WO2009067501A2

WO2009067501A2 - Infrared thermography for monitoring brown adipose tissue

Info

Publication number: WO2009067501A2
Application number: PCT/US2008/084010
Authority: WO
Inventors: Aaron M. Cypess
Original assignee: Joslin Diabetes Center, Inc.
Priority date: 2007-11-19
Filing date: 2008-11-19
Publication date: 2009-05-28
Also published as: WO2009067501A3

Abstract

Methods for evaluating BAT, e.g., BAT function or mass, in humans using IR thermography are described.

Description

Infrared Thermography for Monitoring Brown Adipose Tissue

CLAIM OF PRIORITY

This application claims the benefit of U.S. Patent Application Serial No. 61/003,803, filed on November 19, 2007, the entire contents of which are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos. DK070722 and R01DK33201 awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

This invention relates to methods for using infrared thermography to evaluate brown adipose tissue and its impact on metabolism.

BACKGROUND

Obesity, and disorders associated with obesity such as diabetes, are a major global health concern. Obesity, which is generally associated with an abnormal accumulation of fat cells, develops when energy intake exceeds energy expenditure. Adipose tissues play an important role in obesity, insulin resistance and diabetes. Two functionally different types of fat tissues are present in mammals: white adipose tissue (WAT), which is the primary site of depot of triglycerides and release of fatty acids, and brown adipose tissue (BAT), which is specialized in thermogenic energy expenditure through the expression of uncoupling protein- 1 (UCP-I).

The most commonly known fat cells are white fat cells, also known as white adipose tissue (WAT) cells, which have a thin ring of cytoplasm surrounding a lipid or fat droplet. WAT is found underneath the skin and provides heat insulation, cushioning against shock and jarring, and energy reserves. An average lean person has roughly 20 to 40 billion WAT cells. An obese person can have up to ten times more WAT than the average lean person. The less common fat cells are the brown fat cells, also known as brown adipose tissue (BAT) cells. Energy expenditure for thermogenesis in BAT serves either to maintain body temperature in the cold or to waste food energy. It has roles in thermal balance and energy balance, and when defective, is usually associated with obesity. BAT is typically atrophied in obese animals. The importance of BAT in overall energy homeostasis is underscored by the finding that ablation of BAT in mice results in severe obesity accompanied by insulin resistance, hyperglycemia, hyperlipidemia, and hypercholesterolemia (Lowell at al, Nature 366(6457):740-2 (1993); Hamann et al, Diabetes. 44(11): 1266-73 (1995); Hamann et al., Endocrinology 137(l):21-9 (1996). Increasing the relative proportion and function of BAT may increase whole body energy expenditure, preventing the development of obesity. In fact, the role of BAT as a defense against obesity has been clearly demonstrated through targeted ablation of this tissue in mice and the BAT-less mice become more susceptible to diet-induced obesity, diabetes, and hyperlipidemia (Lowell et al., Nature 366:740-742 (1993); Hamann et al., Endocrinology 137:21-29 (1996).

BAT also features the presence of abundant and large mitochondria (Nedergaard et al., in Brown Adipose Tissue, Trayhurn and Nicholls, Eds. (Edward Arnold, Baltimore, 1986)), which serve as the center site for oxidative phosphorylation, intermediary metabolism, adaptive thermogenesis, generation of reactive oxygen species and apoptosis. In BAT, mitochondrial biogenesis has been long known to accompany brown adipocyte differentiation. During the past decade, it has become increasingly evident that the integrity of mitochondria contribute to a variety of human diseases, including obesity, diabetes, cancer, neurodegeneration, and aging (Duchen, Diabetes 53 (Suppl l):S96-102 (2004); Taylor and Turnbull, Nat. Rev. Genet. 6:389-402 (2005); Lowell and Shulman, Science 307:384-387 (2005)).

Adipose tissues contain a potential mitotic compartment, which can allow for growth and differentiation of WAT or BAT cells. Adipose tissue can be readily assayed using routine techniques. An exemplary assay for adipose cells is the Oil Red O lipophilic red dye assay. The dye is used to stain neutral lipids in cells. The amount of staining is directly proportional to the amount of lipid in the cell and can be measured spectrophotometrically. The amount of lipid accumulation is determined as a parameter of differentiation. WAT and BAT can be distinguished by routine techniques, e.g., morphologic changes specific to WAT or BAT, or evaluation of WAT-specific or BAT- specific markers. For example, BAT cells can be identified by expression of uncoupling protein (UCP), e.g., UCP-I.

SUMMARY

The United States and most of the world's countries are in the midst of an epidemic of obesity and diabetes that is growing into a global public health emergency. Worldwide there are more than 1.1 billion adults who are overweight, and there are at least 155 million children worldwide are overweight or obese. Because obesity often leads to diabetes, cardiovascular disease, stroke, and other health problems, being overweight may account for up to 16% of the global burden of disease (expressed as a percentage of disability-adjusted life-years), and that 2-7% of health care costs in developed nations are attributable to obesity (Hossain et al., (2007) New Eng J Med. 356:213-215). Estimates of the market for anti-obesity pharmaceuticals products vary, but most published reports estimate the world market at somewhere near $1 billion/year (reportbuyer.com/pharma_healthcare/therapeutic/obesity/anti_obesity_treatments_2007_ 2012_blockbuster_target_market.html). These markets will certainly grow as a greater percentage of the population becomes overweight or obese. In spite of the enormous market potential, the development of pharmaceutical products for obesity has not yet been particularly fruitful. There continues to be an urgent need for obesity and diabetes treatments, but existing pharmaceuticals and other products are not currently meeting this need.

Brown adipose tissue (BAT) affects whole-body metabolism, capable of altering insulin sensitivity and modifying insulin secretion (Guerra et al., (2001) J Clin Invest 108, 1205-1213). However, in spite of its potential physiological impact, there are currently no non-invasive methods to evaluate BAT mass or function. The few previous attempts to use thermography for this purpose have not reached the proof-of-concept phase (Rothwell and Stock, (1979) Nature 281 :31-5; Astrup et al., (1984) Clin Sci (Lond) 66:179-86; Astrup, (1986) Acta Endocrinol Suppl 278:1-32; Satomura et al., (2001) Metabolism. 50:1181-5). Therefore, there are currently no known documented systematic, protocol-based uses of IR thermography to monitor BAT mass and function.

In one aspect, the invention provides methods for evaluating brown adipose tissue (BAT) function or mass in a human. The methods include ensuring that the ambient room temperature is below 70 ⁰F; setting a region of interest (ROI) that covers the cervical, supraclavicular, and superior mediastinal areas of the human; scanning the region of interest with an infrared camera to obtain an infrared thermographic image of the region of interest in the human; and processing the image to provide a measure of temperature information. The temperature information provides information regarding BAT mass or function in the human, e.g., the temperature information is correlated to BAT mass or function, thus the method can include a step of correlating the temperature information to BAT mass or function. This correlation can be used to produce a value, e.g., a score, that is descriptive of the BAT mass or function in subject (i.e., in the ROI in the subject). The value can be compared to a reference value, e.g., a reference value that represents a predetermined amount of BAT mass or function, e.g., an ideal amount, an overabundance, or a dearth of BAT mass or function.

Thus in some embodiments the methods can further include comparing the measure of the temperature information to a predetermined value. In another example, the predetermined value can be a measure of BAT mass or function in the ROI of either the same subject or the same ROI in one or more control subjects.

In some embodiments, the predetermined value is a measure of BAT mass or function in the ROI of the same subject at a different time of the year.

In some embodiments, the predetermined value is a measure of BAT function in the ROI of the same subject before or after administration of a test compound; the comparison can be used to identify an effect of the test compound on BAT mass or function.

In some embodiments, the methods include providing a dose of a sympathomimetic drug such as ephedrine, BMP-7, or a small molecule mimetic of BMP- 7, e.g., to induce increased blood flow to BAT or to increase BAT mass or activity, and thereby increase its thermogenic capacity, prior to scanning the selected anatomical structures. In some embodiments, the methods include ensuring that the study subject has not taken any of the following classes of medications or nutritional products shown to reduce BAT glucose uptake or activity: sympatholytics such as α- and β-adrenergic receptor blockers, anxiolytics such as benzodiazepines, anti-thyroid medications. The methods can include instructing the subject not to take any of those medications or nutritional products.

In some embodiments, the scanning is preferentially performed during a month wherein the average temperature is less than about 60°, e.g., less than about 55°, 50°, 45°, or 40°.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is an IR thermographic image of female 129 mice. The two mice were treated i.p. for 6 days with either lmg/kg β3-adrenergic agonist CL-316,243 (top) or vehicle (bottom) and then once more 30-60 minutes before thermographic imaging. Average surface temperature of the regions (rectangles) overlying the interscapular BAT is labeled.

FIG. 2 is an IR thermographic image of two adult humans. A 25 year old human female (left) and a 21 year old human male (right) applied to their skins packs of either hot (filled arrows) or cold (open arrows). Temperatures are indicated by the color scale on the right. The absolute difference in average surface temperature between analogous interscapular sites (boxes) is indicated on the top of the image.

FIG. 3 is a graph illustrating the temperature dependence of maximal BAT activity, showing a correlation between BAT (triangles) and average outdoor temperature (bars).

FIG. 4 is a plot showing that the rate of maximal BAT decreased with increasing outdoor temperature. This effect persisted over the three year study period.

FIG. 5 A is a set of four photographs showing that BAT volume and activity can be quantified using a preselected ROI. In the left-most images, regions of interest (red outline) include the principal cervical, supraclavicular, and superior mediastinal BAT depots. In the right panel, the PET/CT Viewer software is shown quantifying BAT volume and activity using the tool.

FIG. 5B is a 3-dimensional reproduction of the principal BAT depots in a 60 year- old woman who underwent PET/CT scanning for surveillance of thyroid cancer. The green arrows are pointing to the BAT, which is colored light yellow in this image.

FIGs. 5C-i-iv and 5D-i-iv show via PET/CT imaging the location of the principal adult human BAT depots in a woman (5C) and a man (5D). Going from left to right are the coronal images, attenuation corrected PET images, CT images, and on the right is the fused PET/CT image. Fig. 5C, a 60 year-old woman is evaluated for thyroid cancer. Fig. 5D, a 32 year old male is re-staged for non-hodgkins lymphoma. 5C-i and 5D-i, attenuation-corrected coronal PET images; 5C-ii and 5D-ii, attenuation corrected axial PET images; 5C-iii and 5D-iii, CT images; 5C-iv and 5D-iv, fused PET/CT images. Note that the FDG uptake corresponds to fat density on the CT (green arrows).

FIGs. 6A-6C are bar graphs showing BAT rate, volume, and activity show a sexual dimorphism. 6A shows the rates of definitive BAT after patients were subcategorized by sex. 6B and 6C show, respectively, BAT volume in mL and BAT activity in mL*SUVmean. For both panels 6B and 6C, the box-plots of males and females indicate the lower quartile (lower line of boxes), median (middle line of boxes), upper quartile (upper line of boxes), 1.5x interquartile range (lower and upper whiskers), and outliers (circles). FIG. 7 is a graph showing the relationship between BAT expression, and anthropometric and metabolic factors. The BAT-negative and BAT-positive patients were grouped together into one series and then divided into tertiles of age, BMI, and fasting glucose. Multivariate logistic analysis with the corresponding odds ratios and 95% confidence intervals are shown.

FIGs. 8A-8D are photomicrographs showing the results of immunohistochemistry experiments demonstrating the presence of BAT in a typical supraclavicular depot. A 48 year-old woman underwent a parathyroidectomy, and the surrounding tan-yellow tissue was submitted as part of routine for permanent section. Histological analysis showed the presence of brown adipocytes as stained by either hematoxylin and eosin (8 A and 8C) or antibody to UCP-I (1 :50) and counterstained with hematoxylin in 8B and 8D. Magnifications are at 200X (8A and 8B). Panels 8C and 8D show the regions identified by the black arrowheads at 600X.

DETAILED DESCRIPTION

The present invention is based, at least in part, on the development of a noninvasive modality that can evaluate BAT mass or function. Described herein are new methods for the use of infrared (IR) thermography to quantify brown adipose tissue (BAT) mass and thereby monitor the both the potential and effectiveness of treatments for obesity and diabetes. BAT is specialized for energy expenditure and thermogenesis through high levels of glucose uptake, numerous mitochondria, and the expression of the unique uncoupling protein 1 (UCP-I) that dissipates the protonmotive force, generating heat. BAT affects whole-body metabolism, capable of altering insulin sensitivity and modifying pancreatic β-cell function. In mice, different diets and food additives can affect BAT activity and prevention of obesity. It was recently shown that thermogenic ectopic deposits of BAT in intermuscular depots provide a genetically based mechanism for protection from weight gain and metabolic syndrome between the 129Sv and C57B1/6 strains of mice (Almind et al. (2007) Proc Natl Acad Sci U S A. 104:2366-2371).

In humans, BAT involutes after birth and had been thought to be metabolically irrelevant in the adult. Nevertheless, there now is increasing evidence of BAT in adults in both the normal and pathological states. Although it is unclear what role BAT might play in adult human energy balance, it has been estimated that as little as 50 g of BAT could, if maximally stimulated, account for up to 20% of daily energy expenditure (Rothwell and Stock, (1983) Clin Sci (Lond.) 64, 19-23.

Characterizing BAT mass, function, and induction in humans has great potential for measuring whole -body metabolism and ultimately assessing different treatments of obesity and diabetes.

General methods for using IR thermography are known in the art, see, e.g., U.S. Pat. Nos. 7,277,744; 6,983,753; and 6,881,584. IR thermographic cameras are also known and are commercially available, e.g., the ThermaCAM® EX320, available from FLIR Systems, Inc., North Billerica, MA.

The few previous attempts to use thermography for evaluating BAT did not demonstrate proof-of-concept (see Rothwell and Stock, (1979) Nature 281 :31-5; Astrup et al, (1984) Clin Sci (Lond) 66:179-86; Astrup, (1986) Acta Endocrinol Suppl 278:1-32; Satomura et al., (2001) Metabolism. 50:1181-5). Therefore, there are currently no known documented systematic, protocol-based uses of IR thermography to monitor BAT mass and function.

The new uses for IR thermography described herein include two general categories: (1) to evaluate BAT mass and function in particular and (2) to serve as a biomarker for the efficacy of treatments of obesity and diabetes.

1. Evaluating BA T Mass and Function

Brown fat is a metabolically important tissue in rodents and is believed to also have a functional role in humans. Since there is presently no non-invasive modality that can evaluate BAT mass or function, it is technically difficult to characterize new chemical entities designed to increase BAT mass. This deficiency is particularly acute in humans in whom there is no identifiable interscapular brown fat pad as seen in rodents, preventing anatomical biopsy as a confirmatory methodology.

The non-invasive methods using IR thermography as described herein can be used, e.g., to screen potential compounds for efficacy and thereby function as a proximal biomarker. For example, the methods can be used to evaluate test compounds for their effect on BAT mass or function, e.g., compounds intended to or suspected to increase BAT mass, e.g., anti-obesity treatments and diabetes treatments; compounds that are known or suspected to decrease BAT mass or function, e.g., toxins, can also be evaluated. The technology enables the measurement of the effectiveness of diets and pharmaceutical agents to increase BAT mass, e.g., to treat obesity or diabetes. The methods can be used to provide an in vivo assessment of BAT inducers.

In addition, IR thermography can be used in the clinical setting as a non-invasive mechanism to gauge the potential for response to and effectiveness of anti-obesity or diabetes regimens. For example, subjects who have more BAT already present may be more likely to respond to certain treatments than others whose BAT has atrophied. Thus, IR thermography can be used as a tool analogous to dual-energy x-ray absorptiometry (DXA) as a measurement of bone mineral density and a biomarker for osteoporosis.

2. BA T as a Biomarker for the Efficacy of Treatments of Obesity and Diabetes

IR thermography can be used to demonstrate that enhanced BAT function is a distal, disease-related biomarker for obesity and diabetes. The methods can be used to reflect whole-body insulin sensitivity, facilitate the screening of drugs designed to reduce obesity or increase insulin sensitivity, and even predict the potential of given animal or human to respond to a treatment for obesity and diabetes, permitting the development of more individualized therapies. Currently, there is an increasing multitude of whole-body distal biomarkers for obesity and diabetes, including hemoglobin AIc, oral glucose- tolerance test, hyperinsulinemic-euglycemic clamp, inflammatory cytokines, endothelial dysfunction, and genetic polymorphisms. Each of these has a role to play in the characterization of new chemical entities, and IR thermography can be used alone or in combination with any of these other methods of evaluation.

In some embodiments, the methods described herein can include administering to the subject a dose of a sympathomimetic drug, such as ephedrine, sufficient to induce increased blood flow to BAT and thereby increase its thermogenic capacity, prior to scanning the selected anatomical structures. The methods described herein can also include administering to the subject a dose of bone morphogenic protein 7 (BMP-7), or any of the class of small-molecule drugs that recapitulate the actions of BMP-7, which increases brown fat mass and activity (see, e.g., Tseng et al., Nature 454:1000-1004 (2008)), and thereby increase its thermogenic capacity. Alternatively or in addition, the methods described herein can further include ensuring that the study subject has not taken the following classes of medications or nutritional products shown to reduce BAT glucose uptake or activity: sympatholytics such as α- and β-adrenergic receptor blockers, anxiolytics such as benzodiazepines, antithyroid medications (Nedergaard et al., (2007) Am J Physiol Endocrinol Metab. 293:E444-52).

Finally, the methods described herein preferably include performing the thermographic scanning during a month wherein the average temperature is less than about 60°, e.g., less than about 55°, 50°, 45°, or 40°. Alternatively or in addition, the subject can be scanned in a cold room (e.g., less than about 60°), and/or maintained in a cold environment (e.g., less than about 60°) for a period of time prior to the scanning.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1

This example describes the use of IR thermography to identify BAT sites in rodents. Three weight-paired female 129 mice, all 8 months old, were selected. One mouse from each pair was injected intraperitoneally (i.p.) each day for 6 days with lmg/kg of BAT-inducing CL-316,243; the other mice were injected with drug vehicle. Each day, the weights and core body temperatures were recorded. On day 7, the final i.p. injection was given, and the pairs of mice were visualized by IR thermography.

In two of the 3 pairs, the mouse treated with CL-316,243 had a higher surface temperature overlying main interscapular BAT depot compared with the control mouse (Fig. 1). Body weights, core temperatures, and mass of representative subcutaneous WAT, visceral WAT, and BAT all did not differ significantly from each other.

Molecular determination of UCP-I expression in the extracted BAT can be performed using standard molecular biological techniques, see, e.g., (Tseng et al., (2005) Nat. Cell Biol. 7: 601-611). Alternatively or in addition, guanosine 5 '-diphosphate (GDP)-binding assays and fluorescent dyes (e.g., JC-I or rhodamine derivatives) can be used to provide a direct measure of UCP activity (Nedergaard and Cannon, Am. J. Physiol. 248(3 Pt 1):C365-C371 (1985); Reers et al, Biochemistry 30:4480-4486 (1991)).

The thermogenic capacity of murine BAT is characterized after injecting 4 sets of 129-strain intraperitoneally with either the commercially available β3 -adrenergic agonist CL 316,243 9 (Almind et al., (2007) Proc Natl Acad Sci U S A. 104:2366-2371), ephedrine (Baba et al., (2007) J Nucl Med. 48:981-6), or saline control. The subcutaneous supraclavicular and interscapular BAT depots are identified via IR thermography (ThermaCAM® EX320, FLIR Systems, Inc., North Billerica, MA). Known sites of BAT (i.e., the interscapular BAT pad) and WAT (posterior subcutaneous depots, as a control) are sampled with a 25 gauge needle, and tissue is transferred to QIAZOL® lysis reagent for RNA isolation before being prepared for quantitative RT- PCR. Initially, the genetic identity of the biopsy samples is confirmed by measuring levels of BAT-specific UCP-I as well as genes expressed in BAT more than WAT: cidea, type 2 iodothyronine deiodinase, transmembrane glycoprotein Cig30, PPARα, and PGC- lα. The signals are compared with the levels of predominantly WAT genes: leptin, nuclear corepressor RIP140, and the matrix protein fibrillin- 1 (Gesta et al., (2007) Cell 131 :242-56).

In addition, 3 groups of obesity-prone B6 mice (4 per group) are fed for 28 days with regular diet, high-fat diet, or a low-protein. A fourth group, the leptin-deficient ob/ob strain is fed on the low-protein. Food intake, weight gain, glucose, insulin, and leptin levels will be measured. The mice are then placed individually in indirect calorimetry chambers (Oxymax OPTO-M3 system; Columbus Instruments, Columbus, OH), and 02-consumption is measured for 24 hours. Finally, after an overnight fast, the four groups are imaged via IR thermography, and changes in BAT mass are measured. Other diets that have been associated with affecting BAT differentiation are also evaluated.

These results indicate that infrared thermography is an expedient, cost-effective way to measure of the effectiveness of BAT-modifying agents. Example 2

This example describes the use of IR thermography to identify BAT sites in humans.

In subjects who have demonstrated via PET/CT the presence of 18F-FDG-avid adipose tissue that is likely to be BAT are studied. The subjects are pretreated with either saline or ephedrine, a sympathomimetic shown to increase BAT thermogenicity (Soderlund et al, (2006) Eur J Nucl Med MoI Imaging. 33:785-91). The subcutaneous supraclavicular and interscapular BAT depots will then be identified via infrared thermography (ThermaCAM® EX320, FLIR Systems, Inc., North Billerica, MA) followed by biopsy and RT-PCR as described above. To show that human BAT has significant metabolic function, a parallel set of studies is conducted with human volunteers demonstrating thermogenic BAT and compare them to BAT-less controls. Both groups will undergo hyperinsulinemic-euglycemic clamps with indirect calorimetry as a way to precisely quantify insulin sensitivity and whole-body thermogenesis. The measurements will be correlated with the IR thermographic data to determine how effective non-invasive thermography is as a predictor of insulin sensitivity.

Fig. 2 shows the results of using IR thermography to identify BAT sites in humans. In this image, a 25 year old human female (left) and a 21 year old human male (right) had previously applied to the exposed skin of their upper thoraces packs of either hot (filled arrows) or cold (open arrows). In addition, the female subject had just consumed a mixed meal, while the male subject had been fasting for >4 hours. The image demonstrates that areas of greatest surface temperature in both the female and male are in the cervical, suprascapular, and thoracic paraspinal regions, consistent with the sites demonstrated previously (Rothwell and Stock, (1979) Nature 281 :31-5). In addition, the female's surface temperature is considerably higher than the male's. These findings demonstrate that the infrared technology is sensitive enough to detect even small changes in skin surface temperature. (The methods described herein focus the detection of temperature changes over the regions shown herein to contain human BAT, the anterior surface of the neck and shoulders as depicted in Fig. 5C). Example 3

This example describes the temperature dependence of BAT expression. Specifically, even among people believed to have BAT (the prevalence of macroscopic sites is 5-10% in the general population), BAT is most frequently detectable via PET/CT scans only when the outside temperature is low. BAT+ scans occur less frequently in the summertime, supporting an association between BAT mass and outdoor temperature and the sexual dimorphism connected to it. For example, in Boston, it is difficult if not impossible to detect BAT between April and September (see Fig. 3), an effect that persists over several years (see Fig. 4). This finding has significant implications on methods for monitoring the presence and changes in BAT mass and function.

The results shown herein demonstrate a distinct pattern of BAT distribution, whereby it first appears in the neck and then proceeds downward as one has more BAT, suprascapularly and then through the thorax and abdomen.

These two particular observations explain why other groups had difficulty identifying BAT by biopsy after looking for it using thermography — it was either too warm outside or they weren't looking in the right place. PET/CT technology can be used to identify with high resolution where much of the deeper BAT tissue can be found.

Example 4

The presence of BAT in adult humans was further confirmed using ¹⁸F-FDG positron emission tomography/computerized tomography (PET/CT). 3,640 ¹⁸F-FDG PET/CT were analyzed scans in 1,972 different patients completed over a 3 -year period. An updated version of the PET/CT Viewer analysis program (Barbaras et al., Am J Roentgenol 2007; 188(6):W565-W568), was used to quantify both BAT volume and activity in a user-defined region of interest covering the principal cervical, supraclavicular, and superior mediastinal BAT depots. (Fig. 5A). Patients were determined to have BAT if there were areas >4 mm within specific regions of tissue that had both the CT density of adipose, defined by a Hounsfield Unit (HU) density (of -250 to -50 (this is the x-ray attenuation unit used in CT Scan interpretation that characterizes the relative density of a substance), and also a SUVmax of ¹⁸F-FDG of > 2.0. This cutoff was chosen because (a) it was the lower bound of activity in BAT-positive patients as seen in a previous study (Williams, Am J Roentgenol 2008; 190(2):2409), and (b) 2.0 was over two standard deviations higher than the average background seen in our patients' typical WAT depots.

Using these criteria to define BAT, 106 of the 1,972 patients (5.4%) had PET/CT positive tissue consistent with BAT, similar to what has been reported in prior smaller studies (Hany et al, Eur J Nucl Med MoI Imaging 2002; 29(10): 1393-1398; Cohade et al, J Nucl Med 2003; 44(8): 1267-1270; Truong et al., Am J Roentgenol 2004; 183(4):1127-1132).

BAT deposits in an exemplary subject are shown in a 3D rendering shown in FIG. 5B, which was obtained by taking the DICOM images from the fused PET/CT scan and reconstructing them to make the 3D image shown. Figures 5C-i-iv and 5D-i-iv show via PET/CT imaging the location of the principal adult human BAT depots in a woman (5C) and a man (5D). In Fig. 5C, a 60 year-old woman was being evaluated for thyroid cancer; in Fig. 5D, a 32 year old male was being re-staged for non-hodgkins lymphoma. These results demonstrate that in a normal human, BAT is present in a distinct fascial plane: in the neck, ventralside, bilaterally, superficial and lateral to the sternocleidomastoid neck muscles.

There was a pronounced sexual dimorphism in this grossly detectable BAT. Of the 106 patients with BAT, the prevalence in males was only 3.2% (31/959), whereas the prevalence in females was 7.4% (75/1013), resulting in a female-to-male predominance of 2.3 to 1 (Fig. 6A). These proportions were significantly different from that seen in the entire population of patients who underwent PET/CT, which was 49% male and 51% female (P<0.001). The rest of the BAT+ patients' anthropometric and metabolic data are shown in Table 1.

Table 1. BAT+ Patient Anthropomorphic Data

Characteristic mean ± SD

Number M :F 31 :75

Age, M / y 49.3 ± 16.6 Age, F / y 50.0 ± 16.2

^Height, M / cm 174.8 ± 8.5 Height, F / cm 162.9 ± 6.9

^Weight, M / kg 81.1 ± 14.8 Weight, F / kg 67.3 ± 14.7

BMI, M / kg*m^~2 26.4 ± 3.6 BMI, F / kg*m^~2 25.4 ± 5.7

%SA^§ , M / m² 1.96 ± .20 BSA, F / m² 1.72 ± .17

Fasting glucose, M / mg/dL 102.9 ± 32.5 Fasting glucose, F / mg/dL 96.6 ± 24.5

^Difference between males and females P<0.001

§BSA calculated according to the method of DuBois and DuBois.

Table 1 shows that there is a significant sexual dimorphism in the frequency of BAT, with it occurring in females over 2x more frequently than males. Splitting the BAT± series into males and females, several anthropomorphic indices were the same: age, BMI, and fasting glucose. Males and females differed, however, in terms of average height, weight, and body surface area (BSA). Based on quantification of volume in the cervical, supraclavicular, and superior mediastinal regions of greatest activity, and assuming a density of CT-defined fat of 0.90 g/mL (39), the median amounts of detectable BAT in these defined collections in both the male and female BAT-positive patients was 10-15 g with ranges of 0.5 to 42 g in males and 1.1 to 89 g in females (Fig. 6B). Median volumes and activity were similar in both sexes (P>0.05), and both showed a positive skew. However, for females, maximal BAT volumes and activities were more skewed to higher levels than in males, suggesting that compared with males, human females had a much greater capacity to increase their BAT mass and activity (Figs.6B andβC).

Example 5

To identify how BAT correlated with anthropomorphic indices and fasting glucose and minimize any seasonal or date dependent variability, 204 date-matched, BAT- negative patients were selected to compare with the 106 BAT-positive patients (Table 2). Oncologic diagnosis did not distinguish these two series of patients (Table 3), allowing the study to focus on more general metabolic parameters.

Table 2. BAT- and BAT+ Patient Anthropomorphic Data*

Characteristic hBAT- hBAT± Significance

Age, M / y 59.4 ±14.1 49.3 ± 16.6 p<.001 Age, F / y 60.1 ±15.7 50.0 ±16.2 p<.001

^Height, M / cm 175.9 ±6.9 174.8 ±8.5 p<.47 Height, F / cm 164.2 ±9.1 162.9 ±6.9 p<.69

^Weight, M / kg 82.9 ±14.6 81.1 ±14.8 p<.55 Weight, F / kg 69.5 ±16.5 67.3 ± 14.7 p<.38

BMI, M / kg*m^~2 26.7 ±3.9 26.4 ±3.6 p<.70

BMI, F / kg*m^~2 26.4 ±6.2 25.4 ±5.7 p<.28

%SA^§, M/m² 1.99 ±.18 1.96 ±.20 p<.49

BSA, F / m² 1.74 ±.20 1.72±.17 p<.53

Fasting glucose, M / mg/dL 105.9 ±29.2 102.9 ±32.5 p<.63 Fasting glucose, F / mg/dL 101.7 ±24.5 96.6 ±24.5 p < .11

*A11 values are mean ± standard deviation

^Indicates the difference between males and females had a p<.001

§BSA was calculated according to the method of DuBois and DuBois. In Table 2, the anthropomorphic indices and metabolic parameters of the BAT+ and BAT- series of patients were compared. After splitting them into males and females, it was found that both BAT+ males and females differed significantly from the BAT- patients in terms of age. However, the series' average heights, weights, BMIs, BSAs, and fasting glucoses were not different. Table 3 shows the comparison of the different oncologic diagnoses between the BAT+ and BAT- series of patients. Oncologic diagnosis did not affect the frequency of BAT.

Table 3. Comparison of BAT+ and BAT- Oncologic Diagnoses

Males, P<0.63 Females, P<0.20

Cancer Diagnosis BAT- BAT + BAT- BAT +

Lymphoma 49 13 33 36 Other Malignancies 51 14 57 35 No malignancy 8 4 6 4

Total 108 31 96 75

To understand the relationship between BAT expression, and the relevant anthropometric and metabolic factors, multivariate logistic regression was performed using gender, age, BMI, and fasting glucose (Fig. 7). The BAT -negative and BAT- positive patients were grouped together into one series and then divided into textiles of age (low- (23 to 5Oy), mid- (50 to 65y), and high-ranges (64 to 9Oy)), BMI (low- (18.3 to 23.5 kg/m²), mid- (23.5 to 27.8 kg/m²), and high-ranges (27.8 to 43.3 kg/m²)), and fasting glucose (low- (59 to 92 mg/dL), mid- (92 to 103 mg/dL), and high-ranges (104 to 281 mg/dL). Of note, cancer diagnosis and smoking history were also investigated and found to have no significant effect on the presence of BAT, nor were they confounding variables (not shown). After female predominance, the presence of detectable BAT was greatest in the youngest group and progressively decreased in the middle-age and older groups (P<0.001). An intriguing interaction between age and BMI was also identified: in the youngest strata, increasing BMI was associated with more BAT. However, as people age past 50 years, increasing obesity is associated with less BAT (P<0.01). In summary, the results showed a very distinctive pattern - BAT was found most frequently in individuals who were young, and as people age, obesity was associated with less BAT.

Example 6

Previous pathological studies of individuals without hibernomas have shown that on careful pathological examination, otherwise normal human adults have cells with characteristics of brown adipocytes mixed with white adipose tissue collected into small depots in the neck and supraclavicular regions (Heaton, J Anat 1972; 112(Pt l):35-39). To confirm that the ¹⁸F-FDG PET-evident adipose tissue (PEAT) seen in our ROI was BAT, adipose tissue taken from these regions at neck surgery for other causes such as parathyroidectomy and thyroidectomy was analyzed. Characteristic brown adipocytes mixed together with white adipocytes were identified in different proportions in 33 patients. A representative biopsy is shown in Figs. 8A-8D from a 48 year-old woman who underwent a parathyroidectomy. The gross description was a tan-yellow, lobulated soft tissue mass that on microscopic analysis revealed a mixture of white and brown fat, with the multilocular lipid droplets and central nuclei characteristic of BAT (Figs. 8 A and 8C). Immunohistochemical analysis showed specific staining of these cells with an antibody for UCP-I (Figs. 8B and 8D), confirming that this tissue was in fact brown fat.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of evaluating brown adipose tissue (BAT) function or mass in a human, the method comprising: ensuring that the ambient room temperature is below 70 ⁰F; setting a region of interest (ROI) that covers the cervical, supraclavicular, and superior mediastinal areas of the human; scanning the region of interest with an infrared camera to obtain an infrared thermographic image of the region of interest in the human; processing the image to provide a measure of temperature information; and correlating the temperature information with BAT mass or function in the human, thereby evaluating the BAT mass or function.

2. The method of claim 1, further comprising comparing the measure of the temperature information to a predetermined value, the predetermined value being a measure of BAT mass or function in the ROI of either the same subject or the same selected anatomical structure of one or more control subjects.

3. The method of claim 2, wherein the predetermined value is a measure of BAT mass or function in the ROI of the same subject at a different time of the year.

4. The method of claim 2, wherein the predetermined value is a measure of BAT function in the ROI of the same subject before or after administration of a test compound, wherein the comparison indicates an effect of the test compound on BAT mass or function.

5. The method of claim 1 , further comprising providing a dose of a sympathomimetic drug such as ephedrine to induce increased blood flow to BAT and thereby increase its thermogenic capacity, prior to scanning the selected anatomical structures.

6. The method of claim 1 , further comprising ensuring that the study subject has not taken any of the following classes of medications or nutritional products shown to reduce BAT glucose uptake or activity: sympatholytics such as α- and β- adrenergic receptor blockers, anxiolytics such as benzodiazepines, anti-thyroid medications.

7. The method of claim 1 , wherein the scanning is preferentially performed during a month wherein the average temperature is less than about 60°.