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1 Variable Cold-induced Brown Adipose Tissue Response to Thyroid Hormone Status
2 Alina Gavrila, MD, MMSC1; Per-Olof Hasselgren, MD, PhD2; Allison Glasgow3; Ashley N. 3 Doyle3; Alice J. Lee3, Peter Fox5, Shiva Gautam, PhD1; James V. Hennessey, MD1; Gerald M. 4 Kolodny, MD4; Aaron M. Cypess, MD, PhD, MMSC1,5,6
5 1Department of Medicine, 2Department of Surgery, 3Harvard Catalyst Clinical Research Center, 6 4Radiology, Beth Israel Deaconess Medical Center, Boston, MA, 02215; 7 5Section of Integrative Physiology and Metabolism, Research Division, Joslin Diabetes Center, 8 Harvard Medical School, Boston, MA, 02215; 9 6Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and 10 Kidney Diseases, NIH, Bethesda, MD 20892, USA.
11 Email addresses: agavrila@bidmc.harvard.edu; phasselg@bidmc.harvard.edu; 12 aglasgow02@gmail.com; andoyle2@gmail.com; alee16@bidmc.harvard.edu; 13 pfox1986@gmail.com; sgautam@bidmc.harvard.edu; jhenness@bidmc.harvard.edu; 14 gkolodny@bidmc.harvard.edu; aaron.cypess@nih.gov.
15 Running Title: Thyroid Hormone and Brown Adipose Tissue 16 Keywords: Brown adipose tissue, thyroid hormone, hypothyroidism, thyrotoxicosis, 17 thermogenesis.
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18 Abstract 19 Background: In addition to its role in adaptive thermogenesis, brown adipose tissue (BAT) may 20 protect from weight gain, insulin resistance/diabetes and metabolic syndrome. Prior studies have 21 shown contradictory results regarding the influence of thyroid hormone (TH) levels on BAT 22 volume and activity. The aim of this pilot study was to gain further insights regarding the effect 23 of TH treatment on BAT function in adult humans by evaluating the BAT mass and activity 24 prospectively in six patients, first in the hypothyroid and then in the thyrotoxic phase. 25 Methods: The study subjects underwent 18F-FDG PET-CT scanning after cold exposure to 26 measure BAT mass and activity while undergoing treatment for differentiated thyroid cancer, 27 first while hypothyroid following thyroid hormone withdrawal at the time of the radioactive 28 iodine treatment and then 3-6 months after starting TH suppressive treatment when they were 29 iatrogenically thyrotoxic. We measured thermogenic and metabolic parameters in both phases. 30 Results: All study subjects had detectable BAT under cold stimulation in both the hypothyroid 31 and thyrotoxic state. The majority but not all (4 out of 6) subjects showed an increase in 32 detectable BAT volume and activity under cold stimulation between the hypothyroid and 33 thyrotoxic phase (total BAT volume: 72.0 ± 21.0 vs. 87.7 ± 16.5 mL, P = 0.25; total BAT 34 activity 158.1 ± 72.8 vs. 189.0 ± 55.5 SUV*g/mL, P = 0.34). Importantly, circulating T3 was a 35 stronger predictor of energy expenditure changes compared to cold-induced BAT activity. 36 Conclusions: Iatrogenic hypothyroidism lasting 2-4 weeks does not prevent cold-induced BAT 37 activation, while the use of TH to induce thyrotoxicosis does not consistently increase cold38 induced BAT activity. It remains to be determined which physiological factors besides TH play 39 a role in regulating BAT function.
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40 Introduction 41 In the midst of worldwide obesity and diabetes epidemics, it is crucial to find new avenues to 42 gain insight into the pathophysiology and treatment of these disorders. Brown adipose tissue 43 (BAT) is of particular interest since in addition to its role in adaptive thermogenesis, it may also 44 protect against weight gain, insulin resistance/ diabetes and metabolic syndrome (1-3). Indeed, 45 because of its very high metabolic activity, very little functional BAT can have a profound 46 metabolic impact (4, 5). 47 Studies using PET/CT scanning have shown metabolically active BAT in adult humans 48 whose activity correlated inversely with body fat (5, 7, 8). Significant amounts of functional 49 BAT have been detected in more than 95% of lean subjects in response to acute and chronic cold 50 exposure in controlled prospective studies (5, 8, 9, 10). Since BAT activity is induced by cold 51 exposure, BAT might play an important role in human thermogenesis and energy balance. This 52 is in accordance with studies in small rodents, which showed that cold-exposure stimulates BAT 53 activity by increasing sympathetic nerve activity and by affecting local thyroid hormone (TH) 54 metabolism (11, 12). Cold exposure increases the expression and activity of tissue deiodinase 2 55 (DIO2), which stimulates the conversion of thyroxine (T4) to active triiodothyronine (T3), 56 resulting in increased local T3 production from T4 and TH receptor saturation (13). Both 57 catecholamines and local T3 act synergistically to stimulate uncoupling protein 1 (UCP-1) 58 expression and through this thermogenesis in BAT. UCP-1 uncouples oxidative 59 phosphorylation, thus representing the key protein in BAT thermogenesis (12). TH also has 60 direct effects on oxidative phosphorylation and regulates the mitochondrial proliferation, 61 differentiation and maturation through genomic and non-genomic mechanisms (14, 15). In 62 addition, TH is known to play an important role in brown adipocyte differentiation (16, 17).
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63 Thus, TH has been shown to have an important role in cold adaptation in rodents, and it may be 64 important for temperature adaptation in humans (18). Understanding cold adaptation in adult 65 humans is important, since factors that increase energy dissipation through facultative 66 thermogenesis could potentially be used as anti-obesity agents (18, 19). If TH can influence 67 BAT mass and activity and through this energy expenditure, then TH or its analogues might be 68 useful in the treatment of obesity and the metabolic syndrome. 69 A few published human studies have shown contradictory results reporting increased BAT 70 volume and activity in either hypothyroid or thyrotoxic subjects (20-23). To gain further insight 71 regarding the effect of TH status on BAT in adult humans and whether it could be a feasible 72 treatment strategy for activating BAT thermogenesis, we evaluated the BAT mass and activity 73 prospectively in six patients who were undergoing treatment for thyroid cancer, first in the 74 hypothyroid and then in the thyrotoxic phase. The study patients underwent thyroidectomy 75 followed by TH withdrawal to induce hypothyroidism prior to the radioactive iodine (RAI) 76 treatment and then started TH suppressive treatment with the goal of maintaining mild 77 thyrotoxicosis long-term to prevent recurrence. The study patients underwent two PET-CT scans 78 to measure BAT mass and activity, first while profoundly hypothyroid at the time of the RAI 79 treatment and then three to six months after starting TH suppressive treatment when they were 80 thyrotoxic. We compared the BAT mass and activity measured on these two scans. We also 81 evaluated the relationship between BAT, TH status, and calorimetric and metabolic parameters.
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82 Materials and Methods 83 Study Participants 84 Eight patients diagnosed with papillary thyroid cancer who underwent thyroidectomy and 85 were scheduled to receive RAI treatment after TH withdrawal were recruited for the study. The 86 study was approved by the Institutional Review Board of Beth Israel Deaconess Medical Center 87 (BIDMC) and Joslin Diabetes Center, and patients provided written informed consent. 88 Exclusion criteria included use of medications that can affect BAT, such as beta blockers, 89 adrenergic agonists, benzodiazepines; use of medications that can affect thyroid function test 90 interpretation, including estrogen, aspirin, dilantin; use of systemic, oral or intravenous 91 corticosteroids or other medications known to cause insulin resistance in the previous 6 weeks. 92 Patients with a history of any local or systemic infectious disease with fever or requiring 93 antibiotic within four weeks of the PET/CT scans were not enrolled in the study.
94 Study Protocol (Figure 1) 95 The patients participated in a short screening visit to evaluate eligibility prior to the RAI 96 treatment followed by two longer study visits when the whole-body BAT volume and activity 97 were measured by 18F-FDG PET/CT under cold stimulation at 15-16°C, first in the hypothyroid 98 state at the time of the RAI treatment and then in the thyrotoxic state 3-6 months after starting 99 TH suppressive treatment. Six out of the eight patients who started the study completed all study 100 visits and were included in the analysis. Prior to participating in the study, one patient 101 underwent completion thyroidectomy for a thyroid nodule positive for papillary thyroid 102 carcinoma on fine needle aspiration cytology, while five patients underwent hemithyroidectomy 103 followed by completion thyroidectomy for thyroid nodules with fine needle aspiration showing
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104 indeterminate cytology (2 patients), for a large thyroid nodule with benign cytology (1 patient), 105 for a large thyroid nodule with non-diagnostic cytology (1 patient), and for recurrent thyroid 106 cancer after undergoing lobectomy in the remote past (1 patient). Preparation for the 107 hypothyroid phase of the evaluation occurred at least 6 weeks postoperatively for 5 patients and 108 consisted of a 6 week L-thyroxine (LT4) withdrawal with a 4 week liothyronine bridging support 109 (12.5 mcg twice daily) for minimization of clinical symptoms followed by 2 weeks of complete 110 thyroid hormone withdrawal and a low iodine diet prior to the clinically indicated 131-I 111 administration. One study patient was already hypothyroid at the time of her initial endocrine 112 clinic visit with a TSH of 22 (reference range 0.27-4.2 µIU/mL); the levothyroxine was 113 discontinued at that time and the patient received RAI treatment 2 weeks later without the 114 liothyronine bridging support. 115 The patients were prepared for the study visits as previously described (24, 25). Baseline 116 vital signs and laboratory tests (thyroid function tests, insulin, HbA1c, and lipid panel) were 117 obtained. Baseline indirect calorimetry was performed during both the hypothyroid and 118 thyrotoxic phase after the patients were placed in a supine position and quiet environment for 30 119 minutes in the Clinical Research Center (CRC). The room temperature was maintained above 120 23°C throughout the stay in the CRC. Core body temperature was monitored during the study 121 visits. 122 To maximize the BAT activity/FDG uptake and PET-CT scan BAT visualization, the 123 patients were then transported to a room set at 20ºC and wore a surgeon's cooling vest (Polar 124 Products, Inc.) set to 15-16°C, as described previously (24, 25). Sixty minutes after wearing the 125 vest, a second set of vital signs and laboratory tests (thyroid function tests, glucose, insulin, free 126 fatty acids) were obtained and an intravenous bolus of 592 MBq (16 mCi) of 18F-FDG was
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127 administered; this dose was calculated based on the sensitivity of the scanner available at the 128 time of the study. The patients remained in the semi-darkened quiet room for another 60 minutes 129 while wearing the cooling vest, and at this time, a second measure of indirect calorimetry was 130 performed. Following 120 minutes of cold exposure, the vest was removed, and BAT images 131 were acquired using a 4-MDCT scanner (Discovery/LightSpeed PET/CT, GE Healthcare) 132 following a previously described imaging protocol (24). BAT volume, metabolic activity and 133 maximum standardized uptake value (SUVmax) were measured in the cervical, supraclavicular, 134 and anterior thoracic depots from vertebral level C3 to T7 using the PET-CT Viewer shareware, 135 as described previously (24). The BAT metabolic activity represents the primary endpoint of 136 this study and is calculated in a three-step process. First, in each axial slice on CT, a voxel is 137 defined as containing adipose tissue if it has a HU density between -250 and -10. Second, the 138 amount of retained FDG in each voxel is quantified by measuring the mean amount of positron139 derived gamma radiation in the voxel compared to the injected dose per body mass (MBq/mL of 140 voxel)/ (MBq/g of body mass), giving the “meanSUV*g/mL”. Finally, the total-body metabolic 141 activity is calculated by taking the sum of the volume of BAT from each voxel 142 =“mL*meanSUV*g/mL.” As expected, according to the method used to quantify BAT 143 parameters, there was a strong positive correlation between the cold-induced BAT volume and 144 activity of the study subjects in the hypothyroid phase (rho = >0.99, P = 0.01) as well as 145 thyrotoxic phase (rho = 0.94, P = 0.005). SUVmax reflects the highest amount of retained FDG 146 anywhere within the tissue. We used this metric to compare BAT activity with more 147 homogeneous tissues such as liver and skeletal muscle. The SUVmax of the FDG uptake in the 148 right lobe of the liver and skeletal muscle (erector spinae) was also measured.
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149 Physiological and Laboratory Parameters 150 Blood pressure and heart rate were measured using a SureSigns VS3 vital signs monitor 151 (Philips Healthcare). Core body temperature was measured with a temporal artery thermometer 152 (Exergen Co.). Indirect calorimetry measurements including the resting metabolic rate (RMR), 153 energy expenditure (EE), respiratory quotient (RQ), and substrate utilization were calculated 154 using Sensormedics VMAX Encore equipment (VIASYS Respiratory Care Inc.) as well as 155 techniques well established in the laboratory and standardized values. Insulin and TSH were 156 measured according to standard procedures at the Harvard Catalyst CRC 157 (http://catalyst.harvard.edu/services/hccrc-lab/). Total T4, total T3, free T3, T3 uptake, free T4 158 index, thyroxin binding globulin (TBG), free fatty acids (FFAs), lipid panel and HbA1c were 159 measured using standard procedures at the Laboratory Corporation of America. Glucose was 160 measure at the BIDMC clinical laboratory.
161 Statistical Analysis 162 This pilot study was designed to evaluate whether the TH status had an effect on BAT mass 163 and activity in adult humans, specifically whether treatment with TH increases BAT mass and 164 activity as the subjects transitioned from hypothyroidism to thyrotoxicosis. We anticipated 165 consenting and screening up to 10 patients, so that 6-8 patients would complete all segments of 166 the study (completion rate of 80%). 167 Data were analyzed using Statistical Package for the Social Science (SPSS, IBM, Armonk, 168 New York) and Statistical Analysis System (SAS, SAS Institute, Inc.). All P values presented 169 are two-tailed, and values ≤ 0.05 were considered to indicate statistical significance, while values 170 < 0.10 were considered to represent a trend to achieve significance. Changes in BAT volume,
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171 activity and maximum standardized uptake value (SUVmax), as well as hormonal and metabolic 172 changes in the study patients between the hypothyroid and thyrotoxic state were evaluated using 173 the non-parametric Wilcoxon signed-rank test. Associations between BAT volume and activity, 174 TH and metabolic parameters were evaluated by Spearman correlation.
175 Results 176 The baseline characteristics of the six patients who completed the study are summarized in 177 Table 1. Five patients had a normal baseline TSH prior to being enrolled in the study. One 178 patient who was enrolled at the time of her completion thyroidectomy, had a low baseline TSH 179 of 0.19 (reference range 0.27-4.2 µIU/mL) on TH treatment prior to starting the study. All 180 patients were hypothyroid after TH withdrawal at the time of the first study PET/CT scan. In 4 181 out of the 6 study patients, the final TSH was in the thyrotoxic range, while the fifth patient had a 182 TSH in the low reference range at 0.38 µIU/mL 3-6 months after starting TH suppressive 183 treatment at the time of the second PET/CT scan. However, the sixth patient had a final TSH of 184 5.46 µIU/mL. Therefore, although the mean was in the euthyroid range, all patients but one were 185 thyrotoxic at the time of the second PET/CT scan.
186 Thyroid function tests and metabolic parameters. TSH was higher while total T4, free T4 187 index, free T3 and T3 uptake were lower in the hypothyroid compared to the thyrotoxic state 188 (trend only for free T3) (Figure 2A-C, Table 2). There was no significant difference in the total 189 T3 between the hypothyroid and thyrotoxic state. There was no significant change in any thyroid 190 function test after 60 min of cold exposure during each state. TBG levels did not change 191 between the hypothyroid and thyrotoxic states. The total and LDL cholesterol were higher in the
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192 hypothyroid compared to the thyrotoxic state. There were no significant changes in fasting 193 triglyceride, free fatty acids, glucose, insulin, and HbA1c between the hypothyroid and 194 thyrotoxic state (Table 2).
195 Vital signs. The systolic BP did not change significantly during the study. The diastolic BP 196 increased after 60 min of cold exposure during both the hypothyroid and thyrotoxic states, the 197 levels after cold exposure being higher in the hypothyroid compared to the thyrotoxic state The 198 HR decreased significantly after 60 min of cold exposure in the hypothyroid but not the 199 thyrotoxic phase. There were no other significant changes in vital signs measured at room 200 temperature and after 60 min of cold exposure during the hypothyroid as well as thyrotoxic state 201 or between the two states (Table 2).
202 Energy expenditure. The baseline RMR of the study subjects measured at room temperature 203 was higher in the thyrotoxic compared to the hypothyroid state. The RMR increased after 60 204 min of cold exposure during both the hypothyroid and thyrotoxic states. There was no difference 205 between the RMR measured in the two states after cold exposure. There were no other 206 significant changes in RMR or RQ measured at room temperature and after 60 min of cold 207 exposure during the hypothyroid as well as thyrotoxic state or between the two states (Table 2). 208 There was a positive correlation between the RQ measured at room temperature and after 60 min 209 of cold exposure in the hypothyroid state (Spearman rho = 0.83, P = 0.04). There was no 210 correlation between the RMR measured at room temperature and after 60 min of cold exposure 211 during each state.
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212 Glucose uptake in BAT and other tissues. All patients had detectable BAT after acute cold 213 exposure in both the hypothyroid and thyrotoxic state (Figure 3A-D, Table 3). Other tissues 214 showed significant but small changes in glucose uptake. The SUVmax of the liver decreased 215 (3.39 ± 0.27 vs. 3.03 ± 0.25 g/mL, P = 0.01), while the SUVmax of the skeletal muscle increased 216 (0.62 ± 0.06 vs. 0.84 ± 0.10 g/mL, P = 0.02), when going from the hypothyroid to the thyrotoxic 217 phase (Figure 4A-C). 218 The majority but not all (4 out of 6) patients showed an increase in detectable, acute, cold219 induced BAT volume and activity between the hypothyroid and thyrotoxic states. The increases 220 were not correlated with the seasonal trends in outdoor temperatures the patients experienced 221 while they were taking thyroid hormone. The change in BAT volume, activity, and SUVmax 222 between the two states were not significant. In the two patients who did not show an increase in 223 BAT parameters between the hypothyroid and thyrotoxic state, the RMR also did not change 224 after 60 min of cold exposure in both the hypothyroid and thyrotoxic state. However, there was 225 a positive correlation between the BAT volume as well as BAT activity measured after cold 226 exposure in the hypothyroid and thyrotoxic state (BAT volume: rho = 0.83, P = 0.04; BAT 227 activity: rho = 0.89, P = 0.02). 228 There was a negative correlation between the cold-induced BAT volume as well as activity 229 and the subjects’ weight and BMI in both the hypothyroid and thyrotoxic state. There was no 230 correlation between the BAT parameters and the subjects’ age and core body temperature. The 231 baseline systolic BP measured in the hypothyroid state correlated strongly and positively with 232 the BAT mass and activity measured in both states.
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233 Inter-relationships among metabolic measures. Total T4, free T4 index, total T3 and free T3 234 correlated positively with the cold-induced BAT SUVmax in the hypothyroid state (all variables 235 in hypothyroid state: rho = 0.88, P = 0.02) (Figure 5A). Free T3 correlated negatively with the 236 cold-induced BAT volume and activity in the thyrotoxic state (free T3 at room temperature/BAT 237 volume: rho = -0.83, P = 0.04; free T3 at room temperature/BAT activity: rho = -0.77, P = 0.07; 238 freeT3 after cold exposure/BAT volume: rho = -0.99, P = 0.01; free T3 after cold exposure/ BAT 239 activity: rho = -0.99, P = 0.01) (Figure 5B). Total T3 also correlated negatively with the cold240 induced BAT volume and tended to correlate negatively with the BAT activity in the thyrotoxic 241 state (total T3 at room temperature/BAT volume: rho = -0.83, P = 0.04; total T3 after cold 242 exposure/BAT volume: rho = -0.83, P = 0.04; total T3 at room temperature/BAT activity: rho = 243 0.77, P = 0.07; total T3 after cold exposure/ BAT activity: rho = -0.77, P = 0.07). 244 Several facets of thyroid status correlated with RMR. There was a significant inverse 245 relationship between the baseline TSH and RMR in the hypothyroid state (rho = -0.89, P = 0.02). 246 In addition, there was a significant positive correlation between the change in RMR after 60 min 247 of cold exposure and the change in total T3 as well as free T3 between the hypothyroid and 248 thyrotoxic state (change in TT3 after 60 min of cold exposure: rho = 0.94, P = 0.005; change in 249 FT3 after 60 min of cold exposure: rho = 0.94, P = 0.005) (Figure 5C). 250 For exploratory analyses, we looked at the relationships between detectable cold-induced 251 BAT metabolism and other metabolic parameters. Serum TG levels correlated negatively with 252 BAT mass and activity in each state (hypothyroid state, BAT volume and activity: rho = -0.89, P 253 = 0.02; hyperthyroid state, BAT volume: rho = -0.83, P = 0.04 and BAT activity: rho = -0.94, P 254 = 0.005), and HbA1c correlated negatively with BAT volume and activity in the hypothyroid 255 state (rho = -0.90, P = 0.015). Total and LDL cholesterol correlated negatively with BAT
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256 activity (rho = -0.83, P = 0.04), while HDL cholesterol correlated positively with BAT mass and 257 activity (BAT volume: rho = +0.90, P = 0.015, BAT activity: rho = +0.84, P = 0.04) in the 258 thyrotoxic state.
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259 Discussion 260 Given the central role that thyroid hormone plays in regulating whole-body energy 261 expenditure and BAT function, we designed this pilot study to evaluate how promising treatment 262 with TH would be toward increasing BAT thermogenesis. However, because of the numerous 263 adverse effects of TH supplementation in healthy humans, a novel study population is required in 264 order to evaluate the full capacity of dosing with TH. We therefore chose as our model a series 265 of patients with thyroid cancer because their standard course of treatment leads to iatrogenic 266 hypothyroidism followed by TH treatment to the thyrotoxic state. All six study patients 267 demonstrated detectable BAT under cold stimulation in both the hypothyroid and thyrotoxic 268 state. The majority but not all (4 out of 6) patients showed an increase in BAT volume and 269 activity under cold stimulation between the hypothyroid and thyrotoxic phase. In addition, there 270 was a positive correlation between the cold-induced BAT volume as well as BAT activity in the 271 hypothyroid and thyrotoxic phase for each patient.
272 It is important to consider that all study patients showed detectable cold-activated BAT in the 273 hypothyroid state, even at TSH levels higher than 100 µIU/mL, which indicates that even low 274 levels of TH are adequate for BAT activation in adult humans. In our study, serum T3 levels 275 correlated negatively with cold-induced BAT mass and activity in the thyrotoxic state in adult 276 humans. These apparently counterintuitive findings suggest that circulating T3 may be a marker 277 of thyroid function without direct effects on BAT. Studies in rodents have shown that the BAT 278 type 2 deiodinase (DIO2) is stimulated by the sympathetic nervous system (SNS) and inhibited 279 by its substrate T4 (11, 12, 26). Excessive circulating T4 levels could result in inhibition of the 280 DIO2 and proportionally lower local T3 concentrations and UCP1 activation in hyperthyroid
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281 subjects (12), while low circulating T4 levels could activate DIO2, increase the local T3 282 production and activate the UCP1 in hypothyroid patients (27, 28). In addition, we need to take 283 into consideration that the SNS and TH act synergistically to respond and adjust the body to 284 different environmental factors, such as cold exposure. Studies in small rodents have shown that 285 cold-exposure stimulates BAT activity by increasing central sympathetic nerve activity and by 286 affecting local TH metabolism (11, 12). Both catecholamines and local T3 act synergistically to 287 stimulate uncoupling protein 1 (UCP-1) expression and through this thermogenesis in BAT. In 288 hypothyroidism, there is an increase, while in thyrotoxicosis there is a decrease in the central 289 sympathetic outflow, as measured by plasma and urinary norepinephrine (NE) levels as well as 290 NE turnover in different tissues such as BAT. However, there is also a reduced/exaggerated 291 response to catecholamines in different tissues, because of decreased/ increased beta-adrenergic 292 receptor density as well as postreceptor mechanisms, which usually overrides the effect of TH on 293 the central SNS outflow and explains the clinical manifestations in hypothyroidism and 294 thyrotoxicosis, respectively (12, 34). Thus, the regulatory mechanisms involved in regulating 295 BAT are complex and one of these mechanisms may be predominant, which could explain the 296 different BAT responses to cold exposure noted in different patients.
297 We did not find a correlation between the change in thyroid function or the change in RMR 298 and the change in cold-induced BAT volume and activity between the hypothyroid and 299 thyrotoxic state. This may be explained by the fact that TH influences both the basal/obligatory 300 and facultative thermogenesis through different mechanisms (11, 29). A treatment that would 301 selectively stimulate facultative thermogenesis could be beneficial for weight loss and 302 improvement in metabolic parameters without the side effects of elevated circulating TH levels.
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303 Our study showed that circulating T3 is a stronger predictor of the change in energy 304 expenditure compared to cold-induced BAT activity. However, the response to TH treatment led 305 to opposite responses among two of the principal thermogenic organs: glucose uptake in the 306 liver was lower, but skeletal muscle uptake was higher. Therefore, it cannot be assumed that all 307 tissues respond the same to chronic treatment with TH. Our findings also support the hypothesis 308 that TH contributes significantly to cold-induced thermogenesis by affecting multiple organs 309 including skeletal muscle in addition to BAT (30, 31, 21, 22). In our study, there was a more 310 consistent increase in the skeletal muscle activity as compared to the BAT activity between the 311 hypothyroid and thyrotoxic state. This may be explained by the fact that the skeletal muscle 312 activity is mainly regulated by thyroid hormone, while the synergistic activity with the SNS may 313 be less important in this tissue (32). In addition, the difference between the two tissues could be 314 potentially explained by the fact that our study patients took high-dose levothyroxine at the time 315 of the second PET-CT scan, which can decrease DIO2 activity; this enzyme has more important 316 effects in BAT than skeletal muscle (33).
317 The few published human studies looking at the relationship between thyroid status and BAT 318 function have shown contradictory results (20-23). Skarulis et al. reported detectable BAT 319 measured by PET-CT in a hypothyroid patient and an increase in BAT parameters when the 320 patient became thyrotoxic upon resuming TH treatment as part of follow-up for thyroid cancer 321 (20). In contrast, a severely hypothyroid child with Hashimoto’s thyroiditis was found with a 322 significant supraclavicular BAT volume measured by MRI and BAT activity measured by 323 infrared thermal imaging, which both decreased after two months of TH replacement (23).
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324 Lahesmaa et al. reported a three-fold increase in glucose uptake measured by FDG-PET in ten 325 hyperthyroid patients compared to euthyroid patients (21), while Zhang et al. reported no 326 detectable BAT in ten patients with newly diagnosed Graves’ hyperthyroidism before starting 327 treatment (22). Additional human studies are needed to gain insight into the factors that 328 moderate the effect of TH on BAT volume and activity. It may be that TH directly stimulates 329 BAT, but the general increase in the resting metabolic rate may obviate the need for additional 330 BAT in some patients. The end result of these two simultaneous but opposite effects of TH on 331 BAT may explain why some patients increase their BAT while others have less. In addition, as 332 described above, the complex interaction between TH and sympathetic nervous system in 333 regulating BAT may play a role.
334 We recruited thyroid cancer patients for this pilot study. This represents an excellent study 335 model for BAT evaluation since treatment for thyroid cancer involves several steps including 336 thyroidectomy, followed by induction of short-term hypothyroidism prior to the radioactive 337 iodine treatment and then long-term suppressive TH treatment to prevent recurrence. Therefore, 338 we can evaluate the changes in BAT mass and activity between the hypothyroid and thyrotoxic 339 state in this patient population.
340 This is a pilot study designed with the goal to obtain preliminary data in a small but 341 physiologically informative group of patients with thyroid cancer whose clinical course involves 342 treatment with high-dose levothyroxine to change their thyroid status from severe 343 hypothyroidism to mild thyrotoxicosis. The signal from this cohort would be a best-case 344 scenario for any studies in healthy subjects or patients with milder hypothyroidism. Our data
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345 show that based on the mean change in BAT metabolic activity, we would have to study more 346 than a total of 50 pairs of subjects to demonstrate a significant effect of thyroid hormone 347 treatment on detectable BAT. Therefore, this pilot study has achieved one of its essential goals 348 and demonstrated that is not very feasible to use levothyroxine treatment in most populations as 349 a way of increasing BAT mass or metabolic activity.
350 As known from prior animal as well as human studies, chronic cold exposure is probably the 351 strongest determinant of BAT volume and activity (9,10). Based on our study design, we cannot 352 easily distinguish the effects of chronic cold from the chronic administration of high doses of 353 thyroid hormone. However, the change in BAT activity was not correlated with the season of 354 thyroid hormone treatment, so the effects of cold exposure were not likely to have substantially 355 confounded the modest effect we saw from chronic exposure to high doses of thyroid hormone. 356 In our study, we measured cold-induced BAT, since significant amounts of functional BAT have 357 been detected in more than 95% of lean subjects in response to acute cold exposure (5, 8), while 358 significantly fewer subjects showed detectable BAT via PET-CT scanning when studied at room 359 temperature. With the current study design we were able to evaluate the effect of TH on cold360 induced BAT by studying the same patients after cold exposure in both the hypothyroid and 361 thyrotoxic state. Therefore, we can infer that the change in the BAT mass and activity between 362 the two phases is related to the thyroid hormone status change.
363 In conclusion, in this pilot study we found that all 6 study patients had detectable BAT after 2 364 hours of cold exposure in both the hypothyroid and thyrotoxic state, and the majority but not all 365 (4 out of 6) patients showed an increase in cold-induced BAT volume and activity between the 366 hypothyroid and thyrotoxic state. Therefore, iatrogenic hypothyroidism lasting 2-4 weeks does
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367 not prevent cold-induced BAT activation. At the same time, the use of TH to induce 368 thyrotoxicosis does not consistently increase cold-induced BAT activity. Although the use of 369 levothyroxine is not feasible, using a BAT-specific TH analog may be still feasible to increase 370 BAT thermogenesis as a treatment for obesity and metabolic disease. Further studies are needed 371 to evaluate this and also determine which physiological factors besides TH are involved in 372 stimulating BAT growth and thermogenesis.
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373 Acknowledgments: This work was supported by the 2012 Harvard Catalyst Clinical Research 374 Center (HCCRC) Resources and Services Funding Award; the 2013 Harvard Catalyst Clinical 375 Research Center (HCCRC) Laboratory Support and Genotyping Award; National Institutes of 376 Health (NIH) grants K23 DK081604, the Intramural Research Program of the National Institute 377 of Diabetes and Digestive and Kidney Diseases (NIDDK); and the Clinical Translational Science 378 Awards UL1RR025758, UL1TR000170 and UL1TR001102 to Harvard University and its 379 affiliated academic health care centers from the National Center for Research Resources 380 (NCRR) and the National Center for Advancing Translational Science and financial 381 contributions from Harvard University and its affiliated academic healthcare centers. The content 382 is solely the responsibility of the authors and does not necessarily represent the official views of 383 Harvard Catalyst, Harvard University and its affiliated academic healthcare centers, or the 384 National Institutes of Health.
385 We thank the Beth Israel Deaconess Medical Center (BIDMC) Clinical Research Center nursing 386 team, Bionutrition Core, and nuclear medicine technologists for the excellent support they 387 provided; Michelle Beck for supervising the study development and progression; Anthony 388 Hollenberg for reviewing the manuscript; Matthew R Palmer for reviewing the PET-CT study 389 protocol; Lauren Weiner for her assistance in coordinating the clinical studies; and our 390 volunteers for their commitment to the studies.
391 Disclosures: The authors have nothing to disclose.
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392 Corresponding author: 393 Alina Gavrila, M.D., M.M.Sc. 394 Division of Endocrinology and Metabolism 395 Beth Israel Deaconess Medical Center 396 330 Brookline Avenue GZ6 397 Boston, MA 02215 398 Phone: 617-667-9344 399 Fax: 617-667-7060 400 agavrila@bidmc.harvard.edu
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422 [8]. Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JM, Kemerink GJ, 423 Bouvy ND, Schrauwen P, Teule GJ 2009 Cold-activated brown adipose tissue in healthy men. N 424 Engl J Med 360(15):1500-1508. 425 [9]. van der Lans AA, Hoeks J, Brans B, Vijgen GH, Visser MG, Vosselman MJ, Hansen 426 J, Jörgensen JA, Wu J, Mottaghy FM, Schrauwen P, van Marken Lichtenbelt WD 2013 Cold 427 acclimation recruits human brown fat and increases nonshivering thermogenesis. J Clin Invest 428 123(8):3395-403. 429 [10]. Yoneshiro T, Aita S, Matsushita M, Kayahara T, Kameya T, Kawai Y, Iwanaga T, Saito M 430 2013 Recruited brown adipose tissue as an antiobesity agent in humans. J Clin Invest 431 123(8):3404-8. 432 [11]. Silva JE 2006 Thermogenic mechanisms and their hormonal regulation. Physiol Rev 433 86(2):435-64. 434 [12]. Silva JE, Bianco SD 2008 Thyroid-adrenergic interactions: physiological and clinical 435 implications. Thyroid 18(2):157-65. 436 [13]. Bianco AC, Silva JE 1988 Cold exposure rapidly induces virtual saturation of brown 437 adipose tissue nuclear T3 receptors. Am J Physiol 255(4 Pt 1):E496-503. 438 [14]. Harper ME, Seifert EL 2008 Thyroid hormone effects on mitochondrial energetics. Thyroid 439 18(2):145-56. 440 [15]. Lee JY, Takahashi N, Yasubuchi M, Kim YI, Hashizaki H, Kim MJ, Sakamoto T, Goto 441 T, Kawada T 2012 Triiodothyronine induces UCP-1 expression and mitochondrial biogenesis in 442 human adipocytes. Am J Physiol Cell Physiol 302(2):C463-72. 443 [16]. Obregon MJ 2008 Thyroid hormone and adipocyte differentiation. Thyroid 18(2):185-95. 444 [17]. Obregon MJ 2014 Adipose tissues and thyroid hormones. Front Physiol 5:479.
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445 [18]. Laurberg P, Andersen S, Karmisholt J 2005 Cold adaptation and thyroid hormone 446 metabolism. Horm Metab Res 37(9):545-9. 447 [19]. Wijers SL, Saris WH, van Marken Lichtenbelt WD 2009 Recent advances in adaptive 448 thermogenesis: potential implications for the treatment of obesity. Obes Rev 10(2):218-26. 449 [20]. Skarulis MC, Celi FS, Mueller E, Zemskova M, Malek R, Hugendubler L, Cochran 450 C, Solomon J, Chen C, Gorden P 2010 Thyroid hormone induced brown adipose tissue and 451 amelioration of diabetes in a patient with extreme insulin resistance. J Clin Endocrinol Metab 452 95(1):256-62. 453 [21]. Lahesmaa M, Orava J, Schalin-Jäntti C, Soinio M, Hannukainen JC, Noponen T, 454 Kirjavainen A, Iida H, Kudomi N, Enerbäck S, Virtanen KA, Nuutila P 2014 Hyperthyroidism 455 increases brown fat metabolism in humans. J Clin Endocrinol Metab 99(1):E28-35. 456 [22]. Zhang Q, Miao Q, Ye H, Zhang Z, Zuo C, Hua F, Guan Y, Li Y 2014 The effects of 457 thyroid hormones on brown adipose tissue in humans: a PET-CT study. Diabetes Metab Res Rev 458 30(6):513-20. 459 [23]. Kim MS, Hu HH, Aggabao PC, Geffner ME, Gilsanz V 2014 Presence of brown adipose 460 tissue in an adolescent with severe primary hypothyroidism. J Clin Endocrinol Metab 461 99(9):E1686-90. 462 [24]. Cypess AM, Chen YC, Sze C, Wang K, English J, Chan O, Holman AR, Tal I, Palmer 463 MR, Kolodny GM, Kahn CR 2012 Cold but not sympathomimetics activates human brown 464 adipose tissue in vivo. Proc Natl Acad Sci USA 109(25): 10001–10005. 465 [25]. Cypess AM, Weiner LS, Roberts-Toler C, Elía EF, Kessler SH, Kahn PA, English 466 J, Chatman K, Trauger SA, Doria A, Kolodny GM 2015 Activation of human brown adipose 467 tissue by a β3-adrenergic receptor agonist. Cell Metab 2015; 21(1):33-8.
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468 [26]. Mullur R, Liu YY, Brent GA 2014 Thyroid hormone regulation of metabolism. Physiol 469 Rev 94(2):355-82. 470 [27]. Bianco AC, McAninch EA 2013 The role of thyroid hormone and brown adipose tissue in 471 energy homoeostasis. Lancet Diabetes Endocrinol 1(3):250-8. 472 [28]. Bianco AC, Maia AL, da Silva WS, Christoffolete MA 2005 Adaptive activation of thyroid 473 hormone and energy expenditure. Biosci Rep 25(3-4):191-208. 474 [29]. Cannon B, Nedergaard J 2004 Brown adipose tissue: function and physiological 475 significance. Physiol Rev 84(1):277-359. 476 [30]. Silva JE, Bianco SD 2008 Thyroid-adrenergic interactions: physiological and clinical 477 implications. Thyroid 18(2):157-65. 478 [31]. Laurberg P, Andersen S, Karmisholt J 2005 Cold adaptation and thyroid hormone 479 metabolism. Horm Metab Res 37(9):545-9. 480 [32]. Solmonson A, Mills EM. 2016 Uncoupling proteins and the molecular mechanisms of 481 thyroid thermogenesis. Endocrinology 157(2):455-62. 482 [33]. Werneck-de-Castro JP, Fonseca TL, Ignacio DL, Fernandes GW, Andrade-Feraud CM, 483 Lartey LJ, Ribeiro MB, Ribeiro MO, Gereben B, Bianco AC 2015 Thyroid hormone signaling 484 in male mouse skeletal muscle is largely independent of D2 in myocytes. Endocrinology 485 156(10):3842-52. 486 [34]. Young JB, Bürgi-Saville ME, Bürgi U, Landsberg L 2005 Sympathetic nervous system 487 activity in rat thyroid: potential role in goitrogenesis. Am J Physiol Endocrinol Metab. 488 288(5):E861-7.
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1 Figure Legends 2 Figure 1. Study Protocol.

Page 26 of 35

3 Figure 2. Changes in thyroid function in individual subjects measured at room temperature and 4 after 60 min of cold exposure (15-16°C) in the hypothyroid and then thyrotoxic state. (A) 5 Changes in serum TSH. (B) Changes in serum free T4. (C) Changes in serum free T3.
6 Figure 3. Changes in BAT volume, activity and SUVmax in individual subjects measured after 7 120 min of cold exposure (15-16°C) in the hypothyroid and then thyrotoxic state. (A) Brown fat 8 FDG uptake in a study subject (53 years old female, BMI 19.6 kg/m2) illustrated using coronal 9 representations of PET in the hypothyroid state (TSH 70.90 µIU/mL) and then thyrotoxic state 10 (TSH 0.03 µIU/mL), the green arrows pointing to the right and left supraclavicular BAT depots. 11 (B) Changes in BAT volume. (C) Changes in BAT activity. (D) Changes in BAT maximum 12 standardized uptake value (SUVmax).
13 Figure 4. Changes in 18F-FDG uptake between the hypothyroid and hyperthyroid state 14 measured in different tissues in individual study subjects. (A) BAT. (B) Liver. (C) Skeletal 15 muscle.
16 Figure 5. Correlations of circulating T3 with BAT and with RMR. (A) Negative correlation 17 between FT3 and BAT in the hypothyroid state. (B) Positive correlation between FT3 and BAT
1

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18 in the hyperthyroid state. (C) Positive correlation between the change in RMR and the change in 19 FT3 between the hypothyroid and hyperthyroid state.
2

Table 1. Baseline characteristics of the study subjects

Mean ± SEM Range

Gender

1M/5F

Age (years)

43.5 ± 4.7

22-53

Weight (kg)
2
Body Mass Index (kg/m )
Baseline TSH (µIU/mL)

75.3 ± 5.7 25.45 ± 2.5 1.36 ± 0.66

60.6-98.8 19.6-35.85 0.19-2.09

Thyroid Cancer TNM Stage

I-IVA

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Table 2. Changes in the subject characteristics measured at room temperature and after cold exposure (15-

16°C) in the hypothyroid and then thyrotoxic state.

Hypothyroid

Room

Thyrotoxic

Characteristic (units)

Temperature

Cold Exposure

Room Temperature Cold Exposure

Temperature (°C)

36.7 ± 0.1

36.2 ± 0.2

36.5 ± 0.1

36.5 ± 0.2

Systolic BP (mmHg)

118.3 ± 4.0

127.6 ± 3.2

118.5 ± 4.3

126.2 ± 5.8

Diastolic BP (mmHg) Heart Rate (bpm) Free Fatty Acids (mEq/L)

75.5 ± 3.5 66.8 ± 3.5 --

a
84.0 ± 2.9
a
60.0 ± 1.5
0.63 ± 0.09

71.2 ± 2.5 64.5 ± 4.4 --

bd
78.7 ± 2.5 65.2 ± 5.7
0.62 ± 0.07

Triglycerides (mg/dL) Total Cholesterol (mg/dL) LDL Cholesterol (mg/dL) HDL Cholesterol (mg/dL)

99.8 ± 28.4 279.7 ± 46.3 173.2 ± 39.6 86.5 ± 6.8

-----

84.2 ± 20.0
c
201.3 ± 24.6
c
110.7 ± 23.3 74.2 ± 6.6

-----

Glucose (mg/dL)

--

81.5 ± 4.8

--

84.7 ± 3.8

Insulin (µU/mL)

6.5 ± 2.0

5.6 ± 1.9

6.4 ± 2.5

5.4 ± 1.6

Hemoglobin A1c (%)

5.6 ± 0.2

--

5.6 ± 0.03

--

TSH (µIU/mL) Total T4 (µg/dL) Free T4 index

60.7 ± 14.9 2.7 ± 1.4 0.77 ± 0.4

60.8 ± 15.0 2.9 ± 1.5 0.81 ± 0.43

c
1.2 ± 1.1
c
10.6 ± 0.8
c
3.8 ± 0.3

d
1.0 ± 0.9
d
11.0 ± 0.9
d
3.9 ± 0.25

Total T3 (ng/dL)

49.7 ± 24.3

53.3 ± 26.1

102.2 ± 14.5

106.0 ± 12.3

Free T3 (pg/mL)

1.2 ± 0.7

1.3 ± 0.8

c
3.2 ± 0.3

3.2 ± 0.2

T3 Uptake (%)

25.8 ± 1.7

25.7 ± 1.5

c
36.2 ± 1.6

d
35.3 ± 1.5

Thyroxin Binding Globulin --

18.2 ± 2.7

--

14.2 ± 1.6

(µg/mL)
Resting Metabolic Rate (Kcal/day)

1299.0 ± 57.7

a
1497.2 ± 106.7

c
1470.0 ± 81.9

b
1612.8 ± 46.9

Respiratory Quotient (RQ) 0.82 ± 0.02

0.80 ± 0.02

0.84 ± 0.03

0.81 ± 0.02

Values represent mean ± SEM. Vitals, laboratory tests and indirect calorimetry were measured after 60 min of cold

exposure, while BAT activity, volume and SUVmax were measured after 120 min of cold exposure at 15-16°C.

a = P ≤ 0.05, hypothyroid state room temperature vs. hypothyroid state after cold exposure.

b = P ≤ 0.05, thyrotoxic state room temperature vs. thyrotoxic state after cold exposure.

c = P ≤ 0.05, hypothyroid state room temperature vs. thyrotoxic state room temperature.

d = P ≤ 0.05, hypothyroid state after cold exposure vs. thyrotoxic state after cold exposure.

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Table 3. Changes in BAT volume, activity and SUVmax measured after 120 min of cold exposure

(15-16°C) in the hypothyroid and thyrotoxic state in the six study subjects.

Mean ± SEM

Range

BAT Volume (mL)

Hypothyroid

72.0 ± 21.0

20 - 150

Thyrotoxic

87.7 ± 16.5

23.5 - 136.9

Change

15.7 ± 13.9

-39.8 - 55.3

% Change

53.35 ± 35.9%

-26.5 - 203.4%

BAT Activity (mL x mean SUV* g/mL)

Hypothyroid

158.1 ± 72.8

23.1 - 495.8

Thyrotoxic

189.0 ± 55.5

27.85 - 398.8

Change

30.9 ± 31.5

-97 – 125.85

% Change

74.35 ± 44.5%

-23.8 - 218.2%

BAT SUVmax (g/mL)

Hypothyroid Thyrotoxic

7.3 ± 2.6 7.9 ± 2.95

2.0 - 19.5 2.1 - 22

Change

0.6 ± 0.95

-2.15 - 3.9

% Change
% Change from the baseline hypothyroid value.

25.6 ± 35.1%

-42.8 – 195%

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190x254mm (96 x 96 DPI)