CATECHOLAMINES INDUCE ENDOPLASMIC RETICULUM STRESS VIA BOTH ALPHA AND BETA RECEPTORS

Abstract

Severely burned patients suffer from a hypermetabolic syndrome that can last for years after the injury has resolved. The underlying cause of these metabolic alterations most likely involve the persistent elevated catecholamine levels that follow the surge induced by thermal injury. At the cellular level, endoplasmic reticulum (ER) stress in metabolic tissues is a hallmark observed in patients following burn injury and is associated with several detrimental effects. Therefore, ER stress could be the underlying cellular mechanism of persistent hypermetabolism in burned patients. Here, we show that catecholamines induce ER stress and that adreno-receptor blockers reduce stress responses in the HepG2 hepatocyte cell line. Our results also indicate that norepinephrine (NE) significantly induces ER stress in HepG2 cells and 3T3L1 mouse adipocytes. Furthermore, we demonstrate that the alpha-1 blocker, prazosin, and beta blocker, propranolol, block ER stress induced by NE. We also show that the effects of catecholamines in inducing ER stress are cell type-specific, as NE treatment failed to evoke ER stress in human fibroblasts. Thus, these findings reveal the mechanisms used by catecholamines to alter metabolism and suggest inhibition of the receptors utilized by these agents should be further explored as a potential target for the treatment of ER stress-mediated disease.

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) (2). Severe burn injury induces metabolic disturbances such as insulin insensitivity, hyperlipidemia, and hyperglycemia, which can last for years in most patients(3–5) (6). These changes can partially be attributed to severe liver dysfunction, including steatosis and hepatomegaly (7)(8). 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 (3,9). 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 (5). 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 (10) (11).

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 (6). ER stress is also observed in diabetes and atherosclerosis and is proposed to play a causative role in the onset of these diseases (12) (13) (14). 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 to 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 mg of fat explants were seeded in 2 mL of DMEM (1 g/L glucose) supplemented with antibiotics and treated with various reagents for 24 hours.

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, Canada), whereas C3H/10T½ adipocytes were purchased from ATCC (cat#CCL-226). 3T3L1 and C3H/10T½ 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 (15). HepG2 cells were seeded in wells 24–48 hours 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, USA) alone or in combination with prazosin, propranolol and yohimbine (100 μM) (Sigma Aldrich, St. Louis, MO, USA) diluted in medium. Differentiation of adipocytes were initiated two days post-confluency in DMEM containing 10% FBS in presence of a differentiation cocktail (insulin, dexamethasone, IBMX) for two 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). Semi-quantitative 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, IN, USA) and a phosphatase inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Samples were centrifuged, and protein concentrations were measured by BCA assay (Thermo Scientific, Waltham, MA, USA). A total of 25μg 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, USA).

Immunofluorescent staining & 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 hr 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 manufacturers protocol. The TUNEL assays (Promega Dead End Fluoromteric TUNEL system, #G3250) were performed similarly in eight chambers slides with HepG2 treated 24 hours 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 3X at different locations within each sample and 4 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 hours to attach. Media was then changed to treatment media and incubated for 24 hours. 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’s 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 to control cells (Figure 1A–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.

Figure 1: Catecholamines induce ER stress in HepG2 cells.

A) Representative Western blot of BiP in HepG2 cells exposed to different levels of norepinephrine for 24 hours. B) Representative Western blot of BiP in HepG2 cells exposed to different levels of epinephrine for 24 hours. C) Quantification of BiP protein levels in cells exposed to norepinephrine. D) Quantification of BiP protein levels in cells exposed to epinephrine. Each bar shows the relative ratio of BiP level normalized to α- tubulin. The ER stress inducer Thapsigargin (Tg, 1.5 μM) is shown as positive control. E) Immunostaining of cleaved-ATF6 (red), ER (green), and nuclei (blue) upon stimulation with norepinephrine (100 μM) and Thapsigargin (1.5 μM) is shown alongside the quantification of ATF6 + cells. Experiments were repeated 3 separate times with a total sample size (n=8–9) per group included in the analysis. *p<0.05, ** p<0.01, *** p<0.001 vs CTL.

To better define the complex adrenoreceptors involved in mediating the ER stress effects of catecholamines, we co-treated these cells with the alpha-1, alpha-2, and beta-blockers, prazosin, yohimbine, and propranolol respectively. Adrenergic receptors have 5 known subtypes: α1, α2, β1, β2, and β3. Each has different affinities for catecholamines and mediate different activities. When HepG2s were treated with norepinephrine there were approximately 4 times more (p<0.05) BiP positive cells in response to norepinephrine treatment compared to untreated cells (Figure 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 to untreated conditions (P <0.05) (Figure 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 co-treatment with norepinephrine (P <0.05). However, the beta-blocker propranolol did not effectively attenuate the number of BiP-positive cells (Figure 2A). These findings were also evident at the protein level when BiP protein expression was assessed via Western blot analysis (Figure 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). Together, these findings suggest that catecholamines induce ER stress in HepG2 cells primarily via α-adrenergic receptors, which have a higher potency towards norepinephrine.

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

A) Representative immunofluorescence staining and quantification of BiP in HepG2 cells treated with 100 μM norepinephrine and the alpha-1, alpha-2, and beta-blocker (100 μM) (prazosin, yohimbine and propranolol, respectively) for 24 hours. B) Western blot and quantification showing the expression of BiP in response to the treatments indicated. NE, Norepinephrine. Arrows indicate positive cells. Experiments were repeated 3 separate times with a total sample size (n=8–9) per group included in the analysis. *p<0.05 vs NE or CTL respectively.

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 (16–17). ATF6 showed the greatest up-regulation (approximately 5 fold; P<0.05) in HepG2s treated with norepinephrine (Figure 3A–B). Adrenergic blockers added (100 μM each) in conjunction with the norepinephrine decreased ATF6 levels (Figure 3A–B). Specifically, the alpha-1 and alpha-2 blockers prazosin and yohimbine were most effective in preventing ATF6 induction by norepinephrine (P<0.01) (Figure 3A–B). Beta-blocker propranolol- treated cells also had a lower ATF6 level compared to norepinephrine-treated cells, but did not reach significance (Figure 3A–B). Norepinephrine also increased the up-regulation of the downstream target of IRE-1α, XBP-1s, compared to untreated cells (Figure 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) (Figure 3C). However, propranolol-treated cells had no effect in reducing XBP-1s in norepinephrine treated cells. Moreover, norepinephrine also induced a three-fold up-regulation of IRE-1α compared to untreated cells (P<0.05) (Supplementary Figure 2A–B). 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). The third trans-membrane receptor PERK involved in halting protein translation, however, showed no response to norepinephrine in HepG2s (Supplementary Figure 3A–B). Treatment with adrenergic blockers in conjunction with norepinephrine also had no effect on PERK expression (Supplementary Figure 3A–B). 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.

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

A) Representative immunostaining of cleaved ATF6 in HepG2 cells treated with 100 μM norepinephrine and the alpha-1, alpha-2, and beta-blocker (100 μM) (prazosin, yohimbine and propranolol, respectively) for 24 hours. B) Quantification of cleaved-ATF6 positive (+) cells obtained from the immunostaining performed in (A). C) qPCR expression profile of XBP-1s in HepG2s exposed to norepinephrine (100 μM) alone or in presence of the alpha- and beta-blockers (100 μM) for 24 hours. NE, Norepinephrine. Arrows indicate positive cells. Experiments were repeated 3 separate times with a total sample size (n=8–9) per group included in the analysis. *p<0.05 vs NE or CTL respectively.

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 (18). In fact, burn injury with its associated chronic catecholamine storm has been shown to induce ER stress, CHOP and apoptosis in hepatocytes (19). 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 (Figure 4A). Quantification of apoptosis with TUNEL assay was consistent with the expression of CHOP (Figure 4B). Cyclic GMP has also been associated with ER and mitochondrial dysfunction and has been shown to be a downstream target of norepinephrine (20), (21). In cells treated with norepinephrine, there was a significant increase in cGMP (Figure 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.

Figure 4: Norepinephrine does not induce CHOP and apoptosis.

A) Immunostaining of CHOP (Green) in HepG2 cells treated for 24 hours with norepinephrine (NE, 100 μM) or Thapsigargin (1.5 μM). Arrows indicate CHOP-positive cells. B) TUNEL assay was performed on HepG2 cells treated in the same conditions described in A). Arrows indicate TUNEL-positive cells. C) Averaged relative levels of cGMP in HepG2 treated with norepinephrine (100 μM) alone or in presence of the alpha- and beta-blockers (100 μM) for 24 hours. Experiments were repeated 3 separate times with a total sample size (n=9) per group included in the analysis.** p<0.01, *** p<0.001 vs CTL. ND, Not detected.

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 (6,13). 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 (Figure 5A). This effect was similar to the effect observed in response to the ER stress inducer, tunicamycin (Figure 5A). Furthermore, all three adrenoceptor blockers tested, prazosin, yohimbine and propranolol, were able to prevent the up-regulation of ATF6 in response to norepinephrine (Figure 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 (Figure 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) (Figure 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 (Figure 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.

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

A) Western blots of cleaved-ATF6 in human fat explants untreated (Ctrl) or treated for 24 hours with the indicated concentrations of norepinephrine alone or in presence of the alpha- and beta-blockers (100 μM). The ER stress inducer tunicamycin (Tun, 5 μg/ml) is shown as a positive control. Quantification of band intensity (A.U., arbitrary unit) is shown on the panel on the right. B) Immunostaining of cleaved-ATF6 in mature 3T3L1 adipocytes untreated (Ctrl) or treated for 24 hours with norepinephrine (100 μM) or tunicamycin (Tun, 5 μg/ml). Quantification of the number of +cells for cleaved-ATF6 is shown on alongside the staining. C) Immunostaining of cleaved-ATF6 in mature 3T3L1 adipocytes untreated (Ctrl) or treated for 24 hours with norepinephrine (100 μM) alone or in presence of the alpha- and beta-blockers for 24 hours. D) ATF4 expression was quantified by Western blot in 3T3L1 mature adipocytes lysates untreated (Ctrl) or exposed to norepinephrine (100 μM) alone or in presence of the alpha-1,2, and beta-blockers (100 μM) for 24 hours. Experiments were repeated 3 separate times with a total sample size (n=6–10) per group included in the analysis. Arrows indicate positive cells. *p<0.05, ** p<0.01, *** p<0.001 vs CTL, # p<0.01 vs blockers.

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 hours of norepinephrine did not exert an increase in the number of fibroblasts expressing BiP, ATF6, and or PERK (Figure 6A–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 (Figure 6D). Together, these findings suggest that the effects of catecholamines in inducing ER stress are cell type specific.

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

A-C) Isolated fibroblasts from human skin were treated for 24 hours with 100 μM norepinephrine and immunostaining of (A) BiP, (B) ATF6, and (C) PERK were performed and quantified respectively. D) Adrenoceptors α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, β3 expression in human fibroblasts were assessed as described in (A-C). Experiments were repeated 3 separate times with a total sample size (n=5–9) per group included in the analysis.

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 (10), (22). Indeed, in burn patients, norepinephrine levels are elevated for months after the injury when compared to epinephrine (5). 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 (23), (24), (25). 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 (7), (26).

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 (5). 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 (27–28). 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 (10–11). 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 (13,15). 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 (23), (29), (30), 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 (26), (31). 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 25–30% of children acutely after burn and 10–20% many years post-burn (32). 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 (33).

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.