Regulation of body temperature and brown adipose

Articles in PresS. Am J Physiol Endocrinol Metab (January 22, 2014). doi:10.1152/ajpendo.00615.2013
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Regulation of body temperature and brown adipose tissue thermogenesis by bombesin
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receptor subtype-3
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Dalya M. Lateef,1 Gustavo Abreu-Vieira,2 Cuiying Xiao,1 Marc L. Reitman1
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Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and
Kidney Diseases, NIH, Bethesda, Maryland
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Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University,
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Stockholm, Sweden
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Running head: BRS-3 regulation of body temperature
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Address for correspondence: M. L. Reitman, Building 10-CRC, Room 5-5940, 10 Center Drive,
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Bethesda, MD 20892-1453 (e-mail: [email protected]).
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5 Figures, 1 Table, Abstract 187 words
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Abbreviations: BAT, brown adipose tissue; iBAT, interscapular BAT; BRS-3, bombesin
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receptor subtype-3; WAT, white adipose tissue; Tb, body temperature
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Copyright © 2014 by the American Physiological Society.
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Abstract
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Bombesin receptor subtype-3 (BRS-3) regulates energy homeostasis, with Brs3 knockout
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(Brs3-/y) mice being hypometabolic, hypothermic, and hyperphagic and developing obesity. We
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now report that the reduced body temperature is more readily detected if body temperature is
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analyzed as a function of physical activity level and light/dark phase. Physical activity level
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correlated best with body temperature four minutes later. The Brs3-/y metabolic phenotype is not
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due to intrinsically impaired brown adipose tissue function or in the communication of
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sympathetic signals from the brain to brown adipose tissue, since Brs3-/y mice have intact
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thermogenic responses to stress, acute cold exposure, and β3 adrenergic activation, and Brs3-/y
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mice prefer a cooler environment. Treatment with the BRS-3 agonist MK-5046 increased brown
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adipose tissue temperature and body temperature in wild type, but not Brs3-/y mice.
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Intrahypothalamic infusion of MK-5046 increased body temperature. These data indicate that
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the BRS-3 regulation of body temperature is via a central mechanism, upstream of sympathetic
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efferents. The reduced body temperature in Brs3-/y mice is due to altered regulation of energy
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homeostasis affecting higher center regulation of body temperature, rather than an intrinsic
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defect in brown adipose tissue.
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Keywords
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BRS-3, obesity, sympathetic nervous system, brown adipose tissue, thermoregulation,
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CL316243, MK-5046
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Introduction
Obesity, an imbalance between energy intake and energy expenditure, is associated with
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several comorbidities, including diabetes mellitus and cardiovascular disease. Current treatments
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for obesity are inadequate, stimulating research to better understand the physiology of energy
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homeostasis and to develop safe and effective pharmacologic treatments. Study of bombesin
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receptor subtype-3 (BRS-3) can advance both of these objectives.
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BRS-3 is a G-protein coupled receptor for which the natural ligand is unknown, and
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despite its name, BRS-3 has a low affinity for bombesin and related natural peptides (7, 17, 21).
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In addition to other locations, BRS-3 is present in the central nervous system, including the
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hypothalamus and caudal brainstem (10, 16, 26, 37), regions involved in the regulation of
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feeding, energy expenditure, and body weight (32). Genetic and pharmacologic studies
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demonstrate a role for BRS-3 in energy homeostasis. Brs3 knockout (Brs3-/y) mice exhibit
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hyperphagia, reduced metabolic rate and obesity (18, 27), and pharmacological blockade of
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BRS-3 in rats increases food intake and body weight (10). Conversely, BRS-3 agonists reduced
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food intake, increased metabolic rate and body temperature (Tb), and reduced body weight in
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mice, rats, and dogs (10, 11, 22).
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Currently, the mechanisms underlying Tb regulation by BRS-3 are unclear. Brown
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adipose tissue (BAT) is the major site of facultative thermogenesis, dissipating chemical energy
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via UCP1, thereby generating heat and maintaining Tb (2). BAT activity is regulated by
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sympathetic neural input, with upstream regulation from hypothalamic, preoptic, and other brain
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regions (1, 23). Thus, the lower Tb in Brs3-/y mice could arise from intrinsic defects at a number
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of sites including BAT (5), sympathetic neuronal transmission (35), or the brain sites at the core
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of thermal regulation (20). Alternatively, the mild hypothermia could be due to an altered input
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into what is otherwise a normally-responding thermal regulatory system.
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We now evaluate the consequences of disrupting BRS-3 signaling on BAT. We also
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examine the effects of BRS-3 agonism on BAT thermogenesis and the involvement of the central
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nervous system. Together these approaches allow us to suggest a mechanism for how the BRS-3
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system affects Tb.
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Materials and Methods
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Compounds. MK-5046 (33) was generously provided by Merck Research Laboratories (Rahway,
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NJ) and CL316243 and lipopolysaccharide were purchased from Sigma-Aldrich (St. Louis, MO).
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Mice. C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Brs3-/y
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mice were provided by Dr. James Battey (18) and back-crossed at least eight generations onto a
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C57BL/6J background. Mice were housed at ~21-22°C in a humidity-controlled environment
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with a 12:12-h light-dark cycle and with water and chow (NIH-07 diet) available ad libitum. All
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experiments used male mice, typically 12-16 weeks of age. Pentobarbital sodium (80 mg/kg ip)
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was used for anesthesia for physiological measurements. Survival surgery anesthesia used
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ketamine 100 mg/kg and xylazine 10 mg/kg ip, with Banamine analgesia (2.2 mg/kg sc daily for
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3 days). At least 7 days were allowed for recovery. All animal studies were approved by the
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NIDDK/NIH Animal Care and Use Committee.
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Body and interscapular brown adipose tissue (iBAT) temperature measurement in anesthetized
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mice. Mice were anesthetized (pentobarbital), placed on a heated (35ºC) table, and a thermistor
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probe (YSI 427; Measurement Specialties, Shrewsbury, MA) was sutured into place underneath
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the iBAT via an incision caudal to the fat pad (4). A rectal temperature probe (YSI 455,
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Measurement Specialties) was inserted and secured with tape. The mouse was then positioned
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supinely and a tracheal catheter was inserted, allowing free movement of room air. At least 30
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minutes were allowed for stabilization prior to drug infusion.
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Body and iBAT temperature measurement by telemetry in ambulatory mice. Mice were
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anesthetized, an IPTT-300 transponder (BioMedic Data Systems, Seaford, DE) was sutured into
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place under the iBAT depot. Another IPTT-300 was inserted intraperitoneally via a midline
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abdominal incision, and sutured to the omentum. Body and iBAT temperature were recorded
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with a scanner (DAS-7007, BioMedic Data Systems). To avoid confounding temperature
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increases due to handling stress, all readings were taken within 60 seconds of initially handling a
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mouse with at least one hour (and typically two hours) between serial measurements.
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Alternatively, Tb and activity were continuously measured by telemetry (Mini Mitter/Philips
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Respironics, Bend, OR) using ER4000 energizer/receivers, G2 E-mitters implanted
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intraperitoneally, and VitalView software with data collected each minute. The Tb vs activity
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cross-correlation analysis was performed with NeuroExplorer (Nex Technologies, Madison, AL).
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Ambulatory tests of thermal regulation. Temperature preference was measured by placing mice
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in a stainless steel pan (64 x 15 x 20 cm, length x width x height) spanning two hot/cold plates
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set at 20 ºC and 45 ºC. Ninety-minute sessions were conducted on two successive days, with the
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last 60 minutes of the second session used for analysis. Position was tracked with an overhead
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camera and video tracking software (Ethovision 9.0, Noldus, Leesburg, VA). In the cage-switch
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assay, stress was induced by placing a mouse in a cage previously occupied by a different male
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mouse (19) with Tb and activity monitored by Mini Mitter. The febrile response at 21 ºC
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ambient temperature caused by lipopolysaccharide (10 μg/kg ip) (28) was measured by Mini
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Mitter. Indirect calorimetry was performed with a 12-chamber Environment Controlled CLAMS
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(Columbus Instruments, Columbus, OH) with ad libitum access to food and water during the
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entire testing period.
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Intravenous infusions. Mice were anesthetized and the jugular vein catheterized using PE-10
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tubing. CL316243 and MK-5046 were dissolved in saline or 5% dimethylacetamide in saline,
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respectively, and delivered in a volume of 1 ml/kg.
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Intrahypothalamic infusions. Mice were anesthetized, cannulas (5.25 mm, 26 gauge, Plastics
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One, Roanoke, VA) stereotaxically implanted bilaterally (1.34 mm posterior, 0.75 mm lateral to
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bregma, 4.75 mm below the surface of the skull (8)), and secured with dental cement (Parkell,
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Inc., Edgewood, NY). Intrahypothalamic infusions used saline as vehicle and were given via a
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33-gauge internal cannula protruding 0.5 mm past the tip of the guide cannula using a syringe
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pump (KD Scientific, Holliston, MA) infusing 1 μl per cannula over 60 seconds. The infusion
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targets the entire hypothalamus. Cannula position was verified by postmortem histological
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analysis.
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Statistics. Data are mean ±SEM. Two-way ANOVA with or without repeated measures followed
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by Holm-Šídák’s post test was used for comparing genotypes vs. the treatment groups. Student’s
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t-test was used when two groups were compared. Analyses used two-tailed P <0.05 as
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statistically significant.
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Results
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Thermal biology in Brs3-/y mice
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Tb in Brs3-/y mice was slightly lower than in controls, reaching statistical significance in the
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light but not the dark phase; there was no difference in physical activity levels (Table 1). To
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better understand the relationship between physical activity and Tb, we quantified the timing of
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the effect of activity on Tb by looking for the lag time that gives the best correlation between
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activity and Tb in a cohort of wild type C57BL/6 mice. This cross-correlation analysis revealed
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that increased activity causes Tb increases with a 4-minute (range, 2- to 6-minute) lag (Figure
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1A). Based on this result we analyzed the Brs3-/y Tb and activity data using 10-minute averages.
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The Tb histogram showed the lower Tb in the light phase and the slight shift in Brs3-/y mice but
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a similar overall pattern to the controls (Figure 1B). The activity histogram showed the
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expected higher level in dark than light phase with no difference between Brs3-/y and control
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mice (Figure 1C). Next we examined Tb as a function of activity and observed a clear
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difference between Brs3-/y and control mice, with a greater difference at lower physical activity
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levels (Figure 1D).
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To further understand Tb regulation in Brs3-/y mice, a number of interventions that change Tb
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were tested. Brs3-/y mice increased Tb normally during the first hour in response to the stress of
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being handled and had a comparable febrile response to lipopolysaccharide, with the
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hyperthermia resolving more rapidly in the Brs3-/y mice (Figure 2A). There was a normal
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circadian rhythm and Tb and physical activity increase in response to the social stress of a cage
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switch (19) (Figure 2B). When Brs3-/y mice were placed in a thermal gradient, they dispersed
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over the gradient much more widely than the controls and chose cooler environmental
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temperatures (Figure 2C). The lower preferred environmental temperature was not explained by
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the slightly higher body weight.
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Brs3-/y mice have intact, functioning BAT
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We next examined BAT function. Brs3-/y mice (12-16 weeks old), while of comparable body
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weight to controls (wild type, 22.0 ±0.8 g and Brs3-/y 22.6 ±0.4 g), showed increased iBAT,
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inguinal white adipose tissue (WAT), and gonadal WAT weights (Figure 3A). The increased
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iBAT weight is likely due to increased triglyceride content as Brs3-/y mice have larger lipid
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droplets (Figure 3B). Ucp1 mRNA and protein levels were similar between Brs3-/y and control
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mice (data not shown). Body and iBAT temperatures in overnight fasted Brs3-/y mice were
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reduced compared to wild type mice (Figure 3C). The iBAT was slightly warmer than the body
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core, but this was not statistically different in this experiment.
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To measure thermogenic capacity, mice were treated with a maximal dose of a β3-
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adrenoreceptor agonist, CL316243 (2). The Brs3-/y mice showed a greater increase in Tb,
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energy expenditure, and fat oxidation (indicated by the reduced respiratory exchange ratio,
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RER) than the control mice (Figure 3D-F). Thus, the intrinsic thermogenic capacity in response
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to a β3-adrenoreceptor agonist is clearly intact in Brs3-/y mice.
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To assess an integrated thermogenic response (sensory information to brain to BAT), the
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body and BAT temperature response to cold exposure was measured. The slight reduction in
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Tb was similar in Brs3-/y and control mice (Figure 3G) as was the rise in iBAT temperature
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(Figure 3H). Together, these results demonstrate that Brs3 deficient mice have functional,
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inducible BAT, despite their lower body and BAT temperatures, and point to a possible
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reduction in neural activation of BAT.
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BRS3 agonist activates BAT via a central mechanism
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BRS3 agonists can increase Tb (10, 22), so we next examined the effect of a peripherally
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administered, brain-penetrant BRS3 agonist, MK-5046 (11), on iBAT temperature, using
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anesthetized mice to eliminate the effect of changes in physical activity. Mice were treated with
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MK-5046, CL316243, or vehicle. MK-5046 increased iBAT temperature in wild type but not
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Brs3-/y mice (Figure 4A). In contrast, as a positive control, CL316243 increased iBAT
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temperature in both wild type and Brs3-/y mice (Figure 4B,C). Similar results were obtained
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measuring rectal temperature (Figure 4D). These data demonstrate that MK-5046 has a
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thermogenic effect on BAT, and that it is acting via BRS-3.
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To determine if the thermogenic effect of MK-5046 was centrally mediated, we measured
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the effect of intrahypothalamic infusion of MK-5046 on Tb. MK-5046 increased Tb by 0.42
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±0.11°C (P<0.001; Figure 5A,B), demonstrating that MK-5046 is acting centrally to increase Tb.
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Discussion
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We have investigated in detail the reduced Tb of Brs3-/y mice. The results show no
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intrinsic defects in BAT or in the CNS’s ability to engage sympathetic efferents to BAT. Rather
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we propose that the reduced Tb is a secondary effect of altered energy homeostasis affecting
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higher center regulation of Tb, as discussed below. Peripherally-administered BRS-3 agonists
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increase Tb and BAT temperature and small doses administered directly to the hypothalamus
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increase Tb, suggesting that CNS BRS-3 contributes to sympathetic outflow to BAT.
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Intact control of Tb in Brs3-/y mice
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The Tb reduction in Brs3-/y mice studied at room temperature is small, averaging ~0.34
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ºC below control mice. Tb differences of this magnitude are difficult to detect, so experimental
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manipulations are often used to increase their size. For example, in Brs3-/y mice fasting
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amplifies the Tb reduction (22). However, such manipulations have other effects on physiology,
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confounding interpretation of the results. Here we measured Tb by telemetry and analyzed Tb as
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a function of physical activity level and light/dark phase. This approach permits monitoring in
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the home cage without potentially confounding manipulations, makes use of the full dataset, and
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markedly increases the ability to detect Tb changes.
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To our knowledge, Tb kinetics after physical activity have not been reported previously
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in mice. While the quantitative details are likely to depend on the level and type of activity and
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methods of Tb and activity measurement, only a brief lag time is expected due to the small body
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size and thus rapid heat loss. We observed a 4-minute lag between activity and the Tb response.
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The energy cost of defending Tb is 2.02 ±0.06 kcal/d/ºC in 27 g mice (Abreu-Vieira,
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unpublished observations). Thus, the ~0.34ºC Tb reduction in Brs3-/y mice saves ~0.70 kcal/d. It
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is not known how this energy savings partitions to fat storage vs reduced food intake. There are
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no reports characterizing Brs3-/y mice housed chronically at thermoneutrality, which would erase
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their Tb reduction, and it is unknown if thermoneutrality would exacerbate the increased Brs3-/y
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adiposity, as it does in other mouse models (3, 25, 31).
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What is the mechanistic basis for the Tb lowering in Brs3-/y mice? Direct stimulation of
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the thermogenesis effector tissues, WAT and BAT, with a β3 adrenergic agonist demonstrated
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full functionality. In fact, the increases in metabolic rate and Tb were actually greater in Brs3-/y
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mice. Possible explanations for the augmented Brs3-/y thermogenic response are increased fatty
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acid availability from the larger WAT and/or BAT stores, providing more fuel for greater heat
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generation in BAT and more responsive adipose tissues due to reduced prior stimulation. More
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upstream, the neural sympathetic control of heat generation by BAT is intact in Brs3-/y mice, as
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demonstrated by the response to handling stress, cage switch stress, and lipopolysaccharide.
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Notably, the response to cold exposure is similar in Brs3-/y and wild type mice, demonstrating
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intact sensing of environmental temperature. Besides the lower Tb, the Brs3-/y mice prefer a
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cooler environment, indicating that the lower Tb is targeted and is not due to an ‘inability’ to
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defend a higher Tb.
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These data strongly suggest that in Brs3-/y mice there is no defect intrinsic to the BAT
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itself or to the circuitry determining sympathetic signals efferents to BAT. A unified way to
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view the thermal physiology of Brs3-/y mice is that the endogenous BRS-3 ligand is a signal of
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the ‘energy replete’ state. In this sense, Brs3-/y mice are similar to Leprdb/db mice (12). The
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inability of Brs3-/y mice to sense the putative endogenous ligand (or of Leprdb/db mice to sense
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leptin (34)) elicits a physiologic drive to increase energy stores and reduce energy expenditure.
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Tb reduction is a normal component of that response--small mammals expend large amounts of
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energy to stay warm, and a reduction in Tb is a common, effective energy-conserving strategy
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(9). The recent identification of a Drosophila BRS-3 homolog and two ligand peptides (14) may
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facilitate discovery of the elusive mammalian endogenous BRS-3 ligand(s). Other examples of
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mutant mice that likely have reduced Tb secondary to altered energy homeostasis include
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Lepob/ob (29), Mc4r-/- (unpublished observations), and Fgf21 over-expressors (15). It is probably
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a general rule in small-sized mammals that modifiers of the regulation of energy homeostasis can
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affect Tb.
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BAT and Tb effect of a BRS-3 agonist, MK-5046
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BRS-3 agonists can increase Tb, particularly in the fasted state and the Tb increase is
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reduced by propranolol, supporting a sympathetic mechanism of action (10, 22). Here, we
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directly show that the Tb increase occurs via BAT activation. Intrahypothalamic injection of
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MK-5046 increased Tb, demonstrating a role for CNS BRS-3. BAT thermogenesis is controlled
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at multiple levels in the CNS. Thermal sensory inputs, some routed via the lateral parabrachial
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nucleus (LPB), and other signals are integrated in the preoptic area, transmitted to the
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dorsomedial hypothalamus (DMH), rostral raphe pallidus, and on to preganglionic sympathetic
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neurons (23). Other regions contributing to regulating sympathetic tone to BAT include the
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paraventricular hypothalamus (PVH), orexinergic lateral hypothalamus (LH), and the nucleus of
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the solitary tract (NTS). BRS-3 binding activity (10) is found in many of these areas (e.g. DMH,
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PVH, LH, LPB, NTS) as is BRS-3 mRNA (e.g., medial and median preoptic area, DMH, PVH,
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LPB) (37). Further studies (such as injection of smaller volumes of agonist and localized
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ablation of Brs3) will be required to localize more precisely which neurons and nuclei are
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driving the Tb increase and their upstream and downstream interactions.
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BRS-3 agonism for the treatment of obesity?
How does mechanistic understanding of BRS-3’s role in thermal biology inform the
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prospects of BRS-3 agonism for the treatment of obesity? First, it is important to recognize that
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smaller homeotherms such as mice have larger surface area to volume ratios, generate much heat
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in BAT, and strive to conserve body heat (9). In contrast large homeotherms such as adult
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humans get most (but not all (24)) of their heat from BAT-independent metabolism and have
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developed mechanisms to facilitate heat loss. Due to these physiologic differences mice, unlike
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adult humans, vary their target Tb greatly in response to environmental changes, such as cold
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exposure or food deprivation. As noted, the BRS-3 system’s effects on Tb are likely secondary
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to its primary role in energy homeostasis. This makes the concern that BRS-3 agonists will
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produce clinical hyperthermia unlikely, and is consistent with the lack of observed Tb changes in
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men treated with MK-5046 (30). The realization that adult humans can and do expend energy
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via BAT activation has brought attention to BAT (24) and highlighted the potential for drugs that
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activate BAT as a treatment for obesity (36). BRS-3 agonists fit this paradigm—they increase
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energy expenditure by BAT. BRS-3 agonists also inhibit food intake, which is crucial since
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increased food intake often accompanies BAT activation and thereby impairs weight loss due to
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BAT activation. However, caution is warranted. Leptin is another ligand that reduces food
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intake and activates BAT and leptin is a near-miraculous drug when given to treat leptin
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deficiency (6). However >99.99% of obese people have a high leptin level, not a deficiency, and
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treatment with leptin in these patients is not effective (13). Until an endogenous BRS-3 ligand is
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identified, it is unknown if obese people have low or high BRS-3 ligand levels and this may
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determine how effective a BRS-3 agonist will be for treating obesity.
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Acknowledgments
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We thank Alexxai Kravitz for advice and the Tb-activity cross-correlation analysis and Oksana
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Gavrilova and Margalit Goldgof for intellectual and technical contributions. This research was
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supported by the Intramural Research Program (ZIA DK075057, ZIA DK075063) of the
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National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH. The visit of
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GAV to NIH was supported within a grant from the Swedish Research Council to Jan
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Nedergaard and from institutional funds from the Department of Molecular Biosciences, The
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Wenner-Gren Institute, Stockholm University.
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Figure legends
385
Figure 1. Effect of activity and light phase on Tb in Brs3-/y mice. (A) Cross-correlation
386
analysis of Tb and activity in C57BL/6 mice was performed with a dataset from ten C57BL/6
387
mice, each monitored for 140 hours in 1-minute intervals. A positive value in the time shift axis
388
means the Tb data are shifted later relative to the activity data by the indicted number of minutes.
389
Data are mean ±SEM. (B-D) The mean Tb and activity in 10-minute intervals was calculated for
390
wild type (WT) and Brs3-/y (KO) mice, n=10/group (n=2760 dark phase and n=1970 light phase
391
data pairs per genotype). The frequency histograms for (B) Tb and (C) activity and (D) the
392
quadratic fit of the Tb to the activity were calculated separately for the dark and light periods.
393
The dotted lines show the 95% confidence intervals of the fitted curves.
394
Figure 2. Regulation of Tb in Brs3-/y mice. (A) Fever elicited by lipopolysaccharide (LPS).
395
Wild type (WT) and Brs3-/y (KO) mice were treated with lipopolysaccharide (10 μg/kg ip) or
396
vehicle (saline) at 9 am (time 0). Data are change from baseline (-90 to -30 minutes) mean
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397
±SEM, n=9-12/group. * indicates p<0.05 WT LPS vs KO LPS of 10-minute averages. Baseline
398
Tb were 36.1 ±0.2 (WT veh), 36.1 ±0.3 (WT LPS), 35.9 ±0.2, (KO veh), and 35.9 ±0.2 (KO
399
LPS) °C. (B) Cage-switch. WT and KO mice were individually placed in a cage vacated by
400
another male. Time is clock time; switch was at 11 am. Data are mean; SEMs are omitted for
401
visual clarity; N=8-10/group. (C) Choice of environmental temperature. Mice were placed in a
402
thermal gradient (nominally 20 to 45 ºC) during the light phase with position monitored
403
continuously by video (n=10/group). Bar graph shows mean ±SEM and * indicates p=0.001.
404
Mean body weights were 27.8 ±1.0 and 31.5 ±1.1 g in WT and KO, respectively (p=0.03).
405
Ambient temperature was 21.6 ºC. The wild type and Brs3-/y mice moved similar distances.
406
Figure 3. Intact BAT function in Brs3-/y mice. Wild type (WT) and Brs3-/y (KO) mice were
407
studied at 12-16 weeks of age. (A) iBAT, inguinal WAT and gonadal WAT weights. (B)
408
Histology of iBAT. The scale bar is 50 μm. (C) Ambulatory telemetry (BioMedic) BAT and
409
body temperature at 9 am after a 16-hour fast. (D-F) Effect of CL316243 on (D) body
410
temperature, (E) energy expenditure, and (F) respiratory exchange ratio, RER. Mice (body
411
weight: WT, 25.0 ±0.4 g and KO, 26.5 ±0.8 g) were acclimatized to the indirect calorimetry
412
chambers at 22ºC for 4 days and then at 30ºC for one day with vehicle or CL316243 treatment
413
(0.1 mg/kg ip) on the sixth or seventh day in a crossover design.
414
difference in temperature between BAT and core body temperature upon exposure to 4°C. Time
415
is time since onset of cold exposure. Data are mean ±SEM, n=5–6/group, *P <0.05.
416
Figure 4. MK-5046 increases iBAT and body temperature. Effects of intravenous treatments
417
were studied in pentobarbital-anesthetized wild type (WT) and Brs3-/y (KO) mice at 12-16 weeks
418
of age. The change in iBAT temperature in response to (A) MK-5046 (MK, 1 mg/kg iv,
(G) Body temperature and (H)
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419
equivalent exposure to 10 mg/kg orally (33), which causes a maximal body temperature increase
420
(11)) and (B) CL316243 (CL, 0.1 mg/kg iv), compared to vehicle (Veh) in wild type (WT) and
421
Brs3-/y (KO) mice. For visual clarity, the vehicle data are repeated in A and B. (C) Mean change
422
in BAT temperature, average of the 5-30 minute data from A and B. Baseline iBAT
423
temperatures were 35.2 ±0.3 (WT Veh), 35.8 ±0.2 (WT CL), 35.0 ±0.3 (WT MK), 35.0 ±0.2
424
(KO Veh), 34.9 ±0.3 (KO CL), and 35.9 ±0.2 (KO MK) °C. (D) Mean change in body
425
temperature, average of the 5-30 minute data from the same experiment. Data are mean ±SEM,
426
n=6/group, *P <0.05.
427
Figure 5. Effect of bilateral intrahypothalamic MK-5046 treatment (1 μg x 2) in pentobarbital-
428
anesthetized C57BL/6 mice. (A) Body temperature change from time baseline and (B) average
429
of the 5-30 minute data. Baseline temperatures were 35.7 ±0.2 (Veh) and 35.3 ±0.1 (MK) °C.
430
Experiment is a crossover design and data are mean ±SEM, n=9/group, *P <0.05.
431
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Table 1. Body temperature and physical activity
433
Dark Tb (ºC) Dark Activity Light Tb (ºC) Light Activity
434
435
Wild Type
37.36 ±0.14
15.7 ±0.9
36.57 ±0.17
8.0 ±0.7
436
Brs3-/y
37.13 ±0.07
15.4 ±0.7
36.11 ±0.09
8.0 ±0.4
437
N
10
10
10
10
438
p
0.17
ns
0.03
ns
439
Tb and activity were measured by Mini Mitter telemetry for four days, omitting data when mice
440
were handled or treated. The means of 2760 (dark phase) or 1970 (light phase) minutes of Tb and
441
activity data were calculated for each of ten mice. Activity is in arbitrary units.
442
B 16
0.5
Frequency (%)
Tb-Activity Correlation
A 0.6
0.4
0.3
0.2
0.0
-40
Dark WT
Dark KO
Light WT
Light KO
12
8
4
0
-20
C 40
0
20
Time Shift (min)
34
40
35
36
37
38
39
Tb (°C)
D 38
Dark WT
Dark KO
Light WT
Light KO
20
15
Tb (°C)
Frequency (%)
35
37
Dark WT
Dark KO
Light WT
Light KO
10
36
5
0
0
10
20
30 40
Activity
50
60
70
Lateef Figure 1
0
10
20
Activity
30
40
1.5
1.0
*
WT veh
WT LPS
KO veh
KO LPS
B
0.5
0.0
-0.5
C 50
40
37
36
WT
KO
35
20
0
0
1
2
3
4
5
Time (h)
Lateef Figure 2
6
7
18
21
24
3
*
30
WT
KO
10
-1.0
-1
warm
38
Position (cm)
**
**
**
Tb (°C)
Temperature change (°C)
A 2.0
6
Time (h)
9
12
15
18
cool
20
25
30
35
Body Weight (g)
40 WTKO
A 500
400
Weight (mg)
*
WT
KO
*
300
B
200
*
100
0
iWAT
gWAT
WT, veh
KO, veh
WT, CL
KO, CL
D
WT
KO
38
36
*
*
0.2
36
0.0
32
Body
F
BAT
WT, veh
KO, veh
WT, CL
KO, CL
0
G37
2
4
6
8
Time (h)
10
12
2
4
6
8
H3
WT
KO
BAT-Tb (°C)
Tb (°C)
0.9
0
10
12
Time (h)
1.0
RER
0.6
0.4
37
34
WT, veh
KO, veh
WT, CL
KO, CL
E
Energy Expenditure (kcal/h)
BAT
38
Tb (°C)
Temperature (°C)
C
36
WT
KO
2
1
35
0.8
0
34
0.7
0
2
4
6
8
Time (h)
10
12
0
2
4
Time (h)
Lateef Figure 3
6
0
2
4
Time (h)
6
'BAT Temperature (°C)
B
WT-Veh
WT-MK
KO-Veh
KO-MK
1.5
1.0
0.5
0.0
'BAT Temperature (°C)
0
10
20
Time (min)
0.5
0.0
**
0.2
*
0.0
-0.2
-0.4
-10
30
Veh
CL
MK
D
0.3
0.2
'Tb (°C)
-10
0.4
1.0
-1.0
-1.0
0.6
WT-Veh
WT-CL
KO-Veh
KO-CL
-0.5
-0.5
C
1.5
'BAT Temperature (°C)
A
0
10
20
Time (min)
Veh
CL
MK
*
0.1
*
0.0
-0.1
-0.2
-0.3
-0.6
-0.4
-0.8
-0.5
WT
KO
WT
Lateef Figure 4
30
KO
A 1.0
B 0.6
Veh
MK
0.8
'Tb (°C)
0.6
'Tb (°C)
*
0.4
0.4
0.2
0.2
0.0
0.0
-0.2
-0.2
-0.4
0
10
20
30
Time (min)
40
Veh
Lateef Figure 5
MK