INTRODUCTION

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PREVIOUS INVESTIGATIONS have demonstrated that older vs. younger rats have blunted cold-induced nonshivering thermogenesis of brown adipose tissue (BAT; 16, 30, 35, 41). This age-related attenuation in cold-induced brown fat nonshivering thermogenesis is associated with reduced mass, protein, and thermogenic capacity, the latter being manifested by total tissue mitochondrial GDP binding and uncoupling protein-1 (UCP₁) content. When GDP binding and UCP₁ content are expressed per unit of mass, rather than as a function of the total mass, differences between the age groups are not, in general, observed (18, 30, 34). We interpret the loss in total but not specific UCP₁/GDP binding as indicating that the amount of BAT is a primary determinant of the age-related reduction of BAT thermogenic capacity. This suggestion is consistent with previous observations of significantly less interscapular BAT (IBAT) mass in old vs. young adult rats (30, 34, 35, 42, 48).

The mechanisms underlying the age-related reduction in brown fat mass remain to be determined. Investigations using other tissues suggest that this alteration may be associated with an age-related decrease in the proliferative capacity of preadipocytes. For example, cultured human skin fibroblasts lose ~30% of their proliferative capacity throughout the lifespan of the donor (27, 28). These cells undergo a decline of ~0.2 cell doublings per year in donors 0-90 yr of age. There is also a significant age-related decline in the proliferative activity of cultured human epidermal keratinocytes (39). We suspect that similar alterations in brown fat cellular proliferation may account for the loss in brown fat mass occurring in older rats.

The age-related decline in cellular proliferative capacity during aging may be associated with altered levels of growth factors involved in the proliferative process and/or blunted responsiveness to these factors. One growth factor is norepinephrine (NE; 13, 15), which is released from sympathetic neural terminals widely distributed throughout BAT. It is thought that NE binds to the ₁-adrenergic receptor on brown adipocyte precursor cells and, through the adenosine 3',5'-cyclic monophosphate (cAMP) cascade, promotes proliferation and maturation of brown preadipocytes (12, 38). Local release of NE is essential for an optimal proliferative response of BAT to chronic cold exposure (5°C for 4 days; 11). In addition to stimulating BAT growth, NE enhances transcription of UCP₁ (the brown fat-specific mitochondrial protein that mediates cold-induced brown adipocyte thermogenesis by dissipating the proton gradient and allowing fatty acid oxidation to proceed at high rates). Removal of the sympathetic neural signal to BAT by surgical denervation significantly blunts the UCP₁ increases in response to chronic cold (38).

The purpose of this study was to test the hypothesis that brown fat in 26-mo-old Fischer 344 (F344) male rats has an impaired capacity to respond to the trophic stimulus of chronic cold exposure with increased cell number, mass, and UCP₁ content. To test this, we measured IBAT mass, cell size and proliferation, and UCP₁ content in IBAT from young and old rats exposed to 10°C for 5 days. In addition, we assessed plasma levels of insulin-like growth factor I (IGF-I) and NE, potential BAT growth factors.

	MATERIALS AND METHODS

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Animals and Animal Care

Experimental Protocol

Rats in each age group were divided into two experimental groups (4-6 rats per group). One group was maintained at 25°C, which is within the thermoneutral zone for these animals (30), for 5 days. The other group was housed at 10°C for 5 days. Other environmental conditions were identical to those described above. We used 10°C for the chronic cold exposure because pilot studies indicated that many 26-mo-old rats were unable to maintain core temperature at 4-8°C for more than 24 h. Preliminary data showed that 10°C provided a significant cold stress demonstrated by increased UCP₁ and IBAT mass in 6-mo-old animals.

One day before the beginning of the thermoneutral or cold exposure (t_pre) and once daily throughout the 5 days of exposure (t₁-t₅), 0.6 ml of arterial blood was collected and body mass and colonic temperature were recorded between 1100 and 1200. Arterial blood was collected in chilled microfuge tubes, centrifuged at 6°C for 20 min at 12,000 g, and stored at 75°C for subsequent analysis. Tubes used to collect arterial blood for the measurement of NE were pretreated with ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid and reduced glutathione to prevent catecholamine oxidation.

After blood collection on the fifth day, the animals were given an intravenous injection of 0.7-1.0 ml of 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg) in saline (0.9% NaCl) for evaluation of IBAT cellular proliferation. Four hours after the BrdU injection, the animals were killed with an overdose of pentobarbital sodium. The IBAT depot was rapidly removed, cleaned of adhering white adipose tissue and skeletal muscle, and divided into two portions by cutting along the midsagittal plane. One portion of IBAT was frozen in liquid nitrogen and stored at 75°C for subsequent measurement of total protein and mitochondrial UCP₁. The other portion of IBAT was fixed in 10% neutral buffered Formalin for 5 days and embedded in Paraplast-plus (Sherwood Medical, St. Louis, MO). The embedded IBAT was sectioned such that two sequential 5-µm sections were collected every 150 µm; they were mounted on slides coated with poly-L-lysine (Sigma, St. Louis, MO). One section was stained with hematoxylin and eosin for determination of brown adipocyte area, while the other sequential section was prepared for evaluation of cell proliferation (see below).

Analytic Procedures

Only 0.6 ml of blood could be safely collected from the cannula at each time period without resulting in a decreased hematocrit (29). In a few instances, blood flow was hindered by clotting, leading to a collection volume less than 0.6 ml. Any volume less than 0.6 ml was insufficient to allow accurate measurement of IGF-I as well as NE at all time points. Because NE is a known trophic factor for brown fat (11), we assigned priority to its assay. Therefore, plasma was analyzed for NE concentration at each blood collection time point, whereas IGF-I was analyzed in plasma collected only at t_pre and t₅.

IBAT UCP. The quantification of IBAT UCP₁ content was performed using an immunoblotting assay modified from the method of Lean et al. (24), as previously described by Gabaldon et al. (10). Protein content of the UCP₁ homogenate was determined by the method of Lowry et al. (25) using bovine serum albumin as the standard. The anti-rat UCP sera utilized did not react with homogenates of liver or muscle, indicating little or no cross-reactivity with UCP₂ or UCP₃ (1, 9).

Brown adipocyte area. Cell area was measured in ~200 mature brown adipocytes per animal. Twenty random locations, measuring 0.02 mm², were selected per slide. Within each location, the projected area of 10 brown adipocytes was measured. Mature brown adipocytes were identified by their characteristic multilocular appearance and a large, centrally located nucleus. One hematoxylin and eosin-stained section of IBAT per rat was analyzed with the use of light microscopy (×600, Olympus IMT-2 inverted microscope, Sunnyvale, CA). The image was captured with a charge-coupled device camera and then displayed on a monitor using NIH Image software, version 1.49 for Macintosh computers (National Institutes of Health, Bethesda, MD).

Cell proliferation. Cell proliferation of IBAT isolated from BrdU-injected animals (see above) was evaluated by measuring the incorporation of BrdU into brown adipocyte nuclei, using the immunofluorescent BrdU Labeling and Detection Kit no. 1 (Boehringer Mannheim Biochemica, Indianapolis, IN). Unstained IBAT sections were deparaffinized in 90% xylene for 10 min and rehydrated with phosphate-buffered saline (PBS, pH = 7.4). Tissue fixation in Formalin necessitated a pepsin digestion to expose the nuclear epitopes in the IBAT section. Pilot studies confirmed that a 30-min digest with 600 µg of pepsin in a volume of 200 µl resulted in maximal immunoreactivity and unaltered tissue morphology. The IBAT sections were then rinsed with PBS and incubated with a mixture of a primary monoclonal mouse antibody to BrdU and nucleases for 30 min at 37°C. The sections were rinsed with PBS and incubated with the fluorescent secondary antibody (anti-mouse-immunoglobulin-rhodamine) for 30 min at 37°C. Sections were then rinsed with PBS and covered with mounting medium for immunofluorescent microscopy (Sigma) before analysis of fluorescence.

Fluorescence of the BrdU-labeled brown adipocyte nuclei was measured at 540/605 nm, excitation/emission, using an extremely low-light silicon intensified target camera attached to an inverted microscope (Olympus IMT-2). The video image of the brown adipocytes was visualized through a computer using NIH image software, version 1.49 for the Macintosh computer.

The validity of the BrdU immunofluorescent assay was tested regularly by including a control section of small intestine collected from each rat. The small intestine was used as a positive control because of the extremely high turnover rate of intestinal crypt cells. Significant fluorescence, and thus proliferation, was observed in the intestinal crypt nuclei in all sections tested.

The amount of brown adipocyte proliferation is described as the number of BrdU-labeled nuclei per 1,000 total brown adipocyte nuclei, counted on a sequential hematoxylin and eosin-stained slide. We have designated this as the labeling index. Anatomic markers, such as connective tissue and large blood vessels, were used to locate the same position on the hematoxylin and eosin section for the BrdU measurement.

Statistics

	RESULTS

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Colonic Temperature

Body Mass: IBAT Mass, Protein, and UCP

Total IBAT mass (mg) was significantly greater in the 12- vs. 6-mo-old rats maintained at thermoneutrality (Table 1), but IBAT mass of the 26-mo-old rats housed at 25°C did not differ from that of either the 6- or 12-mo-old rats. Five days of exposure to 10°C significantly increased total IBAT mass (mg) in the 6- and 12- but not in the 26-mo-old rats compared with age-matched rats maintained at thermoneutrality. When expressed as milligrams per gram body mass, IBAT mass of the 6- and 12-mo-old cold-exposed rats was greater than that of their 26-mo-old counterparts. Although IBAT mass of the 6-mo-old, cold-exposed rats (mg/g) was significantly greater than that of age-matched animals at thermoneutrality, cold exposure did not affect body mass-adjusted IBAT mass (mg/g) in the 12- or the 26-mo-old rats.

Five days of exposure to 10°C produced significant increases in total IBAT protein (mg) in all groups (Table 1). However, this cold-induced effect became progressively smaller with increasing age. That is, total IBAT protein in the cold-exposed 6-mo-old rats was 2.6 times greater than that of the non-cold-exposed 6-mo-old rats, 1.5 times greater in cold- versus non-cold-exposed 12-mo-old rats, and 1.35 times greater in 26-mo-old cold- versus non-cold-exposed animals. When IBAT protein was expressed per mass of IBAT, no significant differences within the age groups were noted for either the non-cold-exposed or cold-exposed rats. Exposure for 5 days at 10°C produced elevated levels of IBAT protein (mg/g tissue) in the 6- and 26- but not the 12-mo-old rats.

The amount of IBAT UCP₁ did not differ significantly among the age groups in the rats maintained at thermoneutrality whether the data were expressed as micrograms UCP₁ per depot, micrograms UCP₁ per milligram of IBAT mass, or micrograms UCP₁ per milligram of protein (Table 1). After 5 days at 10°C, UCP₁ levels in IBAT of the 6- and 12-mo-old rats were consistently greater than those observed in the 26-mo-old rats. Cold exposure significantly increased brown fat UCP₁ in the 6- and 12-mo-old rats compared with non-cold-exposed animals, regardless of the manner of data expression. Conversely, 5 days of exposure to 10°C did not significantly affect the UCP₁ content of IBAT from 26-mo-old rats.

IBAT Cell Area

Although cell area differed significantly with age and ambient temperature, the cold-induced alterations in morphological characteristics were similar. For example, mature brown adipocytes from all rats maintained at thermoneutrality contained primarily large lipid droplets. Conversely, lipid droplets appeared more numerous and smaller in size in all age groups after cold exposure.

IBAT Cell Proliferation

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Cell proliferation in interscapular brown adipose tissue of male Fischer 344 (F344) rats after 5 days at either thermoneutrality (25°C) or cold (10°C). Values are means ± SE. Values sharing a common superscript letter are not significantly different (P  0.05).

Plasma IGF-I

Plasma NE

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Daily mean plasma norepinephrine (NE) concentrations in 6(A)-, 12(B)-, and 26-mo-old (C) F344 male rats. Values (means ± SE) represent blood levels from 1 day before exposure (tpre) through day 5 (t5) of exposure to either thermoneutrality (25°C) or cold (10°C). * Values are significantly different (P  0.05) from age-matched thermoneutral values. tpre plasma samples taken from the 6-mo-old cold-exposed group were destroyed in a freezer malfunction.

When ANOVA was performed on mean NE values averaged over the entire exposure period (t₁ through t₅, without the t_pre control value), there was a main effect of age. That is, when mean NE concentrations (mean ± SE) for both exposure groups (thermoneutral and cold) were combined for each age group, NE levels in the 26-mo-old rats (1,268.7 ± 100.2 pg/ml) were significantly greater (P  0.05) than NE levels observed in the 6-mo-old animals (837.9 ± 84.3 pg/ml). The mean NE levels in the 12-mo-old rats (1,080.9 ± 107.9 pg/ml) were not different from those observed in the 6- or 26-mo-old rats.

As seen in Fig. 2, in the first two blood draws taken, i.e., t_pre and t₁, NE appeared to be elevated, relative to samples collected later in the experiment. This may have resulted from incomplete recovery of the animals from the cannulation surgery and/or a novel and stressful experience of the first blood draws. Either of these may have artificially elevated initial NE values. When these initial values were removed from the data set, i.e., only values between t₂ and t₅ were considered in the analysis and the ages were combined in either thermoneutral or cold groups, there was a significant main effect of temperature. The mean concentration of NE was higher in the cold-exposed (1,203.6 ± 93.8 pg/ml) than in the non-cold-exposed groups (921.7 ± 77.9 pg/ml).

	DISCUSSION

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The data presented here support the hypothesis that brown fat in old F344 rats does not respond to the trophic stimulus of chronic cold exposure to the same degree as in younger animals. That is, 26-mo-old rats did not increase IBAT mass, cell proliferation, or UCP₁ content in response to chronic cold, whereas the 6-mo-old rats had a nearly 2-fold cold-induced increase in IBAT mass, a 26-fold increase in cell proliferation, and a 4-fold increase in UCP₁ content.

Our data indicate that the growth of BAT mass after the 5-day cold exposure reflects primarily cellular hyperplasia rather than hypertrophy. The cold-induced increase in brown adipocyte size is ~20-30% in all age groups and is greatest in brown fat from the 26-mo-old rats, where total BAT mass is smallest and there is no increase in cell proliferation. Brown fat cell size also increased in the cold-exposed 6- and 12-mo-old animals displaying significant increases in BAT mass as well as cellular proliferation. Thus cell hypertrophy alone was not sufficient to produce a significant increase in BAT mass, as demonstrated in the 26-mo-old rats, suggesting a significant role for cold-induced cellular proliferation. Indeed, the level of brown preadipocyte proliferation is 20 times greater in the 6- versus 26-mo-old cold-exposed rats. This is consistent with the findings of Cameron and Smith (5) and Bukowiecki and colleagues (3, 4), who report that BAT growth in rats exposed to several days of cold is due, in large part, to elevated cell proliferation, as determined by an increase in tritiated thymidine in brown fat cells.

The mechanisms underlying the age-related attenuation in cold-induced brown preadipocyte proliferation and maturation are unclear. Previous investigations in young animals suggest that NE may be an important signal for the induction of BAT mass growth and preadipocyte proliferation (2, 38). It is possible that decreased cold-induced sympathetic signaling to BAT may account for the blunted response observed in aged rats. However, arguing against this possibility are several lines of evidence. For example, plasma NE concentrations are consistently elevated, not diminished, in older vs. younger animals during prolonged (see RESULTS) as well as short-term (10) cold exposure. Sympathetic signaling to BAT of male mice (as indicated by neural recordings) and cold-exposed male rats (as measured by NE turnover in BAT) is significantly greater in old vs. young animals (19, 32). Together, these data indicate that diminished NE signaling is unlikely to account for the difference in BAT cell proliferation observed here.

Although the sympathetic neural signal to BAT appears to be intact in the older rat, our data do not rule out the possibility that the diminished cold-induced response in BAT of the 26-mo-old rats is due to age-related alterations in adrenergic-mediated signal transduction in brown adipocyte precursor cells. This suggestion is consistent with evidence in several tissues that demonstrates age-related reductions in adrenergic receptor number and density, adenylyl cyclase activity, and cAMP concentrations after adrenergic stimulation (36, 40, 43, 44, 46). Scarpace et al. (42) reports that -adrenergic receptor numbers decline significantly in brown fat tissue from 24- vs. 3- and 12-mo-old male F344 rats. The greatest decline in adrenergic receptor number occurs in the ₁-subpopulation, the subtype previously shown to be associated most closely with cellular proliferation of preadipocytes in BAT (2, 21, 26, 37). However, it is difficult to conclude from adrenergic receptor studies whether the decline in ₁-adrenergic receptors occurs primarily in preadipocytes, the proliferative cell in BAT, because many investigations use a tissue homogenate rather than an isolated cell preparation. Moreover, results from our laboratory demonstrate that age does not significantly affect the ₃-mediated metabolic response to NE in mature brown adipocytes isolated from 6- and 26-mo-old male F344 rats (17). Studies are currently underway in our laboratory to determine whether this is the case for the -pathway in brown preadipocytes.

Several of our studies indicate that attenuated BAT nonshivering thermogenic capacity of the aged rat, as demonstrated by significantly lower cold-induced mitochondrial GDP-binding and UCP₁ content, is associated with lower amounts of thermogenically active IBAT. In the present investigation, we found that neither IBAT mass nor UCP₁ levels of 6- and 26-mo-old rats maintained at thermoneutrality differed significantly (Table 1). However, chronic cold exposure enhanced brown fat UCP₁ concentration only in the rats that showed significant increases in cell proliferation (and IBAT mass), suggesting the possibility of regulatory factors common to both processes. The nature of these regulatory pathways has not yet been clearly defined, but as with most physiological processes in BAT, one must suspect the involvement of an adrenergically mediated signal. Thus, although further evaluation is necessary to clarify the relationship between cell proliferation and UCP₁ synthesis, the current data suggest that adrenergic-mediated signal transduction in brown preadipocytes is disrupted in the aging rat.

An alternative hypothesis is that the reduced IBAT proliferative response in 26-mo-old rats involves an age-related reduction in levels of IGF-I. However, we found no evidence to support this hypothesis. That is, exposure to chronic cold (5 days at 10°C) in 26-mo-old rats did not lead to increased IBAT mass or IBAT cellular proliferation even though plasma IGF-I levels were unchanged. We cannot eliminate the possibility, however, that IGF-I production at the local level, via paracrine or autocrine release, may be involved in brown adipocyte proliferation. Alterations in tissue growth factor concentrations may yet prove to be an important pathway for cell proliferation in BAT.

Growth factors in addition to those evaluated here may also be involved in the proliferative process in brown fat. Investigations using brown preadipocytes isolated from young and developmentally immature rats suggest that insulin is necessary for proliferation of these cells (20, 47). However, insulin alone is unlikely to be responsible for the cold-induced increase in proliferation, because plasma insulin is significantly reduced in adult rats during acute, as well as chronic, cold exposure (10, 14, 45).

Despite the blunted nonshivering thermogenic capacity of BAT in the 26- vs. 6-mo-old rats, colonic temperature did not differ significantly during 5 days of cold exposure. This finding is in contrast with our observation that 26-mo-old rats are unable to maintain body temperature during more severe bouts of cold exposure (6°C for 4-6 h; 10, 23). We previously demonstrated that shivering thermogenic capacity is not significantly affected by age (8, 22, 33, 35). It is possible that during a relatively mild cold exposure of 10°C, heat generated through skeletal muscle shivering thermogenesis is sufficient to maintain body temperature in the older animal. As the ambient temperature is lowered, significant nonshivering thermogenesis from BAT, in addition to skeletal muscle shivering thermogenesis, is required to prevent the development of hypothermia. Because our data show impaired BAT nonshivering thermogenesic capacity in the 26-mo-old rats, it is possible that during prolonged exposure to temperatures lower than 10°C, body temperature cannot be appropriately maintained. Conversely, the younger animals are able to recruit both shivering and nonshivering thermogenesis to maintain body temperature during both severe and mild cold challenges.

In conclusion, our results indicate that older rats have a reduced capacity to respond to chronic cold with increased brown preadipocyte proliferation and brown adipocyte UCP₁. The observation that circulating levels of IGF-I and NE in 26-mo-old cold-exposed rats were not less than those of 6-mo-old animals indicates that decreased availability of these circulating growth factors cannot explain the attenuation of brown fat proliferation and UCP₁ synthesis in the older animals. Our data do not preclude the possibility of altered transduction of these signals and/or the involvement of other factors.

Perspectives