Tumor necrosis factor ␣ mediates apoptosis of brown adipocytes and defective brown adipocyte function in obesity Enzo Nisoli*†, Luca Briscini*, Antonio Giordano‡, Cristina Tonello*, Sarah M. Wiesbrock§, K. Teoman Uysal§, Saverio Cinti‡, Michele O. Carruba*, and Go¨ khan S. Hotamisligil†§ *Center for Study and Research on Obesity, LITA Vialba, L. Sacco Hospital, University of Milan, Via G.B. Grassi 74, 20157 Milan, Italy; ‡Institute of Normal Human Morphology-Anatomy, University of Ancona, Via Tronto 10͞A, 60020 Ancona, Italy; and §Division of Biological Sciences and Department of Nutrition, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115 Edited by Roger H. Unger, University of Texas Southwestern Medical Center, Dallas, TX, and approved March 21, 2000 (received for review December 14, 1999) Severe quantitative and qualitative brown adipocyte defects are of these animals produced control OB͞OB and ob͞ob mice and common in obesity. To investigate whether aberrant expression of littermates with mutations at one or both TNFR (a detailed tumor necrosis factor ␣ (TNF-␣) in obesity is involved in functional description of these animals is reported in refs. 19–21). All of the brown fat atrophy, we have studied genetically obese (ob͞ob) animal experiments were carried out in accordance with the mice with targeted null mutations in the genes encoding the two highest standards of humane animal care. TNF receptors. The absence of both TNF receptors or p55 receptor alone resulted in a significant reduction in brown adipocyte apo- Terminal Deoxynucleotidyltransferase-Mediated UTP End Labeling ␤ ptosis and an increase in 3-adrenoreceptor and uncoupling pro- (TUNEL) Staining. In situ fragmentation of DNA was investigated tein-1 expression in obese mice. Increased numbers of multilocular in BAT slices derived from 8-week-old lean control, ob͞ob functionally active brown adipocytes, and improved thermoregu- control, and TNFR-deficient female mice by means of a detec- lation was also observed in obese animals lacking TNF-␣ function. tion kit (Boehringer Mannheim) based on direct alkaline phos- These results indicate that TNF-␣ plays an important role in mul- phatase detection of digoxigenin-labeled 3Ј-OH ends of genomic tiple aspects of brown adipose tissue biology and mediates the DNA fragments. The assay was performed according to the abnormalities that occur at this site in obesity. manufacturer’s instructions and the enzymatic reaction was developed by using 5-bromo-4-chloro-3-indolyl phosphate and besity in experimental models is ubiquitously associated nitroblue tetrazolium as chromogenic substrates. Ten high- Owith abnormalities in brown adipose tissue (BAT) (1, 2). In power fields (ϫ100) in each representative section of TUNEL- adult obese animals, the amount of thermogenically active BAT stained interscapular depots for different experiments were ␤ as well as the expression of 3-adrenoceptors in brown adipo- randomly observed and brown adipocyte apoptotic and nonapo- cytes are substantially reduced, potentially resulting in alter- ptotic nuclei were distinguished according to the level of staining ations of metabolic function and thermoregulation (3–5). The and morphological parameters, such as chromatin condensation, molecular basis of these obesity-related changes is poorly un- aggregation along the nuclear periphery, and apoptotic bodies. derstood. It has been demonstrated that expression of tumor necrosis factor ␣ (TNF-␣) in adipose tissue is elevated in a Brown Adipocyte Isolation, Cell Culture, and Treatment. Brown fat variety of experimental obesity models (6–9) and obese humans precursor cells were enzymatically isolated from the interscap- (10, 11). Owing to its ability to inhibit insulin receptor signaling ular BAT of male Sprague-Dawley rats (150–160 g body weight) (12–14), TNF-␣ represents a component of obesity-related in- (Harlan Nossan, Correzzana, Italy), which were kept in standard MEDICAL SCIENCES sulin resistance (15). More recently, TNF-␣ has been shown to laboratory conditions (12 h light͞dark cycle; food and water ad induce brown and white adipocyte apoptosis in vitro (16–18). libitum), as described (16). Three million cells seeded in 24-well Interestingly, in genetic models of obesity, the number of culture plates (Nunclon Delta, Milan, Italy) were cultured under apoptotic cells in the brown fat is dramatically higher than that a water-saturated atmosphere of 6% CO2 in air at 37°C in 2.0 ml in control animals (16). This leads to reduction in the number of of a culture medium, as described (16). In the experiments multilocular thermogenically active brown fat cells and therefore analyzing the effects of TNF-␣, the cultured cells were exposed causes BAT functional atrophy, which is characterized by de- to the culture medium containing murine recombinant TNF-␣ fective thermoregulation in both genetic and dietary obesity. In (freshly diluted in buffer as requested by manufacturer; Gen- addition, because BAT is an important target for insulin action zyme) with specific activities of 5 ϫ 107 units͞mg for 4 h and then and other aspects of energy metabolism, its atrophy could were harvested. The medium was discarded, the wells were further contribute to the abnormal metabolic profile in obesity. washed twice with 2 ml of ice-cold PBS (Sigma), and the cells Here, we have tested whether abnormal TNF-␣ production in were scraped out with a disposable cell scraper (Nunc, Milan, obesity contributes to any of these abnormalities in BAT. Italy) for mRNA extraction and reverse transcription–PCR analysis. Materials and Methods Generation of ob͞ob Mice Deficient in TNF Receptors (TNFR). Obese (ob͞ob) mice deficient in TNFR were generated as described This paper was submitted directly (Track II) to the PNAS office. (19–21). Mice with targeted null mutations at both TNFR 1 and Abbreviations: TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; UCP, 2 loci (p55Ϫ/Ϫ and p75Ϫ/Ϫ, respectively) were crossed with Ob͞ob uncoupling protein; BAT, brown adipose tissue; TUNEL, terminal deoxynucleotidyltrans- ferase-mediated UTP end labeling. mice, to produce animals heterozygous at the TNFR1, TNFR2, † ϩ/Ϫ ϩ/Ϫ ͞ To whom reprint requests should be addressed. E-mail: [email protected] or and ob loci (p55 , p75 , and Ob ob). The resulting triple [email protected]. heterozygotes were then crossbred with each other to produce ͞ The publication costs of this article were defrayed in part by page charge payment. This OB ob mice wild type at TNFR loci or with homozygous null article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. mutations in each or both TNFR. The subsequent intercrossing §1734 solely to indicate this fact. PNAS ͉ July 5, 2000 ͉ vol. 97 ͉ no. 14 ͉ 8033–8038 cAMP Accumulation. Cells were grown for 4 or 8 days (i.e., preconfluent or confluent cells). Two hours before each exper- iment, the medium was replaced by PBS containing 5 mM 3-isobutyl-1-methylxanthine. Cells were then incubated at 37°C for 30 min with or without CGP12177A [(Ϫ)-4-(3-t-butylamino- 2-hydroxypropoxy) benzimidazol-2-one) from CIBA–Geigy]. The reaction was stopped in ice by removing the incubation medium. The cells were harvested with ice-cold PBS, containing 5 mM 3-isobutyl-1-methylxanthine, and after sonication (100 w for 10 s) were centrifuged for 5 min. The resulting infranatants (i.e., liquid between floating fat cake and pelleted cell debris) were stored until cAMP determination (RIA, DuPont͞NEN). Protein content was measured by the BCA Protein Assay (Pierce). Reverse Transcription–PCR. Reverse transcription–PCR was per- formed and its products were quantitated as described (22). Total cytoplasmic RNA was isolated from cultured cells or BAT by using the RNazol method (TM Cinna Scientific, Friendswood, TX). A control experiment without reverse transcriptase was performed for each sample to verify that the amplification was not caused by any residual genomic DNA. The mRNA for ␤-actin was examined as the reference cellular transcript in each PCR reaction. The nucleotide sequences of the amplified prod- ucts were determined, and were found to be identical to the ␣ ␤ mRNA products of the rat and mouse TNF- , 3-adrenorecep- tor, uncoupling protein 1 (UCP-1), and ␤-actin genes. Morphological Evaluation of Brown Adipocytes. Five serial BAT ␮ slices at 200- m distance were prepared from three animals in Fig. 1. TUNEL staining of BAT. (a) Lean control mice acclimated to 28°C, the each group and stained with hematoxylin-eosin. Morphological thermoneutral temperature. (Inset) Intestinal villi showing apoptotic apical evaluation was performed in six optical fields randomly taken in epithelial cells as positive controls. (b) Lean control mice acclimated to cold each slice at ϫ25 magnification, and multilocularity of brown temperature (10°C for 10 days). (c) ob͞ob mice (wild type at TNFR loci). (Inset) adipocytes were counted and scored by two expert independent A negative adipose cell nucleus (arrow) between two apoptotic nuclei. (d) Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ observers. ob͞ob p55 / .(e) ob͞ob p75 / .(f) ob͞ob p55 / p75 / mice. [a–f and Inset a, ϫ125; Inset c, ϫ790.] Body Temperature Measurements. The body temperature of each mouse was first measured at room temperature with a digital ically obese animals was further validated by several other thermometer equipped with a rectal probe of 1-mm diameter. techniques, including agarose gel electrophoresis and transmis- After insertion of the probe 30 mm into the rectum, core body sion electron microscopy (16). In contrast to the ob͞ob animals, temperature is recorded on stabilization of the temperature the number of apoptotic nuclei in the ob͞ob mice lacking p55 reading for 10 consecutive seconds. Mice were then transferred Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ (ob͞ob-p55 / ) or both TNFR (ob͞ob-p55 / p75 / ) were sig- to a cold room and housed in prechilled individual cages at 4°C nificantly lower and approached the ratios observed in the where the body temperature was measured every 2 h. To prevent Ϯ Ϯ heat conduction to the animals, all equipment used in the control lean mice (2 0.3 and 4 1% positive nuclei, respec- tively; P Ͻ 0.005) (Fig.