DIABETICMedicine DOI: 10.1111/dme.12595
Case Report Chronic adrenergic stimulation induces brown adipose tissue differentiation in visceral adipose tissue E. Søndergaard1,2, L. C. Gormsen3, M. H. Christensen3, S. B. Pedersen1, P. Christiansen4, S. Nielsen1, P. L. Poulsen1 and N. Jessen1,5 1 Department of Endocrinology and Internal Medicine, Aarhus University Hospital, 2The Danish Diabetes Academy, 3Department of Nuclear Medicine and positron emission tomography Centre, Aarhus University Hospital, 4Breast and Endocrine Section, Department of Surgery P, Aarhus University Hospital, and 5Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark
Accepted 9 September 2014
Abstract Recruitment of brown adipose tissue is a promising strategy to treat obesity and Type 2 diabetes, but the physiological effects of a large amount of metabolically active brown adipose tissue in humans are unknown.
Background
In the present paper, we report a case of massive brown adipose tissue infiltration of the visceral adipose tissue depot in a person with Type 2 diabetes with a catecholamine-secreting paraganglioma. The patient was evaluated with [18F]-fludeoxyglucose positron emission tomography/computed tomography on three occasions: pre-therapy, during a-blockade and postoperatively. During surgery, biopsies of visceral and subcutaneous adipose tissue were obtained and evaluated for brown adipose tissue. At diagnosis, brown adipose tissue glucose uptake, assessed by [18F]fludeoxyglucose-positron emission tomography, was massively increased. [18F]-fludeoxyglucose uptake was confined to known locations for brown adipose tissue, with additional uptake in the visceral adipose tissue. As a result of increased thermogenesis, resting energy expenditure was doubled. After surgical removal of the tumour, antidiabetic medicine was no longer needed, despite an 8.2-kg weight gain.
Case report
These results show that human visceral adipose tissue holds an unprecedented potential for brown adipogenic differentiation; however, a detrimental effect on glucose metabolism persisted despite massive brown adipose tissue activity, with a doubling of resting energy expenditure.
Conclusion
Diabet. Med. 32, e4–e8 (2015)
Introduction In recent years, the presence and physiological importance of brown adipose tissue has attracted increasing attention [1,2]. It has been hypothesized that recruitment of brown adipose tissue could counteract obesity and its accompanying morbidities, including Type 2 diabetes, but it is not clear whether the potential for brown adipose tissue recruitment in adult humans is sufficient to have a relevant clinical impact on whole-body substrate metabolism [3]. Brown adipose tissue is induced in patients with catecholamine-secreting phaeochromocytoma [1,4–7], and these patients show the extent to which brown adipose tissue recruitment is possible in humans. In these studies, brown adipose tissue recruitment has been in typical locations, such as the neck and mediastinal areas in addition to omental adipose tissue, but the Correspondence to: Niels Jessen. E-mail: [email protected] [The copyright line for this article was changed on 17 February 2016 after original online publication].
e4
amount of induced brown adipose tissue has been limited. In the present study, we report a case of massive infiltration of the visceral adipose depot by brown adipose tissue, associated with a clinically significant increase in thermogenesis and substrate metabolism. Despite brown adipose tissue activation, Type 2 diabetes persisted until removal of the catecholamine-secreting paraganglioma. We evaluated the patient at diagnosis, 2 weeks after initiation of a-blockade and 3 months after surgery with [18F]-fludeoxyglucose (FDG) positron emission tomography/ computed tomography scans to estimate tissue-specific glucose uptake. In addition, a [18F]-L-dihydroxyphenylalanine (DOPA) positron emission tomography scan was performed at diagnosis and 3 months after surgery. Measurement of resting energy expenditure, glucose, free fatty acids and palmitate flux was performed at diagnosis and 3 months after surgery in the morning after an overnight fast. During the surgical procedure, biopsies were obtained from the visceral and subcutaneous adipose tissue for morphological
ª 2014 Diabetes UK
Case report
What’s new? • Increased brown adipose tissue activity cannot prevent a detrimental effect on glucose metabolism in a patient with catecholamine-secreting paraganglioma.
DIABETICMedicine
decreased to 14 g/ml in the perirenal area and to ~3 g/ml in the omental adipose tissue. At follow-up 3 months after surgery, no excess visceral adipose tissue [18F]-FDG uptake
(a)
• Visceral brown adipose tissue differentiation can double resting energy expenditure. • a-adrenergic receptor blockade substantially reduces brown adipose tissue activity. and biochemical characterization. Plasma glucose and free fatty acid levels were measured after an overnight fast. The patient did not take his regular long-acting insulin for 24 h before the measurements. Appendix S1 provides details of the methods applied.
Case report A 61-year-old man with insulin-treated Type 2 diabetes for 4 years, hypertension and chronic obstructive pulmonary disease presented with deteriorating renal function. Computed tomography urography showed a large retroperitoneal tumour, initially suspected to represent a lymphoma, and the patient recalled profuse sweating for the previous 12 months. An [18F]-FDG positron emission tomography/ computed tomography scan performed for suspected lymphoma revealed massive glucose uptake in the visceral adipose tissue, upper chest and neck, suggesting brown adipose tissue differentiation (Fig. 1a,c). [18F]-FDG uptake was markedly increased in the regions normally associated with brown adipose tissue; namely, the neck, periscapular, paravertebral and mediastinal area. In addition, [18F]-FDG uptake was greatly increased below the diaphragm in the perirenal and visceral adipose tissue, as well as the left spermatic cord with the maximum standardized uptake value reaching 23 g/ml (Fig. 1a,c). No excess [18F]-FDG uptake was observed in the subcutaneous adipose tissue. Two weeks after initiation of a-blockade, [18F]-FDG maximum standardized uptake value in visceral adipose tissue had
(b)
(c)
FIGURE 1 (a) Maximum intensity projection [18F]-fludeoxyglucose (FDG)-positron emission tomography images of the patient at diagnosis, during a-blockade and 3 months after surgery. A marked shift in glucose metabolism is evident with initial scans showing massive [18F]-FDG uptake in wide areas of adipose tissue both above and below the diaphragm. a-blockade significantly inhibited [18F]FDG uptake in all areas and glucose metabolism was normalized after tumour removal. (b) [18F]-dihydroxyphenylalanine (DOPA) scans before and after surgery. Only the retroperitoneal tumour (arrow) showed pathological [18F]-DOPA uptake at the initial scan. (c) Transaxial co-registered [18F]-FDG positron emission tomography/ computed tomography images of the periscapular and abdominal region at the initial scan. Intense activity is seen in adipose tissue depots.
ª 2014 Diabetes UK
e5
DIABETICMedicine
was observed and [18F]-FDG maximum standardized uptake value was normalized to 1.5 g/ml in omental fat. As brown adipose tissue differentiation is observed in phaeochromocytoma, an [18F]-DOPA positron emission tomography scan was performed 2 days after the [18F]FDG positron emission tomography/computed tomography scan. 18F-DOPA is an analogue of L-DOPA which is used clinically to trace the dopaminergic pathway in the primary diagnosis of phaeochromocytoma [8]. Two days after the initial [18F]-FDG positron emission tomography scan, an [18F]-DOPA positron emission tomography/computed tomography scan showed intense [18F]-DOPA uptake in the retroperitoneal tumour, consistent with a catecholaminesecreting paraganglioma (Fig. 1b). The diagnosis was confirmed by measurement of greatly elevated plasma-normetanephrine levels (3318 ng/l, normal level< 200 ng/l). Only physiological [18F]-DOPA uptake was observed on the posttherapy control [18F]-DOPA positron emission tomography/ computed tomography scan. Resting energy expenditure at presentation was 184 KJ/ kg/day, more than double the expected resting energy expenditure of 86 KJ/kg/day. The increase was driven by increased thermogenesis. Three months after surgery, the resting energy expenditure had decreased by 50% to 92 KJ/kg/day. The decrease in resting energy expenditure was associated with an increase in body weight from 82.6 to 90.8 kg and an increase in body fat from 27.1 to 30.9%. The increased fat mass was distributed in both the upper and lower body adipose tissue. HbA1c concentration was 50 mmol/mol (6.7%) at diagnosis and decreased to 45 mmol/mol (6.3%) after surgery, despite cessation of an insulin dose of 35 IU a day (Insulatard; Novo Nordisk, Bagsvaerd, Denmark). Fasting plasma glucose concentration fell from 6.6 mmol/l at diagnosis to 5.9 mmol/l 3 months after surgery. Free fatty acid levels decreased from 0.9 mmol/l at the time of diagnosis to 0.2 mmol/l 3 months after surgery. Along with the decrease in resting energy expenditure, the turnover of free fatty acids, measured as palmitate flux, fell from 492 lmol/min at time of diagnosis to 181 lmol/min 3 months after surgery. Adipose tissue biopsies from the visceral adipose depot contained hypercellular capillary-rich areas with a classic multilocular appearance, consistent with brown adipose tissue (Fig. 2a,b). By contrast, subcutaneous adipose tissue contained no multilocular cells (Fig. 2c,d). Uncoupling protein 1 (UCP-1) gene expression was increased ~300-fold in visceral adipose tissue compared with subcutaneous adipose tissue, and the increase in UCP-1 expression in the visceral depot was confirmed at the protein level by immunohistochemical staining of UCP-1 and by western blotting (Fig. 2a, e; Appendix S1). A gene expression array was performed using mRNA isolated from the subcutaneous and visceral adipose tissue from the patient and from a control pool of mRNA isolated from visceral adipose tissue from healthy subjects (Appendix S1).
e6
Adrenergic stimulation and brown fat E. Søndergaard et al.
Discussion The present case report shows that recruitment of large amounts of metabolic active brown adipose tissue is possible in the visceral fat depot in humans. A dysmetabolic condition persisted despite the presence of metabolic active brown adipose tissue and the patient required insulin to regulate glucose levels. After removal of the paraganglioma, glycaemic control improved and the patient was taken off insulin treatment a few days after the surgical procedure. Three months after surgery, the patient did not require antidiabetic medication, despite an 8-kg increase in body weight. The effect of the paraganglioma on glucose metabolism was probably attributable to catecholamine-mediated suppression of insulin secretion [9] and catecholamine-mediated insulin resistance, both directly [10] and as a result of elevated plasma free fatty acids levels caused by increased lipolysis [11]; however, it is evident that the observed brown adipose tissue recruitment had profound effects on whole body metabolism, as it was associated with a doubling of resting energy expenditure, and, despite this hypermetabolic state, the patient maintained normal body weight at the time of diagnosis. This was probably attributable to hyperphagic behaviour, and the weight gain after surgery indicates a habit of increased food intake. As previously reported, up to 90% of excess stored calories have been shown to be deposited as adipose tissue after overeating, explaining the post-surgery increase in fat mass [12]. The hypermetabolic state in phaeochromocytoma has previously been attributed to direct effects of catecholamines on lipolysis and glycogenolysis [13,14], but brown adipose tissue recruitment and activation may contribute substantially. The intriguing concept of brown adipose tissue activation in Type 2 diabetes to induce weight loss has to take into account the balance between stimulation of lipolysis and recruitment of brown adipose tissue when assessing the effects of putative activators of brown adipose tissue activity. In the present case, we observed a massive decrease in [18F]-FDG uptake after initiation of a-blockade with phenoxybenzamine. This could indicate a previously unrecognized role for a-adrenergic signalling in the regulation of brown adipose tissue activity in humans. Despite, the decrease in brown adipose tissue activity, brown adipose tissue was still present in the adipose tissue samples obtained during surgery after 3 weeks a-blockade. This indicates that, although brown adipose tissue activity is short-term regulated, brown adipose tissue differentiation and dedifferentiation occurs over a longer time span. Previously, acute a-adrenergic inhibition has not been observed to affect coldinduced thermogenesis [15]; however, shivering was prominent in this experiment and the effect of a-adrenergic inhibition on brown adipose tissue cannot be estimated. In rodents, the presence of a-adrenergic receptors in brown adipose tissue has been documented [16], and a1-adrenergic stimulation can induce an acute effect on brown adipose
ª 2014 Diabetes UK
Case report
DIABETICMedicine
(a)
(b)
(c)
(d)
(e)
FIGURE 2 (a) Visceral adipose tissue from the omentum stained using anti-Uncoupling protein 1 (UCP-1) antibody. The tissue shows capillary-rich areas with large amounts of cells staining positive for UCP1 and a classic multilocular appearance that resembles brown adipocytes. (b) Visceral adipose tissue stained without primary antibody against UCP1 (negative control). (c) Immunohistochemical staining of subcutaneous adipose tissue from the abdominal region using antibody against UCP1 showed no UCP1 positive cells and did not differ from sections stained without UCP1 antibodies. (d) Subcutaneous adipose tissue stained without primary antibody against UCP1 (negative control). (e) Western blot using anti-UCP1 antibody shows a clear band ~33 kDa in lanes A, rat brown adipose tissue from the interscapular area (positive control), B, omental fat and C, subcutaneous tissue. Stain-Free intensities of the presented western blot is shown in Figure S1.
tissue thermogenesis [17], whereas a2-adrenergic stimulation has been shown to decrease brown adipose tissue thermogenesis [18]. Phenoxybenzamine blocks both a1 and a2 receptors, but, in light of the rodent studies, the effects observed are most likely to be mediated through the a1 receptor. Further investigation of the effects of a-adrenergic stimulation of brown adipose tissue activity in humans appears attractive. In comparison, b-adrenergic stimulation with ephedrine have only shown a modest increase in [18F]FDG uptake in brown adipose tissue compared with coldinduced activation of brown adipose tissue [19], and others have failed to see any effect of b-adrenergic stimulation using comparable designs [4,5]. In the present case, we observed selective brown adipose tissue recruitment in visceral, but not subcutaneous adipose tissue. This could indicate that brown adipose tissue cannot be recruited in subcutaneous adipose tissue because of an absence of brown adipocyte progenitor cells. Notably,
ª 2014 Diabetes UK
autopsy data have shown that subcutaneous, but not visceral, brown adipocytes are lost after the first decade of life [20]. This underlines the fact that there are striking differences between subcutaneous and visceral fat depots, which must be accounted for in investigations on brown adipose tissue. In summary, we report a case of massive brown adipose tissue recruitment associated with chronic adrenergic stimulation in a patient with a noradrenaline-secreting paraganglioma. The increased brown adipose tissue activity was associated with a substantive increase in resting energy expenditure and thermogenesis. Treatment with phenoxybenzamine reduced brown adipose tissue activity to near normal levels, showing a previously unrecognized role of a-adrenergic receptors in the regulation of human brown adipose tissue activity. This case demonstrates a great potential for brown adipose tissue recruitment in humans and suggests that stimulation of brown adipose tissue
e7
DIABETICMedicine
differentiation holds promise in the treatment of obesity. Nevertheless, poor glycaemic control and Type 2 diabetes can persist, despite massive catecholamine-stimulated visceral brown adipose tissue activity. Funding sources
None. Competing interests
None declared.
References 1 Cypess AM, Lehman S, Williams G, Tal I, Rodman D, Goldfine AB et al. Identification and importance of brown adipose tissue in adult humans. N Engl J Med 2009; 360: 1509–1517. 2 Wu J, Cohen P, Spiegelman BM. Adaptive thermogenesis in adipocytes: Is beige the new brown? Genes Dev 2013; 27: 234–250. 3 Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev 2004; 84: 277–359. 4 Cypess AM, Chen YC, Sze C, Wang K, English J, Chan O et al. Cold but not sympathomimetics activates human brown adipose tissue in vivo. Proc Natl Acad Sci U S A 2012; 109: 10001–10005. 5 Vosselman MJ, van der Lans AAJJ, Brans B, Wierts R, van Baak MA, Schrauwen P et al. Systemic ß-adrenergic stimulation of thermogenesis is not accompanied by brown adipose tissue activity in humans. Diabetes 2012; 61: 3106–3113. 6 Lean ME, James WP, Jennings G, Trayhurn P. Brown adipose tissue in patients with phaeochromocytoma. Int J Obes 1986; 10: 219–227. 7 Frontini A, Vitali A, Perugini J, Murano I, Romiti C, Ricquier D et al. White-to-brown transdifferentiation of omental adipocytes in patients affected by pheochromocytoma. 1831; 2013: 950–959. 8 Timmers HJLM, Chen CC, Carrasquillo JA, Whatley M, Ling A, Havekes B et al. Comparison of 18F-Fluoro-L-DOPA, 18FFluoro-Deoxyglucose, and 18F-Fluorodopamine PET and 123I-MIBG Scintigraphy in the Localization of Pheochromocytoma and Paraganglioma. J Clin Endocrinol Metab 2009; 94: 4757–4767. 9 Hamaji M. Pancreatic a- and ß-Cell Function in Pheochromocytoma. J Clin Endocrinol Metab 1979; 49: 322–325.
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Adrenergic stimulation and brown fat E. Søndergaard et al. 10 Turnbull DM, Johnston DG, Alberti KGMM, Hall R. Hormonal and metabolic studies in a patient with a pheochromocytoma. J Clin Endocrinol Metab 1980; 51: 930–933. 11 Baron AD, Olefsky JM, Wallace P. In Vivo regulation of noninsulin-mediated and insulin-mediated glucose uptake by epinephrine. J Clin Endocrinol Metab 1987; 64: 889–895. 12 Bray GA, Smith SR, de Jonge L, Xie H, Rood J, Martin CK. Effect of dietary protein content on weight gain, energy expenditure, and body composition during overeating: a randomized controlled trial. JAMA 2012; 307: 47–55. 13 Petrak O, Haluzikova D, Kavalkova P, Strauch B, Rosa J, Holaj R et al. Changes in Energy Metabolism in Pheochromocytoma. J Clin Endocrinol Metab 2013; 98: 1651–1658. 14 Lenders JW, Eisenhofer G, Mannelli M, Pacak K. Phaeochromocytoma. 2005; 366: 665–675. 15 Frank SM, El-Gamal N, Raja SN, Wu PK. a-Adrenoceptor mechanisms of thermoregulation during cold challenge in humans. Clin Sci 1996; 91: 627–631. 16 Mohell N, Svatengren J, Cannon B. Identification of [3H]prazosin binding sites in crude membranes and isolated cells of brown adipose tissue as a1-adrenergic receptors. Eur J Pharmacol 1983; 92: 15–25. 17 Zhao J, Cannon B, Nedergaard J. a1-Adrenergic Stimulation Potentiates the Thermogenic Action of ß3-Adrenoreceptorgenerated cAMP in Brown Fat Cells. J Biol Chem 1997; 272: 32847–32856. 18 Madden CJ, Tupone D, Cano G, Morrison SF. a2 Adrenergic Receptor-Mediated Inhibition of Thermogenesis. J Neurosci 2013; 33: 2017–2028. 19 Carey AL, Formosa MF, Every B, Bertovic D, Eikelis N, Lambert GW et al. Ephedrine activates brown adipose tissue in lean but not obese humans. 2013; 56: 147–155. 20 Heaton JM. The distribution of brown adipose tissue in the human. J Anat 1972; 112: 35–39.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. The stain-free intensities of the presented Western blot. Appendix S1. Material and Methods section.
ª 2014 Diabetes UK
Case Report Chronic adrenergic stimulation induces brown adipose tissue differentiation in visceral adipose tissue E. Søndergaard1,2, L. C. Gormsen3, M. H. Christensen3, S. B. Pedersen1, P. Christiansen4, S. Nielsen1, P. L. Poulsen1 and N. Jessen1,5 1 Department of Endocrinology and Internal Medicine, Aarhus University Hospital, 2The Danish Diabetes Academy, 3Department of Nuclear Medicine and positron emission tomography Centre, Aarhus University Hospital, 4Breast and Endocrine Section, Department of Surgery P, Aarhus University Hospital, and 5Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark
Accepted 9 September 2014
Abstract Recruitment of brown adipose tissue is a promising strategy to treat obesity and Type 2 diabetes, but the physiological effects of a large amount of metabolically active brown adipose tissue in humans are unknown.
Background
In the present paper, we report a case of massive brown adipose tissue infiltration of the visceral adipose tissue depot in a person with Type 2 diabetes with a catecholamine-secreting paraganglioma. The patient was evaluated with [18F]-fludeoxyglucose positron emission tomography/computed tomography on three occasions: pre-therapy, during a-blockade and postoperatively. During surgery, biopsies of visceral and subcutaneous adipose tissue were obtained and evaluated for brown adipose tissue. At diagnosis, brown adipose tissue glucose uptake, assessed by [18F]fludeoxyglucose-positron emission tomography, was massively increased. [18F]-fludeoxyglucose uptake was confined to known locations for brown adipose tissue, with additional uptake in the visceral adipose tissue. As a result of increased thermogenesis, resting energy expenditure was doubled. After surgical removal of the tumour, antidiabetic medicine was no longer needed, despite an 8.2-kg weight gain.
Case report
These results show that human visceral adipose tissue holds an unprecedented potential for brown adipogenic differentiation; however, a detrimental effect on glucose metabolism persisted despite massive brown adipose tissue activity, with a doubling of resting energy expenditure.
Conclusion
Diabet. Med. 32, e4–e8 (2015)
Introduction In recent years, the presence and physiological importance of brown adipose tissue has attracted increasing attention [1,2]. It has been hypothesized that recruitment of brown adipose tissue could counteract obesity and its accompanying morbidities, including Type 2 diabetes, but it is not clear whether the potential for brown adipose tissue recruitment in adult humans is sufficient to have a relevant clinical impact on whole-body substrate metabolism [3]. Brown adipose tissue is induced in patients with catecholamine-secreting phaeochromocytoma [1,4–7], and these patients show the extent to which brown adipose tissue recruitment is possible in humans. In these studies, brown adipose tissue recruitment has been in typical locations, such as the neck and mediastinal areas in addition to omental adipose tissue, but the Correspondence to: Niels Jessen. E-mail: [email protected] [The copyright line for this article was changed on 17 February 2016 after original online publication].
e4
amount of induced brown adipose tissue has been limited. In the present study, we report a case of massive infiltration of the visceral adipose depot by brown adipose tissue, associated with a clinically significant increase in thermogenesis and substrate metabolism. Despite brown adipose tissue activation, Type 2 diabetes persisted until removal of the catecholamine-secreting paraganglioma. We evaluated the patient at diagnosis, 2 weeks after initiation of a-blockade and 3 months after surgery with [18F]-fludeoxyglucose (FDG) positron emission tomography/ computed tomography scans to estimate tissue-specific glucose uptake. In addition, a [18F]-L-dihydroxyphenylalanine (DOPA) positron emission tomography scan was performed at diagnosis and 3 months after surgery. Measurement of resting energy expenditure, glucose, free fatty acids and palmitate flux was performed at diagnosis and 3 months after surgery in the morning after an overnight fast. During the surgical procedure, biopsies were obtained from the visceral and subcutaneous adipose tissue for morphological
ª 2014 Diabetes UK
Case report
What’s new? • Increased brown adipose tissue activity cannot prevent a detrimental effect on glucose metabolism in a patient with catecholamine-secreting paraganglioma.
DIABETICMedicine
decreased to 14 g/ml in the perirenal area and to ~3 g/ml in the omental adipose tissue. At follow-up 3 months after surgery, no excess visceral adipose tissue [18F]-FDG uptake
(a)
• Visceral brown adipose tissue differentiation can double resting energy expenditure. • a-adrenergic receptor blockade substantially reduces brown adipose tissue activity. and biochemical characterization. Plasma glucose and free fatty acid levels were measured after an overnight fast. The patient did not take his regular long-acting insulin for 24 h before the measurements. Appendix S1 provides details of the methods applied.
Case report A 61-year-old man with insulin-treated Type 2 diabetes for 4 years, hypertension and chronic obstructive pulmonary disease presented with deteriorating renal function. Computed tomography urography showed a large retroperitoneal tumour, initially suspected to represent a lymphoma, and the patient recalled profuse sweating for the previous 12 months. An [18F]-FDG positron emission tomography/ computed tomography scan performed for suspected lymphoma revealed massive glucose uptake in the visceral adipose tissue, upper chest and neck, suggesting brown adipose tissue differentiation (Fig. 1a,c). [18F]-FDG uptake was markedly increased in the regions normally associated with brown adipose tissue; namely, the neck, periscapular, paravertebral and mediastinal area. In addition, [18F]-FDG uptake was greatly increased below the diaphragm in the perirenal and visceral adipose tissue, as well as the left spermatic cord with the maximum standardized uptake value reaching 23 g/ml (Fig. 1a,c). No excess [18F]-FDG uptake was observed in the subcutaneous adipose tissue. Two weeks after initiation of a-blockade, [18F]-FDG maximum standardized uptake value in visceral adipose tissue had
(b)
(c)
FIGURE 1 (a) Maximum intensity projection [18F]-fludeoxyglucose (FDG)-positron emission tomography images of the patient at diagnosis, during a-blockade and 3 months after surgery. A marked shift in glucose metabolism is evident with initial scans showing massive [18F]-FDG uptake in wide areas of adipose tissue both above and below the diaphragm. a-blockade significantly inhibited [18F]FDG uptake in all areas and glucose metabolism was normalized after tumour removal. (b) [18F]-dihydroxyphenylalanine (DOPA) scans before and after surgery. Only the retroperitoneal tumour (arrow) showed pathological [18F]-DOPA uptake at the initial scan. (c) Transaxial co-registered [18F]-FDG positron emission tomography/ computed tomography images of the periscapular and abdominal region at the initial scan. Intense activity is seen in adipose tissue depots.
ª 2014 Diabetes UK
e5
DIABETICMedicine
was observed and [18F]-FDG maximum standardized uptake value was normalized to 1.5 g/ml in omental fat. As brown adipose tissue differentiation is observed in phaeochromocytoma, an [18F]-DOPA positron emission tomography scan was performed 2 days after the [18F]FDG positron emission tomography/computed tomography scan. 18F-DOPA is an analogue of L-DOPA which is used clinically to trace the dopaminergic pathway in the primary diagnosis of phaeochromocytoma [8]. Two days after the initial [18F]-FDG positron emission tomography scan, an [18F]-DOPA positron emission tomography/computed tomography scan showed intense [18F]-DOPA uptake in the retroperitoneal tumour, consistent with a catecholaminesecreting paraganglioma (Fig. 1b). The diagnosis was confirmed by measurement of greatly elevated plasma-normetanephrine levels (3318 ng/l, normal level< 200 ng/l). Only physiological [18F]-DOPA uptake was observed on the posttherapy control [18F]-DOPA positron emission tomography/ computed tomography scan. Resting energy expenditure at presentation was 184 KJ/ kg/day, more than double the expected resting energy expenditure of 86 KJ/kg/day. The increase was driven by increased thermogenesis. Three months after surgery, the resting energy expenditure had decreased by 50% to 92 KJ/kg/day. The decrease in resting energy expenditure was associated with an increase in body weight from 82.6 to 90.8 kg and an increase in body fat from 27.1 to 30.9%. The increased fat mass was distributed in both the upper and lower body adipose tissue. HbA1c concentration was 50 mmol/mol (6.7%) at diagnosis and decreased to 45 mmol/mol (6.3%) after surgery, despite cessation of an insulin dose of 35 IU a day (Insulatard; Novo Nordisk, Bagsvaerd, Denmark). Fasting plasma glucose concentration fell from 6.6 mmol/l at diagnosis to 5.9 mmol/l 3 months after surgery. Free fatty acid levels decreased from 0.9 mmol/l at the time of diagnosis to 0.2 mmol/l 3 months after surgery. Along with the decrease in resting energy expenditure, the turnover of free fatty acids, measured as palmitate flux, fell from 492 lmol/min at time of diagnosis to 181 lmol/min 3 months after surgery. Adipose tissue biopsies from the visceral adipose depot contained hypercellular capillary-rich areas with a classic multilocular appearance, consistent with brown adipose tissue (Fig. 2a,b). By contrast, subcutaneous adipose tissue contained no multilocular cells (Fig. 2c,d). Uncoupling protein 1 (UCP-1) gene expression was increased ~300-fold in visceral adipose tissue compared with subcutaneous adipose tissue, and the increase in UCP-1 expression in the visceral depot was confirmed at the protein level by immunohistochemical staining of UCP-1 and by western blotting (Fig. 2a, e; Appendix S1). A gene expression array was performed using mRNA isolated from the subcutaneous and visceral adipose tissue from the patient and from a control pool of mRNA isolated from visceral adipose tissue from healthy subjects (Appendix S1).
e6
Adrenergic stimulation and brown fat E. Søndergaard et al.
Discussion The present case report shows that recruitment of large amounts of metabolic active brown adipose tissue is possible in the visceral fat depot in humans. A dysmetabolic condition persisted despite the presence of metabolic active brown adipose tissue and the patient required insulin to regulate glucose levels. After removal of the paraganglioma, glycaemic control improved and the patient was taken off insulin treatment a few days after the surgical procedure. Three months after surgery, the patient did not require antidiabetic medication, despite an 8-kg increase in body weight. The effect of the paraganglioma on glucose metabolism was probably attributable to catecholamine-mediated suppression of insulin secretion [9] and catecholamine-mediated insulin resistance, both directly [10] and as a result of elevated plasma free fatty acids levels caused by increased lipolysis [11]; however, it is evident that the observed brown adipose tissue recruitment had profound effects on whole body metabolism, as it was associated with a doubling of resting energy expenditure, and, despite this hypermetabolic state, the patient maintained normal body weight at the time of diagnosis. This was probably attributable to hyperphagic behaviour, and the weight gain after surgery indicates a habit of increased food intake. As previously reported, up to 90% of excess stored calories have been shown to be deposited as adipose tissue after overeating, explaining the post-surgery increase in fat mass [12]. The hypermetabolic state in phaeochromocytoma has previously been attributed to direct effects of catecholamines on lipolysis and glycogenolysis [13,14], but brown adipose tissue recruitment and activation may contribute substantially. The intriguing concept of brown adipose tissue activation in Type 2 diabetes to induce weight loss has to take into account the balance between stimulation of lipolysis and recruitment of brown adipose tissue when assessing the effects of putative activators of brown adipose tissue activity. In the present case, we observed a massive decrease in [18F]-FDG uptake after initiation of a-blockade with phenoxybenzamine. This could indicate a previously unrecognized role for a-adrenergic signalling in the regulation of brown adipose tissue activity in humans. Despite, the decrease in brown adipose tissue activity, brown adipose tissue was still present in the adipose tissue samples obtained during surgery after 3 weeks a-blockade. This indicates that, although brown adipose tissue activity is short-term regulated, brown adipose tissue differentiation and dedifferentiation occurs over a longer time span. Previously, acute a-adrenergic inhibition has not been observed to affect coldinduced thermogenesis [15]; however, shivering was prominent in this experiment and the effect of a-adrenergic inhibition on brown adipose tissue cannot be estimated. In rodents, the presence of a-adrenergic receptors in brown adipose tissue has been documented [16], and a1-adrenergic stimulation can induce an acute effect on brown adipose
ª 2014 Diabetes UK
Case report
DIABETICMedicine
(a)
(b)
(c)
(d)
(e)
FIGURE 2 (a) Visceral adipose tissue from the omentum stained using anti-Uncoupling protein 1 (UCP-1) antibody. The tissue shows capillary-rich areas with large amounts of cells staining positive for UCP1 and a classic multilocular appearance that resembles brown adipocytes. (b) Visceral adipose tissue stained without primary antibody against UCP1 (negative control). (c) Immunohistochemical staining of subcutaneous adipose tissue from the abdominal region using antibody against UCP1 showed no UCP1 positive cells and did not differ from sections stained without UCP1 antibodies. (d) Subcutaneous adipose tissue stained without primary antibody against UCP1 (negative control). (e) Western blot using anti-UCP1 antibody shows a clear band ~33 kDa in lanes A, rat brown adipose tissue from the interscapular area (positive control), B, omental fat and C, subcutaneous tissue. Stain-Free intensities of the presented western blot is shown in Figure S1.
tissue thermogenesis [17], whereas a2-adrenergic stimulation has been shown to decrease brown adipose tissue thermogenesis [18]. Phenoxybenzamine blocks both a1 and a2 receptors, but, in light of the rodent studies, the effects observed are most likely to be mediated through the a1 receptor. Further investigation of the effects of a-adrenergic stimulation of brown adipose tissue activity in humans appears attractive. In comparison, b-adrenergic stimulation with ephedrine have only shown a modest increase in [18F]FDG uptake in brown adipose tissue compared with coldinduced activation of brown adipose tissue [19], and others have failed to see any effect of b-adrenergic stimulation using comparable designs [4,5]. In the present case, we observed selective brown adipose tissue recruitment in visceral, but not subcutaneous adipose tissue. This could indicate that brown adipose tissue cannot be recruited in subcutaneous adipose tissue because of an absence of brown adipocyte progenitor cells. Notably,
ª 2014 Diabetes UK
autopsy data have shown that subcutaneous, but not visceral, brown adipocytes are lost after the first decade of life [20]. This underlines the fact that there are striking differences between subcutaneous and visceral fat depots, which must be accounted for in investigations on brown adipose tissue. In summary, we report a case of massive brown adipose tissue recruitment associated with chronic adrenergic stimulation in a patient with a noradrenaline-secreting paraganglioma. The increased brown adipose tissue activity was associated with a substantive increase in resting energy expenditure and thermogenesis. Treatment with phenoxybenzamine reduced brown adipose tissue activity to near normal levels, showing a previously unrecognized role of a-adrenergic receptors in the regulation of human brown adipose tissue activity. This case demonstrates a great potential for brown adipose tissue recruitment in humans and suggests that stimulation of brown adipose tissue
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DIABETICMedicine
differentiation holds promise in the treatment of obesity. Nevertheless, poor glycaemic control and Type 2 diabetes can persist, despite massive catecholamine-stimulated visceral brown adipose tissue activity. Funding sources
None. Competing interests
None declared.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. The stain-free intensities of the presented Western blot. Appendix S1. Material and Methods section.
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