitory effect of glucosamine on adipocyte differentiation
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PBA reverses the stimulatory effect of glucosamine on cytokine release
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