Caveolin-1 Expression Is Essential for Proper Nonshivering Thermogenesis in Brown Alex W. Cohen,1,2 William Schubert,1,2 Dawn L. Brasaemle,3 Philipp E. Scherer,4 and Michael P. Lisanti1,2

Recently, we have shown that loss of -1 leads to ing and sphingolipids, which impart a relative marked alterations in signaling and lipolysis in insolubility in detergents at low temperatures, as well as white adipose tissue. However, little is known about the the propensity to “float” in sucrose density gradients (2). role of caveolin-1 in brown adipose tissue (BAT), a Unlike generalized lipid rafts, contain an abun- tissue responsible for nonshivering thermogenesis. dance of caveolin family members (caveolin-1, -2, Here, we show that caveolin-1 null mice have a mildly, and -3), the expression of which has been shown to result yet significantly, decreased resting core body tempera- in the characteristic invaginated morphology of these struc- ture. To investigate this in detail, we next subjected the mice to fasting (for 24 h) or cold treatment (4°C for tures (3–5). Via several tissue expression profile studies, it 24 h), individually or in combination. Interestingly, has been shown that caveolin-1 and -2 are coexpressed in caveolin-1 null mice showed markedly decreased body most differentiated cell types, excluding those of muscular temperatures in response to fasting or fasting/cold origin, with particularly high expression in , treatment; however, cold treatment alone had no effect. enothelia, epithelia, and type I pneumocytes of the lung In addition, under these conditions caveolin-1 null mice (6–8). Caveolin-3, on the other hand, is expressed only in failed to show the normal increase in serum nonesteri- striated and cardiac myocytes, where it is thought to be fied fatty acids induced by fasting or fasting/cold treat- the only caveolin family member present (9,10). ment, suggesting that these mice are unable to liberate triglyceride stores for heat production. In accordance The generation of caveolin-deficient mice by several with these results, the triglyceride content of BAT was independent groups has provided evidence for the essen- reduced nearly 10-fold in wild-type mice after fasting/ tial nature of the caveolin in the regulation of cold treatment, but it was reduced only 3-fold in caveo- signaling along several independent pathways (3,4). Phe- lin-1 null mice. Finally, electron microscopy of adipose notypic evaluation of caveolin-1 null mice has resulted in tissue revealed dramatic perturbations in the mitochon- an abundance of reports demonstrating changes in multi- dria of caveolin-1 null interscapular brown adipocytes. ple organ systems and molecular variations, which both Taken together, our data provide the first molecular confirm and refute previous predictions (11). One of the genetic evidence that caveolin-1 plays a critical func- tional and structural role in the modulation of thermo- initial phenotypes identified in caveolin-1 null mice is genesis via an effect on lipid mobilization. resistance to diet-induced obesity, characterized by 54:679–686, 2005 marked lipid imbalances (3). Further examination of this phenotype has revealed that loss of caveolin-1 leads to progressive white adipose tissue (WAT) atrophy, brown adipose tissue (BAT) hypertrophy, postprandial hypertri- aveolae, small flask-shaped invaginations of the glyceridemia, and mild with drastically plasma membrane, represent a morphologically decreased insulin signaling in adipocytes (3,12). Further- identifiable subset of the liquid-ordered do- more, caveolin-1 null mice show marked decreases in Cmains, often referred to as lipid rafts (1). These lipolysis after either physiological (fasting) or pharmaco- structures are enriched in several key constituents, includ- logical stimulation (13). Additionally, isolated fibroblasts from caveolin-1 null mice, stably transfected with the lipid From the 1Department of Molecular Pharmacology, Albert Einstein College of droplet coat protein, perilipin, are markedly defective in Medicine, Bronx, New York; the 2Albert Einstein Cancer Center, Bronx, New their ability to accumulate intracellular lipid stores, com- 3 York; the Department of Nutritional Sciences, Rutgers, the State University of pared with wild-type perilipin-transfected cells (13). These New Jersey, New Brunswick, New Jersey; and the 4Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York. studies suggest that although decreased lipolysis in WAT Address correspondence and reprint requests to Michael P. Lisanti, Depart- should result in obesity, caveolin-1 null mice remain lean, ment of Molecular Pharmacology, The Albert Einstein Cancer Center, 1300 Morris Park Ave., Bronx, NY 10461. E-mail: [email protected]. at least in part, because of an inability to properly store Received for publication 3 August 2004 and accepted in revised form triglycerides in adipose tissue. Another possible contrib- 6 December 2004. uting factor to this leanness is the finding that the BAT of ADRP, adipose differentiation–related protein; AMPK, AMP-activated pro- tein kinase; BAT, brown adipose tissue; HSL, hormone-sensitive lipase; Hsp60, caveolin-1 null mice is markedly hypertrophic, perhaps heat shock protein 60; mDIC, mitochondrial dicarboxylate carrier protein; suggesting that triglycerides are “burned off” in this tissue NEFA, nonesterified ; pAb, polyclonal antibody; PKA, ; UCP, uncoupling protein; WAT, white adipose tissue. at a higher rate than in wild-type mice. © 2005 by the American Diabetes Association. Genetic studies in rodents have implicated BAT both in The costs of publication of this article were defrayed in part by the payment of page the maintenance of body temperature as well as in the charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. regulation of body weight (14–17). BAT functions as a

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thermoregulatory organ, generating heat via “nonshivering (wild-type n ϭ 5, caveolin-1 null n ϭ 5), or further fasted for 24 h at 4°C thermogenesis,” which involves the BAT-specific mito- (wild-type n ϭ 5, caveolin-1 null n ϭ 5), at which time core body temperature was again assessed. The total time of the fasting treatments thus totaled 28 h chondrial uncoupling protein (UCP)-1 (14,18). In BAT, the (4-h baseline fast ϩ 24-h experimental fast). cold-induced activation of lipolysis results in the phos- Serum analysis. Baseline serum was collected from wild-type (n ϭ 10) and phorylation of perilipin and the hydrolysis of stored trig- caveolin-1 null (n ϭ 10) mice by bleeding the tail of each mouse. Mice were lycerides by activated hormone-sensitive lipase (HSL) either further fasted for 24 h at ambient temperature (wild-type n ϭ 5, caveolin-1 null n ϭ 5) or further fasted for 24 h at 4°C (wild-type n ϭ 5, (19–21). Released fatty acids are transferred to mitochon- caveolin-1 null n ϭ 5), at which time serum was again collected from the tail. dria for ␤-oxidation, whereupon they are used to generate Nonesterified fatty acid (NEFA; Half-Micro tests; Roche) and triglyceride heat instead of ATP via the “uncoupling” action of UCP-1 (GPO-Trinder; Sigma) levels were determined colorimetrically. (14). This mechanism is also thought to allow animals to Triglyceride analysis. triglycerides were extracted according to burn off excess caloric intake as heat and thus protect the methods of Dole and Meinertz as modified by Carpe´ne´ (30). Briefly, the interscapular brown fat pads were removed from each mouse (n Ն 5 mice for against obesity. Although the role of BAT in thermoregu- each group), and ϳ50 mg of tissue was homogenized in 2 ml of homogeniza- lation is generally well accepted, its role in obesity is less tion buffer (20 mmol/l Tris, pH 7.3, 1 mmol/l EDTA, 1 mmol/l ␤-mercapto- so. For instance, targeted disruption of the Ucp-1 ethanol). Then, 1 ml of this homogenate was placed in a glass tube, and 2 ml revealed that homozygous knockout mice are unable to of extraction buffer (78% vol/vol isopropanol, 20% vol/vol heptane, and 2% vol/vol 1N sulfuric acid) was added. Next, 2 ml of heptane was added and maintain normal body temperature when exposed to 4°C mixed, and the extracts were allowed to stand until the two phases separated. (15). However, these mice are not obesity prone. Further Then, 2 ml of the upper organic phase was transferred to a glass vial and ␮ complicating the issue, additional studies have shown that evaporated under N2. The sample was redissolved in 250 l of heptane, and complete ablation of BAT in mice, via targeted expression triglycerides were measured colorimetrically (Wako Chemicals, Nuess, Ger- of diphtheria toxin in brown adipocytes, results in obesity many). Results are expressed as a function of total cellular protein content, which was determined using the bicinchoninic acid reagent (Pierce). and insulin resistance (16). Additionally, studies in trans- Immunoblot analysis. Western blotting of interscapular BAT from wild-type genic mice, which overexpress the Ucp-1 gene under and caveolin-1 null mice (n ϭ 5 mice for each group) was performed control of the aP2 promoter, have shown that these mice essentially as previously described (12,13). Quantification of Western blots are resistant to diet-induced obesity (22,23). was performed by densitometry. A P value of Ͻ0.05 was deemed significant. Preparation of paraffin sections. Mice (n Ն5 for each group) were killed, Based on this information, coupled with the findings that and the interscapular brown fat pads were removed, processed, and stained caveolin-1 null mice are resistant to diet-induced obesity with hematoxylin and eosin, as previously described (31). while showing marked hypertrophy of BAT, we hypothe- High-pressure freeze transmission electron microscopy. High-pressure sized that the observed resistance to obesity could be freeze electron microscopy was performed on wild-type (n ϭ 2) and caveo- based on the increased metabolic capacity of BAT and lin-1 null (n ϭ 2) mice at baseline, as previously described (13). Assessment of mitochondrial membrane integrity. Mitochondria were would thus be manifested by an increased core body isolated from BAT at baseline (n ϭ 5 mice for each group) with the aid of a temperature in caveolin-1 null mice. commercially available kit (MITO-ISO1; Sigma, St. Louis, MO). The integrity of the inner membrane was assessed according to the manufacturer’s instruc- tions (MITO-ISO1; Sigma). Outer membrane integrity was assessed with the RESEARCH DESIGN AND METHODS aid of another commercially available kit (CYTOC-OX1; Sigma). Antibodies and their sources were as follows: guinea pig anti-perilipin, guinea Statistical analysis. All results were analyzed using a two-tailed t test. A pig anti–adipose differentiation-related protein (ADRP), and rabbit anti- P value of Ͻ0.05 was deemed significant. prohibitin (all polyclonal antibodies [pAb]; Research Diagnostics, Flanders, NJ); anti–phospho-ser/thr-protein kinase A (PKA)-specific phosphosubstrate, anti–phospho-AMP-activated protein kinase (AMPK)-␣ (thr172), and anti– RESULTS AMPK-␣, (all pAb; , Beverly, MA); polyclonal rabbit mitochon- drial dicarboxylate carrier protein (mDIC), which was produced as previously Caveolin-1 null mice are intolerant to fasting and described (24); rabbit anti–UCP-1 (pAb; Chemicon International, Temecula, cold treatment. The ability of small mammals, such as CA); rabbit anti–heat shock protein 60 (Hsp60; pAb; Stressgen, Victoria, BC, mice, to maintain their body temperature during acute Canada); rabbit anti–CD-36 (pAb; Cayman Chemical, Ann Arbor, MI); rabbit cold exposure or prolonged fasting depends on heat anti–adipocyte-fatty acid binding protein/aP2 (pAb; Alpha Diagnostics, San Antonio, TX); rabbit anti–S3-12, which was produced as previously described production via two sources: 1) shivering thermogenesis in (25); and mouse anti–flotillin-1 (mAb; BD Transduction Laboratories, San muscle and 2) nonshivering thermogenesis in BAT. To Jose, CA). Anti-HSL (pAb) was a generous gift from Dr. Constantine investigate the role of caveolin-1 in heat production via Londos (National Institutes of Health, Bethesda, MD). these two sources, we subjected caveolin-1 null mice to Animal Studies. Caveolin-1 null mice and the corresponding wild-type prolonged fasting (24 h) as well as to acute exposure to cohorts, in the C57BL/6 genetic background, were generated and maintained as previously described (26,27). For these experiments, homozygous caveo- low temperatures (4°C). Because small, captive, nocturnal lin-1 null mice were backcrossed into the parental C57BL/6 genetic back- mammals regularly conserve energy stores by entering a ground eight times. Wild-type and caveolin-1 null littermates used in this study state of daily torpor during the inactive part of their were obtained by interbreeding generation F8 caveolin-1 heterozygous mice. circadian rhythm (second part of the night through early Thus, all mice are on a relatively pure C57BL/6 genetic background. All studies were carried out in mice between the ages of 8 and 10 weeks, a time when it morning), during which body temperature can approach has been shown that body weights of these mice are indistinguishable (26). ϳ25°C (29), we performed all temperature measurements Throughout the article, “baseline” refers to mice that have been fasted for 4 h after the end of this period. Compared with wild-type to limit the effects of recent food consumption on core body temperature mice, caveolin-1 null mice have a marginally, yet signifi- (14,28). cantly, reduced basal core body temperature (37.6 vs. Colonic temperature. The colonic temperature was measured as an indica- Ͻ tion of core body temperature. A lubricated digital probe (VWR International, 37.2°C, respectively; P 0.05) (Fig. 1). Interestingly, when Westchester, PA) was inserted 2 cm into the rectum and held in place until a a cohort of these mice was further fasted for 24 h, we stabilized measurement was achieved. An average of two measurements was found that whereas wild-type mice were capable of main- taken from each mouse. Baseline (4 h fasting) temperatures were collected taining a relatively normal body temperature (37.1°C), from wild-type (n ϭ 15) and caveolin-1 null (n ϭ 15) mice at 10:00 A.M., when Ͻ the mice had returned to the euthermic state after daily torpor (29). Mice were caveolin-1 null mice were not (35.6°C, P 0.05) (Fig. 1). either further fasted for 24 h at ambient temperature (wild-type n ϭ 5, When we next exposed wild-type and caveolin-1 null caveolin-1 null n ϭ 5), placed at 4°C with free access to food for 24 h mice to the cold (4°C, 24 h) with free access to food, we

680 DIABETES, VOL. 54, MARCH 2005 A.W. COHEN AND ASSOCIATES

previously (13), indicate that lipolysis fails to occur in caveolin-1 null mice under either condition. Because the measurement of serum NEFAs predominantly represents fatty acids released from WAT, we next attempted to more directly assess the lipolytic response to fasting and fasting/ cold in BAT, as outlined in subsequent figures. Studies in small mammals and humans have demon- strated that cold exposure leads to dramatic increases in serum NEFA levels, which is accentuated by fasting (32– 35). Additionally, it has been shown that in humans, ingestion of nicotinic acid, which severely limits free fatty acid mobilization from adipocytes, significantly blunts cold-induced heat production via nonshivering thermogen- esis (34). This scenario is, at a basic level, similar to that FIG. 1. Colonic temperature. Caveolin-1 null mice exhibit resting which occurs in caveolin-1 null mice, where attenuated hypothermia and are markedly cold intolerant. At baseline, the colonic mice was significantly higher than lipolysis restricts the availability of free fatty acids as a (15 ؍ temperature of wild-type (n mice (37.6 vs. 37.2°C, P < source for heat production in BAT, leading to resting and (15 ؍ that of caveolin-1 null (knockout, n 0.05). After a further 24-h period of fasting, the colonic temperature of cold-induced hypothermia. ,(decreased significantly (35.6°C, P < 0.05 (5 ؍ caveolin-1 null mice (n was not altered from baseline. In addition to NEFA levels, we also analyzed serum (5 ؍ whereas that of wild-type mice (n After exposure to the cold (24 h, 4°C) with free access to food, the triglyceride levels in wild-type and caveolin-1 null animals. (5 ؍ and caveolin-1 null (n (5 ؍ colonic temperature of wild-type (n mice decreased significantly from baseline values (35.8 and 35.3°C, At both baseline and after fasting/cold treatment, there respectively); however, the resultant temperatures were not statisti- was no difference between the two cohorts of mice, with cally significant from one another. After exposure to the cold (24 h, total serum triglycerides decreasing approximately two- 4°C) with concurrent fasting, the colonic temperature of wild-type mice decreased significantly from fold after exposure to fasting/cold (Fig. 2B). These results (5 ؍ and caveolin-1 null (n (5 ؍ n) baseline (34.2 and 23.4°C, respectively). Additionally, the temperature indicate that loss of caveolin-1 does not effect the com- of caveolin-1 null mice was significantly lower than that of wild-type bustion of circulating triglycerides during cold exposure. mice after exposure to these experimental conditions (P < 0.05). .Values represent the means ؎ SE. *P < 0.05. f, wild type; Ⅺ, knockout found that there was no statistical difference between the resultant core body temperature in these mice (35.8 vs. 35.3°C, P ϭ 0.3) (Fig. 1). However, these values were statistically below those of mice at baseline, indicating that acute cold exposure produces hypothermia in these animals. When we subsequently exposed a cohort of wild-type and caveolin-1 null mice to the cold (4°C, 24 h) with concurrent fasting (fasting/cold treatment), we found that the core body temperature of caveolin-1 null mice fell precipitously (23.4°C), whereas wild-type mice were able to maintain a much higher temperature (34.2°C; P Ͻ 0.05) (Fig. 1). Furthermore, after 24 h of this treatment, caveo- lin-1 null mice were found to be markedly obtunded and shivering intensely, whereas wild-type mice appeared rel- atively normal. Thus, these results indicate that the mech- anisms involved in heat production via both shivering and nonshivering thermogenesis appear intact in caveolin-1 null mice; however, the maintenance of body temperature requires constant access to food, suggesting that the liberation of stored energy is defective in caveolin-1 null mice. NEFA levels fail to rise in caveolin-1 null mice after fasting or cold exposure. Because we have previously shown that pharmacologically mediated lipolysis from WAT is severely defective in caveolin-1 null mice (13), we next sought to investigate whether an analogous defect may exist after fasting or fasting/cold treatment. At base- FIG. 2. Levels of serum NEFAs do not rise significantly in caveolin-1 null mice in response to fasting or fasting/cold treatment. A: Serum line we found that there was no significant difference NEFA. At baseline, serum NEFA levels are not statistically different between serum NEFA levels in wild-type and caveolin-1 between wild-type and caveolin-1 null (knockout) mice. Serum NEFA levels rise normally after fasting or fasting/cold treatment in wild-type null mice (Fig. 2A). However, when mice were subjected mice, but not in caveolin-1 null mice, suggestive of defective lipolysis. to either fasting or fasting/cold treatment, we found that B: Serum triglycerides. At baseline serum triglyceride levels are equiv- serum NEFA levels rose normally and significantly in alent in wild-type and caveolin-1 null mice. After exposure to 24-h fasting/cold treatment, triglyceride levels decrease ϳ50% in both sets ,wild-type mice (32), but they failed to rise in caveolin-1 of mice. Values represent the means ؎ SE. *P < 0.05. f, wild type; Ⅺ null mice. These results, along with those reported knockout.

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lin-1 null BAT (Fig. 3). However, after fasting/cold treat- ment, the triglyceride content of wild-type and caveolin-1 null BAT pads both decreased significantly; on the other hand, levels in wild-type mice fell nearly 10-fold, whereas levels in caveolin-1 null mice were reduced only ϳ3-fold (P Ͻ 0.05). These data indicate that the liberation of stored triglycerides is defective in the BAT of caveolin-1 null mice and, most likely, contributes to the observed phenotype. We next analyzed paraffin-embedded hematoxylin and eosin–stained BAT sections from wild-type and caveolin-1 null mice at baseline and after fasting/cold treatment (Fig. 4). At baseline, the adipocytes of wild-type and caveolin-1 null BAT pads were filled with multilocular lipid droplets FIG. 3. Tissue triglycerides. Mobilization of brown fat triglyceride that, overall, appeared larger in caveolin-1 null mice. In- stores is defective in caveolin-1 null mice. Interscapular brown fat pads terestingly, after fasting/cold treatment, the adipocytes of were removed from wild-type (WT) and caveolin-1 null (knockout wild-type mice were essentially devoid of lipid droplets, [KO]) mice at baseline and after 24-h exposure to the cold (4°C) while fasting (fasting/cold treatment). Total tissue lipids were extracted, whereas those of caveolin-1 null mice appeared more like and triglyceride content was analyzed colorimetrically. Note that at BAT at baseline. In addition, it appears as though the small- baseline, the triglyceride content of wild-type and caveolin-1 null BAT er lipid droplets in caveolin-1 null cells have been utilized, pads is not statistically different. Exposure to fasting/cold treatment caused a significant reduction in the triglyceride content of both perhaps indicating that proteins other than perilipin (i.e., wild-type and caveolin-1 null fat pads from baseline (P < 0.05); S3-12 or adipophilin) surround these smaller droplets however, the triglyceride levels in wild-type BAT fell significantly more than caveolin-1 null BAT (ϳ9.5- vs. ϳ3-fold reduction, respectively; and are not subject to the same caveolin-1–mediated reg- P < 0.05). Values represent the means ؎ SE. *P < 0.05 wild-type (f) vs. ulation as perilipin-coated droplets. Overall, these changes knockout (ᮀ) with cold/fasting. are consistent with those noted above and are further in- dicative of a major lipolytic failure in caveolin-1 null mice. Analysis of BAT triglyceride content and histological Western blot analysis of BAT demonstrates changes examination of hematoxylin and eosin–stained BAT consistent with defective lipolysis in caveolin-1 null sections reveals changes consistent with defective mice. We next analyzed the expression levels of several BAT lipolysis in caveolin-1 null mice. To explore the key proteins involved in brown fat lipid metabolism for notion that defective lipolysis results in cold and fasting any alterations from wild-type. Similar to what we have intolerance in caveolin-1 null mice, we next examined the reported previously in WAT (13), we found that although triglyceride content of the interscapular brown fat pads of the protein levels of perilipin were equal between the two wild-type and caveolin-1 null mice at baseline and after cohorts of mice (P ϭ 0.08), the of perili- combined fasting/cold treatment. Using a protocol de- pin in response to lipolytic stimulation (i.e., fasting/cold) signed to isolate triglycerides from tissue, we noticed that was ϳ4.5-fold less (4.63 Ϯ 0.48, P Ͻ 0.05) in caveolin-1 null at baseline there was no statistically significant difference mice (Fig. 5A). Unlike in WAT (data not shown), it seems between the triglyceride content of wild-type and caveo- that in BAT, both perilipin-A and -B are subjected to major

FIG. 4. Paraffin-embedded hematoxylin and eosin–stained BAT sections demon- strate decreased lipid utilization in caveolin-1 null mice after fasting/cold treatment. Interscapular BAT pads were removed from wild-type (WT) and caveolin-1 null (knockout [KO]) mice at baseline and after 24 h of fasting/cold treatment and processed for staining with hematoxylin and eosin. Note that at baseline, brown adipocytes contain abundant multilocular lipid droplets, which appear as vacant or unstained circular areas in both wild-type and caveolin-1 null animals. After fasting/ cold treatment, the BAT of wild-type mice is essentially devoid of lipid drop- lets, whereas in caveolin-1 null BAT, large circular lipid droplets are still plentiful.

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phosphorylation in response to lipolytic stimulation. In caveolin-1 null mice, phosphorylation of both perilipin isoforms is dramatically decreased, consistent with the idea that lipolysis is defective in the BAT of these mice. Regarding other lipid droplet–associated proteins, such as HSL, S3-12, and adipophilin (ADRP), we noted no statistically significant alterations in caveolin-1 null mice at either baseline or after cold/fasting, except in the case of HSL, which increases approximately twofold in caveo- lin-1 null mice (2.13 Ϯ 0.22, P Ͻ 0.05) but not in wild-type mice, after fasting/cold treatment (Fig. 5B). Several stud- ies have shown that HSL expression in BAT and WAT is altered by dietary manipulations, cold exposure, and chron- ic ␤-agonist stimulation; however, a consensus on whether these conditions lead to an increase or decrease in HSL expression is lacking (21,36–39). Furthermore, the com- bined effects of cold and fasting on HSL expression was not explored in any of these studies. Interestingly, flotil- lin-1, a caveolar resident protein that has recently been shown to associate with lipid droplets (40), was found to be upregulated by about twofold (1.97 Ϯ 0.16, P Ͻ 0.05) in the BAT of caveolin-1 null mice at baseline. After cold/ fasting, the levels of this protein increase above baseline wild-type values approximately fourfold in both cohorts of mice (4.30 Ϯ 0.48, P Ͻ 0.05) (Fig. 5B). Because the role, if any, of flotillin-1 in lipid droplet metabolism is unknown, it is difficult to speculate on the possible functional signifi- cance of these alterations in protein expression. Additionally, two proteins involved in lipid binding and transport, CD36 and adipocyte–fatty acid binding protein/ aP2, were found to be statistically unchanged in caveolin-1 null mice compared with wild-type mice (Fig. 5C). Both of these proteins were upregulated approximately twofold (P Ͻ 0.05) in response to fasting/cold treatment in the BAT of wild-type and caveolin-1 null mice. High-pressure freeze electron microscopy reveals dramatic morphologic defects in the mitochondria of caveolin-1 null brown adipocytes. Using a relatively new technique, high pressure and rapid tissue freezing, we examined the interscapular BAT from wild-type and caveolin-1 null mice by electron microscopy. BAT derived from wild-type mice shows characteristic brown adipo- cytes, with multilocular lipid droplets (Fig. 6, asterisks) and abundant mitochondria (Fig. 6, arrows). Remark- ably, examination of caveolin-1 null BAT reveals dra- matic alterations in adipocyte morphology. In these cells, the mitochondria are markedly larger and dilated (Fig. 6, FIG. 5. Western blot analysis of caveolin-1 null BAT reveals changes arrows). Furthermore, they are much less electron dense consistent with defective lipolysis. Lysates were prepared from wild- than the mitochondria of wild-type mice, thus appearing type (WT) and caveolin-1 null (knockout [KO]) interscapular BAT pads at baseline and after 24 h of fasting/cold treatment. A: Perilipin levels much lighter in color. and phosphorylation state. Immunostaining with an anti-perilipin an- The results shown here are representative of all of the tibody and an antibody that recognizes phosphorylated perilipin iso- forms reveals that 24 h of fasting/cold treatment leads to marked images gathered in which nearly 95% of the mitochondria phosphorylation of both perilipin-A and -B in wild-type, but not in appear altered in caveolin-1 null BAT. This dilation is most caveolin-1 null BAT. B: Levels of HSL, flotillin-1, S3-12, and ADRP. HSL likely the result of an altered osmotic gradient between the levels, which are equal at baseline, increase in caveolin-1 null but not in wild-type BAT with cold/fasting. Flotillin-1 is elevated in the BAT of inner mitochondrial membrane and the , caus- caveolin-1 null animals at baseline. After fasting/cold treatment, flo- ing an influx of fluid. It is possible that caveolin-1 provides tillin-1 protein expression is markedly increased in both wild-type and a necessary scaffolding structure for protein, water, or ion caveolin-1 null BAT. The protein levels of S3-12 and ADRP (adipophi- lin) are unaltered by loss of caveolin-1 or by fasting/cold treatment. C: transport components across the mitochondrial mem- Levels of CD36 and fatty acid binding protein (FABP). The expression brane, the disruption of which leads to the accumulation of the fatty acid binding/transport proteins CD36 and FABP (aP2) increase after exposure to cold/fasting in both wild-type and caveolin-1 of proteins or ions within the mitochondria. This specula- null BAT pads. tion is not without basis, as ion and water channels have been shown to be associated with plasma membrane

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FIG. 6. High-pressure freeze electron mi- croscopy reveals dramatic alterations in the mitochondria of caveolin-1 null mice. Mice were killed in the basal state, and the inter- scapular brown fat pads were removed and processed for high-pressure freeze electron microscopy. Note that the mitochondria (ar- rows) of caveolin-1 null (knockout [KO]) brown adipocytes appear markedly dilated and less electron dense than those of wild- type (WT) mice. Abundant lipid droplets are present in the adipocytes of both genotypes 1 ؍ asterisks) under these conditions. Bar) .␮m; original magnification 5,000؋ caveolae (41,42). In addition, caveolin-1 has been shown to either the basal or induced expression levels of this be targeted to a variety of intracellular locations, including protein (Fig. 7A). Because activation of PKA is known to mitochondria, in a tissue-specific fashion (43). Although be involved in the upregulation of UCP-1 mRNA (45), and the mitochondria of BAT have not been studied directly, it PKA activity is negatively regulated by an interaction with is possible that caveolin-1 normally localizes to the mem- caveolin-1 (13), it is somewhat surprising that loss of brane of this organelle and when absent leads to the caveolin-1 does not affect the expression of UCP-1. In view phenotype described above. of our previous results suggesting an uncoupling of PKA To investigate whether these morphologic changes were activity and perilipin phosphorylation in caveolin-1 null accompanied by alterations in mitochondrial function, we WAT (13), it is possible that the localized activation of next examined the expression of several mitochondrial pro- PKA, which would necessarily effect UCP-1 expression, teins by Western blot. Interestingly, expression of mDIC was may not be altered by loss of caveolin-1. That is, whereas found to decrease ϳ3.5-fold (3.61 Ϯ 0.68, P Ͻ 0.05) after the catalytic subunits of PKA, which would normally be fasting/cold treatment in wild-type mice, as shown previ- sequestered and inactivated within caveolae, become hy- ously (24); however, in caveolin-1 null mice the expression peractivated in the absence of caveolin-1, those catalytic levels of mDIC remain statistically unchanged (Fig. 7A). subunits that are normally associated with the regulatory Expression of mDIC in white adipocytes has been shown subunits in other subcellular locations are not effected by to be affected by cold exposure (downregulation), as well the absence of caveolin-1. as free fatty acid loading (upregulation). mDIC may play a In a further assessment of mitochondrial function and role in fatty acid biosynthesis/lipogenesis and/or glycero- ␤-oxidation, we also analyzed the expression and phos- neogenesis, and its decrease with cold exposure probably phorylation of AMPK subunits. Interestingly, we found reflects a decreased need for these two processes, where- that although the phosphorylation of the AMPK-␣ catalytic as its increase with fatty acid loading may reflect an subunit increases approximately fourfold (4.19 Ϯ 0.67, P Ͻ increase in lipogenesis (24). Failure of mDIC levels to 0.05) in wild-type animals after fasting/cold treatment, its decrease in caveolin-1 null BAT after fasting/cold treat- activation increases only twofold (2.20 Ϯ 0.19, P Ͻ 0.05) in ment may reflect a general uncoupling of the signaling caveolin-1 null mice (Fig. 7B). These alterations occurred cascades involved in the regulation of lipolysis and lipo- without statistically significant changes in the expression genesis, as we have previously characterized in WAT of total AMPK-␣. Because AMPK is normally activated in (12,13). In addition to mDIC, we also analyzed the expres- response to cellular stress (i.e., deprivation) and sion levels of other mitochondrial proteins, i.e., prohibitin, functions to essentially turn on fatty acid oxidation and which is involved in the processing and stabilization of turn off fatty acid synthesis (46), these results suggest that newly transcribed mitochondrial membrane proteins (44), a loss of caveolin-1 leads to decreased ␤-oxidation in BAT and Hsp60, a mitochondrial matrix marker protein, neither after fasting/cold treatment, which could thus help to of which was found to be affected by a loss of caveolin-1. explain the observed cold intolerance. Prohibitin levels remained statistically unaltered after As an assessment of mitochondrial function, we next fasting/cold treatment, whereas Hsp60 levels decreased used two specific techniques that allow one to monitor the nearly twofold (1.91 Ϯ 0.21, P Ͻ 0.05) (Fig. 7A). Western integrity of both the inner and outer mitochondrial mem- analysis of UCP-1 in wild-type and caveolin-1 null BAT branes. Isolated mitochondria were obtained from the BAT samples revealed that this protein is upregulated nearly of wild-type and caveolin-1 null animals in the basal state. 3.5-fold (3.43 Ϯ 0.53, P Ͻ 0.05) after exposure to fasting/ The integrity of the inner membrane, determined via uptake cold treatment and that loss of caveolin-1 did not alter of the fluorescent potential-sensitive dye JC-1, was found

684 DIABETES, VOL. 54, MARCH 2005 A.W. COHEN AND ASSOCIATES

FIG. 7. Western blot analysis of several proteins reveals alterations in mDIC and AMPK-␣ levels in caveolin-1 null animals. A: Lysates derived from wild-type (WT) and caveolin-1 null (knockout [KO]) BAT were subjected to Western blot analysis with antibodies specific to the following four mitochondrial proteins: mDIC (dicarboxylate carrier), prohibitin, Hsp60, and UCP-1. In the absence of caveolin-1, mDIC fails to decrease normally in caveolin-1 null BAT after fasting/cold treatment. Loss of caveolin-1 did not alter the expression patterns of the other proteins analyzed. B: Western blot analysis of wild-type and caveolin-1 null BAT reveals that the activation of AMPK-␣ is defective in the absence of caveolin-1. C and D: Biochemical analysis of outer (C) and inner (D) mitochondrial membrane integrity. The integrity of the outer membrane was assessed by measuring the activity of cytochrome C oxidase in the presence and absence of the detergent n-dodecyl ␤-maltoside. The integrity of the inner membrane was measured by assessing the electrochemical gradient across this membrane with the aid of a fluorescent dye, JC-1. Note ,that isolated BAT mitochondria do not show any drastic functional alterations in caveolin-1 null animals. Values represent the means ؎ SE. f wild type; Ⅺ, knockout. to be unchanged in caveolin-1 null mice (Fig. 7D). Similarly, mice would be expected to routinely experience in their the outer membrane integrity, which can be assayed by natural environment. That is, nondomesticated mice do measuring cytochrome C oxidase activity in the presence not have constant access to food and are often exposed to and absence of detergent, was unchanged in caveolin-1 cold temperatures, especially during the winter months. null BAT (Fig. 7C). Thus, although striking morphologic On a prolonged basis, it is expected that caveolin-1 null abnormalities are present in the mitochondria of caveo- mice would not survive under these conditions and, as lin-1 null animals, no drastic functional alterations in the such, would not necessarily be able to survive outside of membrane integrity of these organelles are apparent. their carefully controlled experimental environment. Thus, caveolin-1 would likely be required for DISCUSSION long-term survival in nature. In summary, this study shows that despite the resistance to diet-induced obesity, no evidence exists for the hyper- ACKNOWLEDGMENTS activation of BAT and consumption of excess caloric in- We thank Dr. Constantine Londos for donating the HSL take via this route in caveolin-1 null mice. Instead, BAT antibodies. This work was supported by grants from the appears relatively inactive because of an inhibition of li- National Institutes of Health (NIH) and the Susan G. polysis in caveolin-1 null mice, in accordance with our Komen Breast Cancer Foundation (to M.P.L) and by NIH findings in WAT (13). In addition, dramatic alterations in grant R01-DK55758 and the American Diabetes Associa- BAT mitochondrial structure were observed. tion (to P.E.S.). A.W.C. was supported by NIH Medical It is interesting to note that the experimental conditions Scientist Training Grant T32-GM07288. M.P.L is the recip- to which these mice were exposed are similar to those ient of a Hirschl/Weil-Caulier career scientist award.

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