key: cord-0890262-etq3z069 authors: Mityukova, Т. A.; Chudilovskaya, E. N.; Basalai, A. A. title: Reactivity of the Thyroid System to Short-Term Stress in Wistar Rats with Visceral Obesity and Restricted Social Activity date: 2022-05-16 journal: J Evol Biochem Physiol DOI: 10.1134/s0022093022020156 sha: 92adbdae8c71585342fb72bf36d752eb324c6973 doc_id: 890262 cord_uid: etq3z069 The obesity problem requires a study of its pathophysiological consequences affecting hormonal regulation and organism’s reactivity to extreme exposures. The study was aimed first to examine the effect of a high-calorie diet and social isolation, as well as their combination for 4 months, on the development of obesity, its metabolic and behavioral sequelae, features of the thyroid status, while at the second stage, to assess the reaction of hormonal indices of the thyroid status to short-term stress in rats. The experiments were carried out on male Wistar rats and at the first stage focused on the effects of a high-calorie diet and social isolation, as well as their combinations for 4 months. At the end of the experiment, behavioral reactions, metabolic syndrome indices, thyroid status, and cortisol levels were evaluated. At the second stage, the animals were exposed to short-term acute stress, and the shifts in the hormonal indices were recorded one hour later versus the initial background. A high-calorie diet led to the development of metabolic syndrome, signs of depression, increased thyroid-stimulating hormone (TSH), thyroxine and triiodothyronine serum levels, as well as iodothyronine deiodinase type 1 (D1) activity, in the rat liver. At the same time, there was a decrease in thyroperoxidase activity and an increase in thyroid levels of triglycerides and malondialdehyde. The physiological response to stress in the control rat group included an increase in cortisol and TSH serum levels, however, against the background of a high-calorie diet, no cortisol release into the bloodstream was recorded. Social isolation did not alter normal reactivity of the adrenal cortex, but reduced TSH release in response to acute stress, since the initial level of this hormone was slightly elevated against the background of chronic social isolation stress. Thus, excessive nutrition and the deficit of social activities in male Wistar rats led to significant changes in the organism’s reactivity to acute stress. Sociological studies suggest that the number of overweight and obese people is increasing world wide, while obesity increases the risk of metabolic syndrome, cardiovascular disease, thyroid dys function, cognitive and psychoemotional disor ders, including depression [1] [2] [3] . The most common causes of obesity are an excessive diet and unhealthy lifestyle, which includes insuffi cient physical and social activities [1] [2] [3] . The state of thyroid status has its own features in obe sity and attracts the attention of researchers, because thyroid hormones are involved in the reg ulation of basal metabolism, thermogenesis and lipid metabolism [2] . Most clinical and epidemio logical studies converge in associating thyroid dysfunction with overweight and obesity. In euthyroid patients, there is a positive correlation between body weight and thyroid stimulating hormone (TSH) indices, although data on peripheral hormone levels in obesity are ambigu ous. It should be admitted that thyroid status plays an important role not only in the pathogenesis of obesity, but also in the system of regulatory pro cesses that provide organism's adaptation to phys ical loads and stress [2, 4, 5] . Nevertheless, the issue of the possible modulating effect of obesity and restricted physical and social activities, as well as their combination, on the reactivity of reg ulatory systems, including the thyroid status, remains poorly understood and requires appropri ate experimental studies. Experimental rat models of obesity and meta bolic syndrome are thoroughly described in the literature, their choice and conduct do not cause difficulties [6] . However, the concept of unhealthy lifestyle is more complex and ambigu ous. At present, such factors as a decrease in social activity due to the development of distance learn ing and remote work practices, as well as the expansion of the virtual entertainment sphere and forced lifestyle changes, are attracting particular attention against the background of the COVID 19 pandemic. This model can be implemented in an experiment by keeping animals in individual cages. According to Mumtaz et al. [7] , the social isolation factor can act as a chronic stressor lead ing to changes in social behavior, the functioning of the neurochemical and neuroendocrine sys tems, and reactivity to acute stress. Based on this, we adopted an individual maintenance as a vari ant of social activity restriction in rats (social iso lation). The study was aimed to explore first the effect of a high calorie diet and social isolation on the development of obesity, its metabolic and behav ioral consequences, as well as the thyroid status features, and then to assess the reaction of thyroid status hormonal indices on short term stress in rats. All experiments with laboratory animals was carried out in compliance with the legislation, in accordance with the provisions of the European Convention for the protection of vertebrate ani mals used for experiments or other scientific pur poses (ETS N 123), as well as the principles and regulations approved by the Bioethics Committee of the Institute of Physiology of the National Academy of Sciences of Belarus (IP NASB). The experiments were performed on sexually mature male Wistar rats. Animals were introduced into the experiment at the age of 2 months and a body mass of 180-200 g. The following types of experi mental exposures were examined: a high calorie diet (HCD) and a social isolation (SI), as well as their combinations. The rats were divided into 4 groups: (1) Control (n = 20)-standard vivarium diet and optimal housing conditions (by 6-7 indi viduals per common cage); (2) Control SI (n = 20)-standard diet and individual cage housing; (3) HCD (n = 36)-a diet was added with animal fat as a lard (45% of food caloric value) and carbo hydrates (drinking water was replaced with 10% fructose solution at an ad libitum access to drink ers), rats were housed under standard conditions (by 6-7 individuals per common cage); (4) HCD SI (n = 36)-a high calorie diet and indi vidual cage housing. HCD included a lard added in accordance with animal's body weight, while observing 45% of an additional contribution to a standard vivarium diet as the body weight increased, and according to a caloric value of the basal diet. Lard consumption per animal was recorded under both group and individual hous ing. Techniplast cages with a floor area of 1500 cm 2 (48 × 38 × 21 cm) were used for a group housing, while for individual housing we used Zoonlab Gmbh cages with a floor area of 360 cm 2 (26 × 20 × 14 cm) for used for an individual hous ing. Both variants complied with the ETS No. 123 requirements, with the floor area per rat being always no less than 200-250 cm 2 . The duration of the experiment was 4 months. In the last week, the Porsolt forced swim test for rodent depressive behavior was performed [8] . Each rat was placed for 6 min in a vessel filled with water up to the mark at a height of 30 cm; water temperature was 24-25°C. The duration of the first act of active swimming, the number and duration of freezes (no swimming movements, full immobility) were recorded. The refusal from active swimming characterized the state of "despair", being a sign of depression [8] . Rat body mass was estimated on Scout Pro scales (China), and systolic blood pressure (SBP) was measured on a PanLab system (Spain). At the final stage of the experiment, half of the animals in each of the four groups were exposed to acute short term stress, forced swimming in cold water (t = 13°C) for 5 min. The animals were withdrawn from the experiment 1 h after stress, under thio pental anesthesia. All other animals were also decapitated using sodium thiopental. The visceral adipose tissue, liver, thyroid, and blood were sam pled for serum separation and further analysis of biochemical and hormonal indices. Blood serum biochemical indices, such as total cholesterol, triglycerides, glucose, and total pro tein, were determined by enzymatic methods using Diasens commercial kits (Belarus) on a BS 200 Automated Biochemistry Analyzer (China) with BS 330 software. Blood serum hormones were determined by enzyme immunoassay: triiodothy ronine (T3), thyroxine (T4), and free T4 (fT4) with Chema kits (Russia), free T3 (fT3) and corti sol with Diagnostic Systems kits (Russia), thy roid stimulating hormone (TSH) with FineTest kits (China). Thyroperoxidase (TPO) activity in thyroid tis sue was assayed as described elsewhere [9] . Thy roid tissue was homogenized using an IKA T10 basic Ultra Turrax homogenizer (Germany) and 0.05 M sodium phosphate buffer (pH 7.4). The homogenate (diluted 1 : 80) was centrifuged at 4°C and 5000 rpm (10700 g) on an Allegra 64R Centrifuge Beckman Coulter (USA). Superna tants were separated, and a protein content was determined by the biuret method on a BS 200 Automated Biochemistry Analyzer (China) using Diasens kits (Belarus). The supernatants for TPO activity assay were kept on an ice bath (melting ice temperature) and used during 3-4 h. Spectropho tometric studies were carried out on a SOLAR CM 2203 spectrofluorimeter. The buffer and other components were placed into a thermostat ted cuvette (1 cm). Incubation mixture composi tion: 0.05 M sodium phosphate buffer (pH 7.4)-2.7 mL, KI solution (0.6 M)-75 μL, superna tant-200 μL. The contents of the cuvette were held for 3 min at 25°C. The reaction was initiated by adding H 2 O 2 (10 μL of 40 mM solution), the sample was stirred, and the optical density was recorded at a wavelength of 353 nm for 3 min. The enzyme activity was expressed in international units per mg of protein per minute of incubation (IU/mg/min). The activity of iodothyronine deiodinase type 1 (D1) in the liver was assayed as described previ ously [10] with some modifications, that of malo nic dialdehyde (MDA)-by the reaction with thiobarbituric acid [11] . The procedure of D1 assay [10] is based on the determining hepatic D1 activity by a conversion of T4 into T3 in the presence of a dithiothreitol (DTT) cofactor. The following reagents were used: 0.05 M Tris HCl buffer (pH 7.8), 0.01 M sodium phosphate buffer (pH 7.6) containing 0.25% bovine serum albumin (BSA), 320 mM DTT in distilled water, T4 (I) standard in methanol-1000 μM added with 0.5 N NaOH (100 μL per 5 mL of solution); T4 (II) standard was obtained by diluting T4 (I) 20 fold on BSA phosphate buffer added with 0.5 N NaOH to pH 8.0 (final concentration 50 μM). Liver homogenate was prepared on Tris buffer (1 : 5) at a melting ice temperature, then centri fuged for 15 min on a BioSan LMC 3000 centri fuge at 3000 rpm (1700 g). Incubation mixture composition: superna tant-700 μL, DTT-40 μL, T4 (II) standard-40 μL. Incubation at 37°C for 1 h was performed in duplicates: 2 experimental and 2 control sam ples (control samples without DTT and T4). The reaction was stopped by adding 700 μL 96% etha nol, while DTT and T4 were added to the control samples. After stirring, the samples were left in a refrigerator for 2 h or overnight for protein precip itation. The supernatant was separated by centrif ugation and used for the determination of T3 by ELISA (storage was possible at -20°C until analy sis). The resulting supernatant contained about 48% ethanol. It was found that, when determining T3 by ELISA in an alcohol containing superna tant, it is necessary to mix it with a "zero serum" (pooled human serum treated with activated char coal), not allowing the ethanol concentration in the sample to exceed 5%. The effect of ethanol concentration on T3 determination by ELISA was tested on pooled human serum. When adding eth anol to this serum with a final concentration of no more than 5%, the T3 level was determined as a baseline, obtained in the native serum. While per forming this technique, the supernatant was added to the "zero serum" at a ratio of 10 : 100 L in such a way that the ethanol content in the sam ple was about 4.8%, which was maximum allow able. Statistical analysis was carried out using Statis tica 6.0. The normality of data distribution was checked using the Shapiro-Wilk test. If the distri bution was normal, the ANOVA was applied using the Fisher exact test (data were presented as M ± SEM). For non normal distributions, the nonparametric methods, Kruskal-Wallace test, were used (data were presented as Me (25th-75th percentile)). Differences were considered signifi cant at p < 0.05. At the first stage, the experiment focused on the effect of a high calorie diet and social isolation. The body mass indices in Control and Control SI rat groups were almost identical at the end of the experiment ( Table 1 ). The high calorie diet led to a statistically significant increase in the body mass by an average of 10% versus Control group, while the combined effect in HCD SI led to a 12% increase on average versus Control SI group. These rat groups (HCD and HCD SI) were prac tically indistinguishable from each other in aver age body mass values. The visceral fat mass did not reveal significant differences in the rats of Control and Control SI groups. The rats of HCD group showed a 4 fold gain in the visceral fat mass versus Control group, while this elevation was less pronounced in HCD SI group: approxi mately 2 fold versus Control SI group. At the same time, the visceral fat mass in HCD group was 2 fold higher versus HCD SI group. Obvi ously, in social isolation, the gain in rat visceral fat mass was less pronounced versus group housing, although fat intake in these rat groups was almost the same (6.7 and 6.8 g per day, respectively). The mean SBP values in both groups fed a standard diet (groups 1, 2) were practically identical, while in the rats fed an excessive diet (groups 3, 4), they significantly increased by 11 and 15%, respec tively, reaching comparable values ( Table 1) . As seen from Table 2 , the values of biochemical blood indices were almost identical in Control and Control SI groups. Feeding a high calorie diet led to an increase in the total serum choles terol level in the rats of HCD SI group. The tri glyceride level increased significantly only in HCD, but not HCD SI, group. The glucose level increased significantly in both groups versus Con trol group. The determination of triglyceride levels in thy roid tissue showed comparable values in the first two groups of rats fed a standard diet and revealed a significant increase in the index with a high cal orie diet regardless of the housing conditions ver sus Control group. However, the accumulation of triglycerides in thyroid tissue was most pro nounced in HCD group, significantly exceeding (1.5 fold) this index in the rats exposed to a com bined effect (HCD SI group). The MDA level in thyroid tissue significantly increased (1.4 fold) with a high calorie diet in group 3 versus Control, while in group 4, there was only an upward ten dency for the its level (Table 2) . Considering the increase in visceral fat mass, SBP elevation and metabolic shifts, we can state that the animals fed a high calorie diet developed an experimental metabolic syndrome. The con sumption of almost equal amounts of lard under conditions of social isolation and in group hous ing led to a more pronounced accumulation of visceral fat and greater metabolic abnormalities under conditions of group housing (Tables 1, 2) . The assessment of the rat psycho emotional status was carried out using the Porsolt forced swim test, which is designed to detect depressive behavior in rodents [8] . As seen from Fig. 1 , the rats of Control SI group differed by a reliable, almost 2 fold, increase in the duration of the first active swimming act versus Control group. The rats fed a high calorie diet showed shifts in the opposite direction, namely a significant decrease in the duration of the first active swimming act and an increase in the freezing (immobility) time, indicative an increasing depression. At the first stage of the experiment, along with hormonal indices, the characteristics of the activ ity of some enzymes of thyroid status were also studied (Table 3) . TPO activity in thyroid tissue was almost indistinguishable in the first two groups. Against the background of a high calorie diet, there was a significant decrease in TPO activity, almost half of the control level, regardless of the housing conditions (Table 3 ). In contrast, hepatic D1 activity significantly increased with excessive nutrition. Serum hormonal indices of the thyroid status are presented in the Figures as light bars (rats without stress) and hatched bars (rats after stress). The differences are significant at p < 0.05: *-vs Control group; #-vs Control SI group; ^-vs HCD group. First of all, it is necessary to consider the effect of a high calorie diet and social isolation on the studied indices. As seen from Fig. 2 , TSH values are not signifi cantly different among the animals of the first two groups (Control and Control SI) without stress, although in group 2, there was an upward ten dency for the TSH level (p > 0.05). A high calorie diet led to a significant (1.8 fold) increase in the TSH level versus Control, while HCD SI group showed a 2 fold increase in the index versus Con trol group and a 1.5 fold increase versus Control SI group. A significant (1.8 fold) increase in the TSH level was observed under the influence of short term stress in Control group; a significant hormone increase (1.5 fold) was also detected in HCD group. However, in the rats kept in isolation (Control SI and HCD SI), the shifts in TSH lev els did not reach statistical significance (Fig. 2) . Against the background of excessive feeding, there were a significant 1.6 fold increase in the total T3 level and a 1.4 fold increase in the total T4 level, which was paralleled by a 1.7 fold eleva tion of hepatic D1 activity versus Control group (Figs. 3, 4 ; Table 3 ). When a high calorie diet was combined with social isolation, T3 and T4 levels remained within control values, despite a signifi cant increase in TSH levels versus Control. Serum T3 and T4 levels in HCD significantly exceeded those in HCD combined with social isolation. The poststress T3 level (Fig. 3) remained rela tively stable in almost all experimental groups, whereas the serum T4 significantly increased in Control SI group against the background of stress (Fig. 4) . As seen from Fig. 5 and 6 , the feeding type and The differences are significant at p < 0.05: *-vs Control group; #-vs Control SI group; ^-vs HCD group. housing conditions had no effect on levels of free thyroid hormone fractions. A poststress elevation of fT3 was only observed in Control group (Fig. 5) , and of fT4 only in HCD SI group (Fig. 6) . A high calorie diet and social isolation caused no significant shifts in serum cortisol levels (Fig. 7) , but there was a downward tendency for its index in Control SI group. A short term stress exposure caused a sharp (1.8 fold) increase in the serum cortisol level in Control group rats versus those not exposed to cold water swimming, hence unstressed (Fig. 7) . It is worthwhile to characterize stress induced changes in the indices in Control group, i.e. an increase in levels of ТSH (1.8 fold) and cortisol (1.8 fold), as well as a slight increase in the serum fT3 level, which appears to be an adequate physi ological reaction to a short term acute stress. In Control SI group, there was also a signifi cant increase in serum cortisol levels (1.8 fold), but there were no changes in such indices as TSH and fT3, which is typical for Control group. The mean serum TSH value in Control SI group after swimming induced stress matched that in Con trol group after swimming. However, at the same time, the magnitude of the response to acute stressor exposure was decreased. In HCD group, there was only observed a 1.5 fold increase in serum TSH levels in the stressed versus unstressed animals. No cortisol release into the bloodstream was detected 1 h after stress. In HCD SI group, there was a poststress increase in cortisol levels similar to that in Con trol group (1.8 fold), but there was no reaction from TSH. The latter may be due to the initially elevated level of the index, which exceeded the control value by more than 2 fold. It is known from the literature that the isolation of rodents in early childhood causes later on a pronounced anxiety and depression accompanied by a decrease in corticosterone blood levels (chronic isolation stress) and changes in neuro plasticity indices [12, 13] . When isolating adult animals, no such characteristic shifts may occur, but more often, a hyperactive phenotype ensues [12, 13] . In our experiments, when tested in the Porsolt test, animals in Control SI group showed a significant increase in the duration of the first swimming act versus Control group. The animals of HCD group showed opposite shifts, namely a decrease in the time of the first swimming act and an increase in the freezing time. These character istics seem to indicate the development of an active phenotype against the background of isola tion stress and the signs of depression in a high calorie diet under conditions of group housing. There is ambiguous information in the litera ture concerning the effect of social isolation on the body mass of animals. Some authors point out that social isolation in overfed adult rodents pro motes an increase in obesity and concomitant alterations of metabolic functions [14] , while oth ers report the opposite results [15, 16] . The social isolation model that we used in our experiments restricted an increase in visceral obesity and char acteristic metabolic and hormonal shifts and also increased animal activity in the Porsolt test. The HCD group rats had a most pronounced visceral obesity and exhibited a tendency toward depres sion in the Porsolt test. As for the peculiarities of the thyroid status in HCD, our results confirmed the clinical observa tions stating an increase in the TSH level in obe sity [2] . This fact can be interpreted as evidence of hypothyroidism in obese patients, however, in our experiments, an increase in the TSH level was recorded against the background of elevated T3 and T4 levels. Current studies indicate that leptin produced by adipocytes has a regulatory effect on TSH secretion as it activates the hypothalamic pituitary adrenal (HPA) axis by affecting the neu rons residing in the paraventricular nucleus and responsible for TSH production [17] . It has been suggested that an increased TSH level in obesity is not a consequence of hypothyroidism, but serves as a mechanism aimed at the activation of basal metabolism [17] . Our work also revealed other adaptive components of the response aimed at the activation of metabolism in excessive high calorie diet and visceral obesity, such as an activation of hepatic D1 and an increase in T3 and T4 blood levels. T3 is known to increase the intensity of lip olysis in adipose tissue through cAMP dependent mechanisms, and thus affects lipolysis synergisti cally with the adrenergic system [17] . However, under conditions of social isolation, serum T3 and T4 shifts characteristic of HCD were absent in spite of elevated TSH levels. The fact of a decrease in TPO activity in thyroid tissue in HCD and HCD SI groups deserves attention. It may be explained by an accumulation of triglycerides in the thyroid gland in obesity and their toxic effect on the thyroid function, which was also noted in other studies [18] . Alternatively, this may be associated with an increase in free radical processes, as evidenced by an increase in the MDA level ( Table 2 ). Inhibition of TPO activity with prolonged overfeeding and visceral obesity may indicate a downward tendency for the thyroid function, which in the long run may entail the development of hypothyroidism. There are few publications in the literature addressing the problem of obesity in the light of its influence on the reactivity of the organism's regu latory systems under stress, although it is well known that a number of neuroendocrine pro cesses are definitely altered in obesity. In our experiments, a study of the reaction to short term stress showed that in the rats with a severest vis ceral obesity (HCD group), there is no stress induced increase in blood cortisol levels after 1 h exposure. It can be assumed that this is due to the neurobiological mechanisms of depression in obese animals. According to the literature, depressed patients are characterized by substantial changes in the HPA axis, with attenuated HPA and sympathetic responses to acute stress [19] [20] [21] being typical for depressed patients. There is evi dence of a biological link between type 2 diabetes, which is characteristic of obesity, depression and HPA axis dysregulation, including stress response [22] . As for the social isolation model, Control SI rat group showed an increased activity in the Porsolt test and an upward tendency for the TSH level, presumably due to chronic isolation stress. Against the background of prolonged social isola tion (Control SI and HCD SI groups), TSH release in response to short term stress, which was characteristic of Control group, did not show up. Thus, the feeding type, as well as the deficit of social activity, are factors that modulate the state of regulatory systems and alter their reactivity in response to acute stressor exposure. CONCLUSIONS 1. A high calorie 4 month diet causes a signifi cant gain in the body and visceral fat mass, as well as an elevation of systolic blood pressure, glucose and triglyceride serum levels, in male Wistar rats. Visceral obesity and triglyceride lev els were increased most pronouncedly when the animals were housed collectively as opposed to their individual housing under social isolation conditions. 2. The results of the Porsolt test indicate an increase in depression in rats fed a high calorie diet and a manifestation of an active phenotype in animals housed in conditions of social isolation. 3. A high calorie diet leads to a significant increase in TSH levels, total serum fractions of thyroxine and triiodothyronine, and hepatic D1 activity, which seems to be an adaptation aimed at the activation of metabolism. Under conditions of combined exposure to a high calorie diet and social isolation, no increase in thyroxine and triiodothyronine blood levels was observed. 4. Against the background of a high calorie diet, there is a decrease in TPO activity and an increase in triglyceride and malondialdehyde lev els in thyroid tissue, which in the long run may lead to a decrease in the thyroid function. 5. The reaction to short term acute stress in animals of Control group includes a sharp eleva tion of cortisol and TSH, as well as a slight increase in the serum level of free triiodothy ronine. 6. The rats fed a high calorie diet also showed an increased TSH release into the bloodstream 1 h after stress, but the cortisol level remained virtu ally unchanged in the meantime. 7. When animals were housed individually (social isolation), regardless of the type of their diet, a typical stress response was observed in the form of a cortisol release into the bloodstream after 1 h exposure. However, in these animals, TSH response to acute stress was not manifested, since the level of this index was already somewhat elevated against the background of chronic stress of social isolation. Thus, social isolation and a high calorie diet evoke oppositely directed changes in behavioral reactions of male Wistar rats. These exposures, as well as their combinations, lead to specific shifts in the functional activity of the pituitary thyroid adrenal axis, which, in turn, corrects the physio logical response to short term stress exposure. The work was supported by the governmental budget funding to the Institute of Physiology of the National Academy of Sciences of Belarus within the SSRP "Fundamental and applied sci ences for medicine", research theme "To investi gate neuroendocrine mechanisms of rat propensity to obesity" (reg. no. 20200320 of 16.03.2020). The definition and prevalence of obesity and metabolic syndrome Thyroid and Obesity: An Intrigu ing Relationship Obesity and Depression: Its Prevalence and Influence as a Prognostic Factor: A Systematic Review The effect of acute exercise session on thyroid hormone econ omy in rats Thyroid hormone regulation by stress and behavioral differences in adult male rats Animal models of meta bolic syndrome: a review Neurobiology and consequences of social isolation stress in animal model A comprehensive review Behavioral despair in mice: a primary screening test for anti depressants Determination of thyroid peroxidase activity in thyroid tissue (experimental study) Iodothyronine metabolism in rat liver homogenates Thiobarbituric acid positive substances as indicators of lipid peroxida tion Social isola tion stress induces anxious depressive like behav ior and alterations of neuroplasticity related genes in adult male mice Social isolation in rats: Effects on animal welfare and molecular markers for neuroplasticity Metabolic effects of social isolation in adult C57BL/6 mice Interplay between diet induced obesity and chronic stress in mice: potential role of FKBP51 Stress induced obesity: lessons from rodent models of stress The crosstalk between thyroid gland and adipose tissue: signal integration in health and dis ease Dietary high fat lard intake induces thyroid dys function and abnormal morphology in rats Stress reactivity in social anxiety disorder with and without comorbid depression The authors declare that they have no evident or potential conflict of interest related to the pub lication of this article.