INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

BROWN ADIPOSE TISSUE (BAT) is a mammalian tissue specialized for heat production and is the major site of nonshivering thermogenesis (3, 8). Blood flow through BAT is directly related to its thermogenic state, and a high rate of blood flow is required for enhanced thermogenesis in BAT to provide oxygen and to transfer heat (6). It has been suggested that norepinephrine (NE) released from sympathetic nerve terminals in BAT is involved in the regulation of blood flow through this tissue. Previous studies indicated that NE significantly increases BAT blood flow, but the mechanism remains to be clarified (5, 6). Subsequently, we demonstrated that NE can mediate vasodilatation through stimulating the production of nitric oxide (NO) in BAT, resulting in an increase in its blood flow (10). NO is produced by NO synthase (NOS), and three isoforms have been identified (4). The isoforms neuronal NOS (nNOS) and endothelial NOS (eNOS) are considered to be constitutively expressed, and their expression can be dynamically regulated by various physiological or pathological conditions. In contrast, inducible NOS (iNOS) is primarily regulated at the transcriptional level, and resting cells express little or no iNOS (4). iNOS has been shown to be expressed in brown adipocytes, suggesting that the NO produced by iNOS is involved in the regulation of BAT function (11). This finding, however, could not explain the physiological changes in BAT blood flow. Because changes in BAT blood flow caused by NE occur very rapidly, NO produced by iNOS is probably not involved in this process. We speculated that the blood flow through BAT may be regulated by NO that is produced by constitutive NOS. However, whether constitutive NOS is expressed in BAT remains unknown. To address this issue, we assessed the expression of nNOS and eNOS in BAT. We also investigated the effect of cold exposure on the expression of these genes.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preparation of animals. Adult Wistar strain rats, weighing ~300 g (aged 12 wk), were given standard laboratory chow (Oriental MF, Oriental Yeast, Tokyo, Japan) and tap water ad libitum under conditions of controlled temperature (25 ± 1°C) and illumination (12-h light cycle starting at 7:00 AM). Animals were either kept at 5°C (cold exposed) or at 25°C (controls). After exposure, the animals were killed at the times indicated in Fig. 3 (i.e., after 4 or 24 h of cold exposure), and then total RNA and total tissue homogenate were prepared from interscapular BAT. Experiments proceeded according to the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences published by the Council of the Physiological Society of Japan.

RT-PCR. Total RNA was obtained from interscapular BAT, liver, heart, and brain using TRI-zol (GIBCO BRL, Life Technologies). Total RNA (50 ng) was amplified by RT-PCR using the Titan One Tube RT-PCR System (Roche) with respective primers. Primers for the nNOS cDNA fragment were 5'-CAAACGCAAAGTGGGAGGTC-3' (forward) and 5'-TTTGCCATCGAGGTCTCTGTC-3' (reverse) based on the rat nNOS cDNA sequence (X-59949, GenBank). Primers for the iNOS cDNA fragment were 5'-ATTCCCAGCCCAACAACACAG-3' (forward) and 5'-AGGCAGCGCATACCACTTCA-3' (reverse) based on the rat iNOS cDNA sequence (U-26686, GenBank). Primers for eNOS cDNA fragment were 5'-CTGGCAAGACCGATTACACGA-3' (forward) and 5'-CGCAATGTGAGTCCGAAAATG-3' (reverse) based on the rat eNOS cDNA sequence (AJ-011116, GenBank). The PCR reaction profile was as follows: denaturation at 94°C for 45 s, annealing at 58°C for 45 s, and extension at 68°C for 1 min, for 30 cycles. The amplified products were resolved by electrophoresis in 2% agarose gels containing ethidium bromide.

Northern blotting. Total RNA (15 µg) was resolved by electrophoresis in a 1.2% agarose gel containing 6.2% formaldehyde and transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech) by capillary blotting. The amplified cDNA fragments were used as cDNA probes for NOS transcripts, after labeling with [-³²P]dCTP using DNA labeling beads (Amersham Pharmacia Biotech). The membranes were hybridized for 1 h at 65°C in QuikHyb solution (Stratagene, La Jolla, CA) and then washed in 2× SSC (1× SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0) containing 0.1% (wt/vol) SDS at 25°C twice for 15 min and in 0.1× SSC/0.1% SDS at 60°C for 30 min. The membranes were exposed to an imaging plate, which was quantified by scanning using a BAS2000 Bioimage analyzer (Fuji Photo Film, Tokyo, Japan). Differences in the amounts of RNA transferred onto the membrane were corrected by hybridizing the membranes with a [-³²P]ATP-labeled synthetic oligonucleotide specific for the 18S rRNA subunit (2).

Western blotting. Brown adipocytes isolated by collagenase digestion, BAT, liver, and heart were homogenized and centrifuged at 12,000 g for 10 min at 4°C. Protein concentrations in the supernatants were determined using the bicinchoninic acid protein assay reagent (Pierce). Total protein (10 µg) was boiled in sample buffer [50 mM Tris (pH 6.8), 10% glycerol, 2% SDS, and 6% -mercaptoethanol] and resolved on a 10% polyacrylamide gel. Proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Hybond-PVDF, Amersham Pharmacia Biotech) using standard procedures. The membranes were incubated with a mouse anti-eNOS antibody (Transduction Laboratories) at a 1/2,500 dilution. We detected eNOS by enhanced chemiluminescence (ECL) using the ECL system (Amersham Pharmacia Biotech) with horseradish peroxidase-conjugated second antibody at a 1/2,500 dilution.

Statistical analysis. Data are expressed as means ± SE. Statistical significance was assessed by one-way ANOVA followed by a Scheffé's F-test. The differences were considered significant for P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Presence of eNOS isoform in BAT. It is well known that the heart and brain express high levels of eNOS and nNOS mRNAs, respectively (4). Figure 1 shows the expression of constitutive NOS mRNAs in various tissues. Specific signals for eNOS mRNA were detected in all tissues by RT-PCR, whereas signals for nNOS mRNA were detected at a high level in the brain, at a very low level in the heart, and not at all in BAT. Northern blotting detected specific signals for eNOS mRNA in all tissues, and the expression levels were high in both heart and BAT. On the other hand, specific signals for nNOS mRNA were detected only in the brain. Figure 2 shows the expression of eNOS protein in several tissues. Specific eNOS signals at 140 kDa were detected in all tissues, and the expression level was higher in BAT than in the heart. The level of eNOS expression was similarly high in isolated brown adipocytes.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Expression of constitutive nitric oxide synthase (NOS) mRNAs in rat tissues. A: RT-PCR analysis. Total RNA (50 ng) from several tissues was amplified by RT-PCR. The PCR products were resolved by electrophoresis in 2% agarose gels containing ethidium bromide. M, molecular size markers; eNOS, endothelial NOS; nNOS, neuronal NOS; BAT, brown adipose tissue. B: Northern blot analysis. Total RNA (15 µg) was Northern blotted to detect constitutive NOS mRNAs. The blots were hybridized with 32P-labeled cDNA probe corresponding to NOS mRNA transcripts. Positions of rRNAs are indicated at right.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Western blots of eNOS protein in BAT and brown adipocytes of rats. Total protein (10 µg) from BAT or isolated brown adipocytes was resolved by 10% SDS-PAGE. Human endothelial lysate (2 µg) was positive control. Representative eNOS signals in several rat tissues (A) and in isolated brown adipocytes (B) are shown.

Influence of acute cold exposure on the gene expression of eNOS and iNOS. Figure 3 shows that acute cold exposure caused a significant increase in eNOS mRNA expression in BAT. Four hours after cold exposure, eNOS mRNA expression was increased up to 2.8-fold, and these levels were maintained for 24 h. To compare the expression levels of eNOS and iNOS mRNAs in BAT, the same membrane was rehybridized with iNOS probe after stripping eNOS signals. The iNOS probe used in this study detected high levels of iNOS mRNAs in both the spleen and the liver 4 h after lipopolysaccharide injection (data not shown), as described elsewhere (1). As shown in Fig. 3, cold exposure did not cause a significant increase in iNOS mRNA in BAT.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Effect of cold exposure on gene expression of eNOS and inducible NOS (iNOS) in BAT. Rats were exposed to cold (5°C) for the indicated periods. A: representative eNOS and iNOS mRNA signals in BAT. C, controls; CE 4h, rats exposed to cold for 4 h; CE 24h, rats exposed to cold for 24 h. B: relative levels of eNOS and iNOS mRNAs quantified in BAT from rats exposed to cold. Membrane was consecutively probed for eNOS mRNA, iNOS mRNA, and 18S rRNA after stripping in 0.1% (wt/vol) SDS. Results are means ± SE from 5 rats. * P < 0.01 vs. control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study shows that eNOS is the only constitutive isoform expressed in BAT and in brown adipocytes and that the level of eNOS gene expression in BAT is significantly increased by cold stimulation. Thus eNOS may be the essential isoform that is responsible for NO production in BAT. The NO produced in BAT is involved in regulation of BAT blood flow. Although we did not examine the regulatory pathway of eNOS activation, it is conceivable that sympathetic nerves control eNOS activity in BAT. This theory is supported by the following lines of evidence. First, intravenous infusion of NE, which is a transmitter used by sympathetic nerves, rapidly increases BAT blood flow within 1 min, and this increased blood flow is completely abolished by N-nitro-L-arginine methyl ester (L-NAME; a NOS inhibitor) (10). Second, electrical stimulation of the ventromedial nucleus of the hypothalamus (VMH), which has been proposed as a central regulatory site of BAT function, leads to a rapid increase in BAT blood flow, probably by increasing sympathetic outflow to BAT (8, 14). We have demonstrated that this VMH stimulation-induced increase in BAT blood flow is also inhibited by L-NAME (unpublished data). Furthermore, the present results indicate that cold stimulation, which enhances sympathetic activity, causes an increase in eNOS gene expression. We therefore conclude that eNOS is the primary isoform that contributes to the physiological regulation of BAT blood flow and that both the activity and the expression of eNOS may be controlled by sympathetic nerve activity.

Although a previous report has suggested that iNOS is involved in NO production in brown adipocytes (11), our findings may imply that the physiological significance of iNOS in BAT is less convincing because eNOS and iNOS are expressed at very different levels in this tissue. The previous study has shown that NE stimulates apparent NO production estimated by nitrite analysis in cultured brown adipocytes; however, very slight iNOS protein expression is observed even though 100 µg of immunoprecipitating protein extract was used in Western blotting. This procedure can cause enrichment of iNOS content in the sample. Thus the signals obtained using this method are amplified and do not reflect the actual physiological expression level. The study presented herein detected strong eNOS signals in BAT and isolated brown adipocytes from control rats using only 10 µg of protein extract without enrichment of eNOS protein. Therefore, we infer that eNOS rather than iNOS may be responsible for the NE-stimulated NO production in brown adipocytes. In contrast to the previous report that demonstrated iNOS expression using immunoprecipitating protein extract in rats exposed to cold for 48 h (11), we did not observe any changes in iNOS mRNA in rats exposed to cold for 4 and 24 h. Thus our results suggest that iNOS may not be the initial isoform responsible for NO production in cold stimulation.

NO derived from brown adipocytes may directly regulate not only blood flow but also thermogenic activity in BAT (12, 13). The administration of L-NAME depresses the in vitro oxygen consumption of BAT (13), and NE stimulates NO production as well as thermogenesis in BAT in vitro (12). Moreover, ₃-adrenoceptor agonist, which is responsible for BAT thermogenesis, increases the BAT temperature as well as blood flow, and these effects are also inhibited by L-NAME (9). We found that the level of the eNOS isoform expression is also high in brown adipocytes. It was recently suggested that the endogenous production of NO is required for the lipolytic activity of white adipocytes (7). Therefore, NO produced in brown adipocytes might be one of the essential regulators of BAT thermogenesis. Further studies are required to elucidate the mechanism of NO action in BAT function.

In summary, we presented evidence that eNOS is the primary isoform that is responsible for the physiological regulation of blood flow as well as for thermogenesis in BAT and suggest that eNOS activity and expression may be controlled by sympathetic nerve activity.

Perspectives