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Basic Science Aspects

Catecholamines Induce Endoplasmic Reticulum Stress Via Both Alpha and Beta Receptors

Abdullahi, Abdikarim; Wang, Vivian; Auger, Christopher; Patsouris, David; Amini-Nik, Saeid∗,†,§; Jeschke, Marc G.∗,†,‡,§

Author Information
doi: 10.1097/SHK.0000000000001394

Abstract

INTRODUCTION

Burn injury is a devastating injury with an annual global incidence of 11 million people with severe enough injury to seek medical attention, ranking fourth in all injuries and higher than the incidence of tuberculosis and HIV combined (1). Severe burn injury induces metabolic disturbances such as insulin insensitivity, hyperlipidemia, and hyperglycemia, which can last for years in most patients (2–5). These changes can partially be attributed to severe liver dysfunction, including steatosis and hepatomegaly (6, 7). Furthermore, burned patients are also affected by hypermetabolism that translates into vast skeletal muscle catabolism and fat tissue lipolysis, further exacerbating patient conditions and contributing to higher mortality (2, 8). Several secreted factors, such as pro-inflammatory cytokines, chemokines, cortisol, and catecholamines, are possible mediators for inducing this hypermetabolic response. Recently, the focus has turned to catecholamines as culprits of the prolonged and increased inflammatory and metabolic responses that characterize burn injury. In fact, catecholamines remain elevated years after the initial injury, suggesting that they might be prominent players (4). In response to stress, the sympathetic nervous system is activated to release catecholamines, which lead to physiological changes such as increased energy expenditure, heart rate, blood pressure, and blood glucose level. Furthermore, we and others have recently shown that this catecholamine surge that persists for months after the initial injury induces white adipose tissue browning in burn patients (9, 10).

On a cellular and molecular level, it is unknown how catecholamines would induce and maintain inflammatory and stress responses for such a prolonged period of time. We have recently shown that endoplasmic reticulum (ER) stress is a hallmark feature in burned patients, which is observed in peripheral blood leukocytes, fat, and muscle in parallel with insulin resistance and lasts for months post-burn (5). ER stress is also observed in diabetes and atherosclerosis and is proposed to play a causative role in the onset of these diseases (11–13). Because the catecholamine release is upstream of cytokines such as interleukin 4 and 6, its involvement in the ER stress response post burn injury was evaluated.

MATERIALS AND METHODS

Human fat tissue and explants

Patients undergoing elective surgery at Sunnybrook Hospital were consented pre-operatively for tissue collection. All experiments were carried out in accordance with the approved guidelines. All experiment protocols were approved by Sunnybrook Research Institute. All consent and procedures for tissue collection were carried out in accordance with the approved guidelines by the Research Ethics Board of Sunnybrook Health Sciences Centre (Study #194-2010). All patients were verbally informed, consented and provided with study packages before any collection of tissues. Subcutaneous white adipose tissue obtained from surgery was either snap frozen in liquid nitrogen for protein expression analysis, and or allocated for ex-vivo experiments. For ex-vivo studies, adipose tissue was dissected and minced in small pieces of approximately 5 mg. Six hundred milligrams of fat explants were seeded in 2 mL of DMEM (1 g/L glucose) supplemented with antibiotics and treated with various reagents for 24 h.

Isolation of human skin-derived fibroblasts

Human skin specimens were obtained, with both informed donor consent and Human Research Ethics Committee approval, and their skin fibroblasts isolated. Briefly, full-thickness skin was dissected to remove any subcutaneous adipose tissue, and cut in 4 mm pieces. Skin fibroblasts were obtained from outgrowth of dermal component of explants cultured in small dishes. After trypsinization, fibroblasts were further cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics.

Cell culture

HepG2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, 4.5 g/L glucose) supplemented with 10% FBS and 1% antibiotics. The 3T3L1 mouse preadipocyte cell line was obtained from Klip (Hospital for Sick Children, University of Toronto, Toronto, Canada), whereas C3H/10T1/2 adipocytes were purchased from ATCC (cat#CCL-226). 3T3L1 and C3H/10T1/2 adipocytes were maintained in DMEM (4.5 g/L glucose) supplemented with 10% FBS serum and differentiated for 7 to 10 days following incubation with a differentiation protocol previously described (14). HepG2 cells were seeded in wells 24–48 h prior to treatments, and treated when cells reached 70% confluence. Medium was removed and replaced with treatments (100 nM to 100 μM) consisting of a combination of epinephrine and norepinephrine ((−) isomer, catalogue #A7257 Sigma Aldrich, St. Louis, Mo) alone or in combination with prazosin, propranolol, and yohimbine (100 μM) (Sigma Aldrich, St. Louis, Mo) diluted in medium. Differentiation of adipocytes was initiated 2 days post-confluency in DMEM containing 10% FBS in the presence of a differentiation cocktail (insulin, dexamethasone, IBMX) for 2 days. In the following days, the cells were maintained in DMEM, 10% FBS until full differentiation was achieved (day 10).

RT PCR

RNA was isolated from human fibroblasts stored in liquid nitrogen. 2 ug of RNA were used to perform a reverse transcription following the manufacturer's recommendation (ABI, #4387406). Semiquantitative PCR was then performed using DreamTaq DNA Polymerase (Thermo Scientific). Primer sequences are available upon request.

Western blotting

Cells were lysed in RIPA lysis buffer, consisting of 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.8, 1% [w/v] Triton X-100, 1 mmol/L EDTA, 0.5 mmol/L phenyl-methanesulfonyl fluoride, 1× Complete protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, Ind) and a phosphatase inhibitor cocktail (Sigma Aldrich, St. Louis, Mo). Samples were centrifuged, and protein concentrations were measured by BCA assay (Thermo Scientific, Waltham, Mass). A total of 25 μg denatured protein from tissues was separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a nitrocellulose membrane, the membrane was blocked with 5% nonfat milk solution and washed three times with buffer (Tris-buffered saline, 0.05% Tween). Western blotting was performed using antibodies recognizing proteins BiP/Grp78 (Cell Signaling, #3183), cleaved-ATF6 (Thermofisher, MA5-16172), CHOP (Cell Signaling, #2895) followed by species-appropriate secondary antibodies conjugated to HRP (BioRad). Proteins were visualized by enhanced chemiluminescence. Band intensities were quantified with the Image J software (National Institutes of Health, Bethesda, Md).

Immunofluorescent staining and microscopy

Cells on eight chamber slides were fixed in paraformaldehyde, blocked with 1% bovine serum albumin in PBS and were stained with antibodies against cleaved-ATF6 (Thermofisher, MA5-16172), BiP (Cell Signaling, # #3183), and CHOP (Cell Signaling, #2895) at a dilution ratio of 1:200 overnight. Slides were washed and incubated for 1 h at room temperature with the appropriate secondary antibodies (Alexa-448 anti-mouse #Z25002, Alexa-647 anti-rabbit #Z25308, Thermofisher Scientific) each at a dilution factor of 1:500. The ER tracker used was from Invitrogen and used according to the manufacturer's protocol. The TUNEL assays (Promega Dead End Fluoromteric TUNEL system, #G3250) were performed similarly in eight chambers slides with HepG2 treated 24 h with the indicated reagents. Slides were washed and counterstained with DAPI before mounting. Slides were stored at 4°C. Slides were imaged using a Zeiss spinning disk confocal microscope. Images for each treatment or condition were taken ×3 at different locations within each sample and four different samples were used per treatment group in the analysis. In the quantifications, a blinded lab technician counted the positive cells in each image for each group of analysis.

Cyclic GMP assay

HepG2 cells were seeded in 6-well plates and allowed 24 h to attach. Media was then changed to treatment media and incubated for 24 h. Cyclic GMP levels were determined using a Cyclic GMP XP Assay Kit (Cell Signaling).

Statistical analysis

Statistical significance was assessed by unpaired, two-tailed Student t test for single comparison. P values of less than 0.05 were considered significant. Statistical differences between 3 or more groups were evaluated using a two-way ANOVA followed by Bonferroni post-hoc tests. Significant results were established for P < 0.05 (∗).

RESULTS

To address these mechanistic questions, we first examined the effects of epinephrine and norepinephrine on ER stress induction in HepG2 cells. Cells treated with either epinephrine or norepinephrine had significant up-regulation of the common ER stress marker BiP (P < 0.05) compared with control cells (Fig. 1, A–D). In order to further confirm the activation of the unfolded protein response (UPR) by catecholamines, we also decided to test the property of norepinephrine to induce the ER stress marker ATF6 and direct it to the nucleus of the cells. As shown in Figure 1E, norepinephrine increased the number of ATF6-positive cells; additionally, ATF6 was abundantly detected in the nuclei of the cells and absent from the ER, indicating UPR activation. Furthermore, treating cells with thapsigargin, a well-established ER stress inducer, showed a similar pattern for ATF6 activation in comparison with norepinephrine-treated cells.

F1
Fig. 1:
Catecholamines induce ER stress in HepG2 cells.

To better define the complex adrenoreceptors involved in mediating the ER stress effects of catecholamines, we cotreated these cells with the alpha-1, alpha-2, and beta-blockers, prazosin, yohimbine, and propranolol respectively. Adrenergic receptors have five known subtypes: α1, α2, β1, β2, and β3. Each has different affinities for catecholamines and mediates different activities. When HepG2s were treated with norepinephrine there were approximately four times more (P < 0.05) BiP-positive cells in response to norepinephrine treatment compared with untreated cells (Fig. 2A). When the alpha-1-specific blocker prazosin was added in combination with norepinephrine, BiP-positive cells were significantly reduced, and reached similar levels when compared with untreated conditions (P < 0.05) (Fig. 2A). The alpha-2 blocker yohimbine had a similar lowering effect as prazosin since the number of BiP-positive cells was similar to control conditions despite the cotreatment with norepinephrine (P < 0.05). However, the beta-blocker propranolol did not effectively attenuate the number of BiP-positive cells (Fig. 2A). These findings were also evident at the protein level when BiP protein expression was assessed via Western blot analysis (Fig. 2B). Additionally, we ruled that the dose used (100 μM) for the three adrenoreceptor blockers did not elicit toxicity and affect cell viability, as assessed by trypan blue staining within the treated HepG2 cells (Supplementary Figure 1A, Supplemental Digital Content, https://links.lww.com/SHK/A899). Together, these findings suggest that catecholamines induce ER stress in HepG2 cells primarily via α-adrenergic receptors, which have a higher potency toward norepinephrine.

F2
Fig. 2:
ER stress is primarily induced via alpha-adrenergic receptors in HepG2 cells.

Norepinephrine activates ATF6 and IRE, but not PERK in the ER stress response

Given the divergent effects of the ER stress pathway, we next examined the activation profile of the three ER stress trans-membrane proteins ATF6, IRE-1α and PERK in hepatocytes exposed to norepinephrine (15, 16). ATF6 showed the greatest up-regulation (approximately 5-fold; P < 0.05) in HepG2s treated with norepinephrine (Fig. 3, A and B). Adrenergic blockers added (100 μM each) in conjunction with the norepinephrine decreased ATF6 levels (Fig. 3, A and B). Specifically, the alpha-1 and alpha-2 blockers prazosin and yohimbine were most effective in preventing ATF6 induction by norepinephrine (P < 0.01) (Fig. 3, A and B). Beta-blocker propranolol-treated cells also had a lower ATF6 level compared with norepinephrine-treated cells, but did not reach significance (Fig. 3, A and B). Norepinephrine also increased the up-regulation of the downstream target of IRE-1α, XBP-1s, compared to untreated cells (Fig. 3C) (P < 0.05). Alpha-1 and alpha-2 blockers were also effective in attenuating this increase in XBP-1s to the baseline level (P < 0.05) (Fig. 3C). However, propranolol-treated cells had no effect in reducing XBP-1s in norepinephrine-treated cells. Moreover, norepinephrine also induced a 3-fold up-regulation of IRE-1α compared with untreated cells (P < 0.05) (Supplementary Figure 2A-B, Supplemental Digital Content, https://links.lww.com/SHK/A899). The Alpha-1 and alpha-2 blockers prazosin and yohimbine were also effective in attenuating this increase in IRE-1α expression (P < 0.05), but not to the baseline level (Supplementary Figure 2A-B, Supplemental Digital Content, https://links.lww.com/SHK/A899). The third trans-membrane receptor PERK involved in halting protein translation, however, showed no response to norepinephrine in HepG2s (Supplementary Figure 3A-B, Supplemental Digital Content, https://links.lww.com/SHK/A899). Treatment with adrenergic blockers in conjunction with norepinephrine also had no effect on PERK expression (Supplementary Figure 3A-B, Supplemental Digital Content, https://links.lww.com/SHK/A899). Together, these findings demonstrate that catecholamines not only induce ER stress in HepG2s primarily via alpha-adregenic receptors, but also evoke specific branches of the ER stress pathway.

F3
Fig. 3:
Norepinephrine evokes specific branches of the ER stress pathway.

Norepinephrine does not induce CHOP and apoptosis

Our findings that norepinephrine was only able to evoke specific branches of the ER stress pathway in HepG2s prompted us to investigate the significance of this selective ER stress activation. Sustained ER stress has been identified as a cause of apoptosis in a CHOP-dependent manner (17). In fact, burn injury with its associated chronic catecholamine storm has been shown to induce ER stress, CHOP, and apoptosis in hepatocytes (18). As shown in Figure 4A, while norepinephrine was able to induce BiP activation earlier, CHOP was not detected in norepinephrine-treated HepG2 cells. Thapsigargin, on the other hand, was effective in inducing CHOP expression (Fig. 4A). Quantification of apoptosis with TUNEL assay was consistent with the expression of CHOP (Fig. 4B). Cyclic GMP has also been associated with ER and mitochondrial dysfunction and has been shown to be a downstream target of norepinephrine (19, 20). In cells treated with norepinephrine, there was a significant increase in cGMP (Fig. 4C) (P < 0.01). The alpha-1 blocker prazosin significantly decreased cGMP levels compared to norepinephrine alone (P < 0.01). However, the alpha-2 and beta blockers had no effect in decreasing cGMP levels in norepinephrine-exposed HepG2 cells. These findings suggest that catecholamine-induced ER stress in HepG2s does induce apoptosis, but the activation of cyclic GMP implies an interference with the metabolic profile of these cells and not survival.

F4
Fig. 4:
Norepinephrine does not induce CHOP and apoptosis.

The beta-blocker propranolol prevents ER stress induced by norepinephrine in human fat explants and adipocytes

In addition to the liver, the adipose tissue appears as a central metabolically active organ that is significantly affected by catecholamines post-burn injury (5, 12). However, little is known about the mechanism by which catecholamines alter adipose tissue metabolism following injury. To investigate this, we used two different but complementary models of adipose tissue (in vitro and ex vivo), to examine the effects of catecholamines on adipose ER homeostasis. Similar to our aforementioned observations in HepG2s, norepinephrine induced ATF6 expression in human fat explants in a dose-dependent manner (Fig. 5A). This effect was similar to the effect observed in response to the ER stress inducer, tunicamycin (Fig. 5A). Furthermore, all three adrenoceptor blockers tested, prazosin, yohimbine, and propranolol were able to prevent the up-regulation of ATF6 in response to norepinephrine (Fig. 5A). Because fat explants are composed of several cells, including mature adipocytes and stromal vascular cells, we also evaluated the response to catecholamines in mature mouse 3T3L1 adipocytes (Fig. 5B). Consistent with our ex vivo findings, norepinephrine potently induced ATF6 expression and was reversed by the adrenoceptor blockers in 3T3L1 mouse adipocytes (P < 0.001) (Fig. 5C). To further support the notion that the UPR was induced by norepinephrine in 3T3L1 adipocytes, the other key ER stress marker ATF-4 was up-regulated by norepinephrine treatment and blocked by the adrenoceptor blockers (Fig. 5D). Taken together, our results indicate that catecholamines primarily induce ER stress in cells that play crucial roles in metabolism, in particular those cells involved in glucose and lipid metabolism.

F5
Fig. 5:
The beta-blocker propranolol prevents ER stress induced by norepinephrine in human fat explants and adipocytes.

Mesenchymal primary fibroblast cells are not responsive to norepinephrine-induced ER stress

Our results thus far have indicated that norepinephrine induces ER stress in cells that play a crucial role in metabolism, in particular glucose and lipid metabolism. We next wanted to investigate if catecholamines also induced ER stress in human primary skin fibroblasts cells that are not primarily involved in metabolic regulation. Unlike in hepatocytes and adipocytes, norepinephrine did not induce ER stress in fibroblasts treated under the same conditions. Fibroblasts exposed to 24 h of norepinephrine did not exert an increase in the number of fibroblasts expressing BiP, ATF6, and or PERK (Fig. 6, A–C). To rule out the possibility that these fibroblasts were not responsive to catecholamines because they did not express the adrenoceptors required, we evaluated the expression of the different adrenoceptor forms in these fibroblasts. Gene expression profiling demonstrated that these fibroblasts expressed significant amounts of the respective receptors, in particular the adrenoceptors β1 and β2, but not the adrenoceptors α1B nor the adipose specific β3 adrenoceptor (Fig. 6D). Together, these findings suggest that the effects of catecholamines in inducing ER stress are cell type specific.

F6
Fig. 6:
Norepinephrine treatment does not induce ER stress in mesenchymal primary human fibroblast cells.

DISCUSSION

In this study, we have shown that catecholamines induce ER stress in hepatocytes and adipocytes, and that these effects can be reversed by adrenergic receptor antagonists. We focused on norepinephrine when studying the effects of adrenergic blockade, as it is the most important catecholamine secreted in the context of burn physiology (9, 21). Indeed, in burn patients, norepinephrine levels are elevated for months after the injury when compared with epinephrine (4). Our experiments further showed that norepinephrine was a more potent inducer of ER stress even at low concentrations. Adrenergic stimulation has also been shown to induce ER stress in other cells such as cardiomyocytes (22–24). However, we did not observe any induction of the ER stress branches of PERK, ATF-6, and BiP in undifferentiated mesenchymal fibroblast cells, suggesting that not all cell types are equally susceptible to catecholamine stimulation. These results support the clinical observation that specific organs, mainly the liver and the adipose tissue, are more involved in the pathophysiological consequences of burn, most probably due to ER stress (6, 25).

Catecholamines play a key role in orchestrating the response to stress and injury. Indeed, production and secretion of these moieties is critical in the response to traumatic injuries like burns, in which there is a catecholamine surge that lasts for years after the initial insult (4). Sustained and elevated catecholamines have also been implicated in compromising the ability to combat infection. For instance, neutrophils incubated with norepinephrine display an immunosuppressive phenotype, and inoculation of mice with these impaired neutrophils increases susceptibility to sepsis and death (26, 27). Furthermore, catecholamines have also recently been implicated in mediating the browning of white adipose tissue in burn patients, thereby facilitating persistent hypermetabolism in these patients (9, 10). However, to date, the effects of catecholamines at the cellular level, particularly, in the context of burns have not been adequately investigated. The ER is an important cellular organelle responsible for posttranslational processing of newly synthesized secretory proteins and in the maintenance of cellular homeostasis during periods of stress (12, 14). Our finding that catecholamines induce ER stress in 3T3-L1 adipocytes might provide an explanation for how catecholamines regulate the adipose browning process that has recently been reported in burn patients.

Previous studies have shown that beta-blockers are effective at reversing the effects of catecholamines on inducing ER stress (22, 28, 29), although most studies were in cardiovascular pathology. Beta-blockers have already been used clinically to reverse the hypermetabolic effects of the catecholamine surge in burns (25, 30). However, these clinical trials did not illustrate the mechanism utilized by these agents to improve metabolic outcome in patients. Our data not only resonates with these clinical trials, but also provides the mechanism by which beta blockers attenuate hypermetabolism in burns. Alpha blockers, on the other hand, have not been well studied. Here, we examined the effects of adrenergic blockers, including alpha blockers and observed that they were more effective than beta blockers at attenuating norepinephrine-induced ER stress in hepatocytes. Future studies, beginning with animal studies, are warranted to examine the effects of alpha-blockers on attenuating ER stress in vivo. This is essential as our data revealed a tissue-specific response to catecholamines. Post-traumatic stress has been diagnosed in 25% to 30% of children acutely after burn and 10% to 20% many years post-burn (31). Prazosin, an alpha-1 receptor blocker, has been shown to successfully decrease the symptoms of post-traumatic stress disorder (PTSD) and anxiety, but our results suggest that alpha blockers may have other added metabolic benefits (32).

Although our study sheds light on the cellular effects of catecholamines and the specific adrenergic receptors that mediate such responses, there are a few limitations to our study. For instance, further studies will be required to confirm our in vitro findings, by investigating whether a similar catecholamine challenge in mice induces ER stress activation and whether inhibiting the adrenergic receptors attenuates ER stress in these mice. In addition, further studies in rodent and clinical trials are required to fully ascertain the therapeutic potential of the alpha and beta-blockers used in our studies. Finally, the effects of catecholamines on inducing ER stress in cells outside of the ones utilized in this study are interesting questions for future research.

CONCLUSION

In conclusion, we have identified the direct receptor-mediated mechanisms by which catecholamines regulate ER stress in hepatocytes and adipocytes. The multiple pathways used by catecholamines to alter metabolism in these cells, although probably advantageous during the initial stages of burn injury, can cause hypermetabolism in chronic scenarios as seen in burn patients. Our findings also suggest that attenuation of ER stress likely explains the metabolic benefits seen in many of the clinical propranolol treatments conducted in burns. Beyond burns, we suggest that the catecholamine-mediated regulation of ER stress has relevance to infections, diabetes, obesity, and other chronic diseases associated with ER stress.

Acknowledgments

Cassandra Belo, Xiaojing Dai, and Peter Qi are acknowledged for their technical support.

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Keywords:

Burns; catecholamines; ER stress

Supplemental Digital Content

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