Regulation of expression in brown adipose tissue

of the lipoprotein

lipase gene

JOHN R. D. MITCHELL, ANDERS JACOBSSON, TODD G. KIRCHGESSNER, MICHAEL C. SCHOTZ, BARBARA CANNON, AND JAN NEDERGAARD The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, S-106 91 Stockholm, Sweden; Departments of Medicine and of Microbiology, University of California, Los Angeles 90024; and Research, Veterans Affairs, Wadsworth Medical Center, Los Angeles, California 90073 Mitchell, John R. D., Anders Jacobsson, Todd G. Kirchgessner, Michael C. Schotz, Barbara Cannon, and Jan Nedergaard. Regulation of expression of the lipoprotein lipase gene in brown adipose tissue. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E500-E506, 1992.-The regulation of lipoprotein lipase gene expression in brown adipose tissue was studied. Rats were preacclimated to 21OC. Exposure to cold (4°C) resulted in a rapid increase in the level of lipoprotein lipase mRNA in the tissue. The level peaked (expressed per pg total RNA) after -8 h and then slowly declined. The increased lipoprotein lipase mRNA level was not due to an increased stability of the mRNA, but, in a transition event from a high to a low expression of the lipoprotein lipase gene, a transcription-dependent process was recruited that accelerated the breakdown of lipoprotein lipase mRNA. Norepinephrine injections increased lipoprotein lipase mRNA levels in the tissue; this effect was mediated via a P-adrenergic receptor. The effect of cold could be mimicked by norepinephrine injections, and these two effects were not additive, indicating that the cold effect was mediated by norepinephrine. The lipoprotein lipase mRNA level was also increased by insulin injections (into fasted animals); thus an increase in lipoprotein lipase gene expression in brown adipose tissue may be induced via two different stimuli, which, intracellularly, would be mediated via different signaling systems. In all investigated conditions, the changes in lipoprotein lipase mRNA levels observed here were parallelled by alterations in lipoprotein lipase activity reported earlier from this laboratory. It was therefore concluded that, under the conditions studied, lipoprotein lipase activity in brown adipose tissue was primarily regulated at the transcriptional level. norepinephrine; mycin

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AND long-standing issue concerning the regulation of lipoprotein lipase activity is the question of the site of the regulation, i.e., whether the activity is transcriptionally, translationally, or posttranslationally regulated (2). Despite the isolation of cDNA clones corresponding to lipoprotein lipase mRNA (15) and the possibility thus achieved to follow mRNA levels in parallel with activity, the question of the site of regulation remains unsettled, with recent authors arriving at opposing conclusions (1, 8, 11, 12, 14, 16, l&33, 39-43, 46, 48, 50, 54). Some of these differences may result from limitations posed by the experimental systems used; in particular, it may be that the behavior of cells in isolated systems is not necessarily identical to that of cells in situ responding to physiological stimuli. In a system (brown adipose tissue) earlier found to respond very markedly to environmental cues and pharmacological treatments with changes in lipoprotein lipase activity (3-7,9,17,20,21,23,24,32,36,45,51),we have in the present investigation analyzed lipoprotein lipase gene expression. We have followed lipoprotein lipase mRNA levels under conditions identical to those AN IMPORTANT

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that we have previously used to measure lipoprotein lipase activity (3-6). We found that all earlier observed changes in lipoprotein lipase activity were parallelled by changes in lipoprotein lipase mRNA. We therefore conclude that, at least in this tissue, lipoprotein lipase activity is primarily regulated at the transcriptional level. The results presented below are discussed in relation to this tenet, which was originally formulated based on studies of the effects of transcriptional and translational inhibitors on lipoprotein lipase activity (6). MATERIALS

AND METHODS

Animals. Adult Sprague-Dawley male rats, - 150 g, were obtained from Eklund (Stockholm, Sweden). On arrival, the rats were initially placed 2/cage at 21°C with a 12:12-h light-dark Cycle (8:oo A.M.-&00 P.M.) for - 1 wk. Unless otherwise stated, the rats had free access to water and to a pellet diet (Ewos R3, Sodertalje, Sweden) containing 27% energy as protein, 12% as fat, and 60% as carbohydrate. For investigations of the acute effect of cold, rats were placed at 4°C at 9:00 A.M. under otherwise similar conditions. They remained at this temperature for 4 h before being killed, unless otherwise stated. For investigations of the effect of insulin, the following regime was followed (4): the food was withdrawn at 5:00 P.M. but was returned at 9:00 A.M. the next day. This protocol was continued for 4-5 nights; on the morning of the experiment, the food was not returned, and one group received an insulin injection. All rats were killed 4 h later. Where indicated, the stated number of rats were injected intraperitoneally at 9:00 A.M. with one of the following solutions: 3 pmol/kg body wt (-)-norepinephrine { 1 mg L-norepinephrine bitartrate [ (-)arterenol (Sigma)]/ml saline; 1 mg/kg body wt}; 3 pmol/kg body wt (-)-isoprenaline { 1 mg L-isoprenaline bitartrate [ (-)isoproterenol (Sigma)]/ml saline; 1 mg/kg body wt}; 3 pmol/kg body wt phenylephrine [0.7 mg L-phenylephrine hydrochloride (Sigma)/ml saline; 0.7 mg/kg body wt]; 4 IU/kg body wt insulin [4 IU (40 IE/ml insulin, Vitrum)/ml saline]; and 0.5 mg/kg body wt actinomycin D [0.5 mg Cosmegen (Merck Sharpe & Dohme)/ml saline]. Saline (1 ml/kg body wt of 0.9% NaCl) was injected in two rats as a control. No effect on lipoprotein lipase mRNA levels was observed, as compared with noninjected rats. The rats were killed 4 h after the injection, if not otherwise indicated. RNA preparation and analysis.The rats were killed with CO, and decapitated, and the interscapular brown adipose tissue was excised; the overlaying white adipose tissue and the surrounding muscle were avoided. Total RNA was isolated from the brown adipose tissue of each rat according to Jacobsson et al. (28). The amount and purity of the RNA solution was determined spectrophotometrically; an extinction coefficient of 0.025 cm-’ . mg RNA- l at 260 nm was used. The ratio 260/280 nm was routinely ~1.8. The RNA preparation was routinely tested for degradation and absence of DNA on agarose minigels. Typically, the yield from one

0 1992 the American

Physiological

Society

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LIPOPROTEIN

LIPASE

animal was -50 fig RNA. The RNA prepared was generally analyzed immediately; some sampleswere stored in 10 mM EDTA solution at -80°C. For slot blots, 4 pg RNA wasdissolvedin 300 ~11.5 M NaCl and 0.15 M sodium citrate (i.e., x10 SSC)/15% formaldehyde, with water addedto yield a total of 400 ~1,incubated for 15 min at 7O”C, and applied to a Zetaprobe filter paper in a slot-blot apparatus;eachwell waswashedwith 400 ~1x10 SSC solution, and the filter paper was dried. The filter paper was then prehybridized with salmonspermDNA (Sigma) and a poly A-poly C mixture. The filters were then hybridized at high stringency, exactly as earlier described(for thermogenin cDNA hybridization) (27) with a nick-translated lipoprotein lipasecDNA probe. The probe usedwas that earlier characterized (31). After washing, the filters were exposedto Kodak X-Omat films at -8O”C, and the intensity of darkening wasevaluated with an LKB laser densitometer. The linear range of this method with varying amountsof RNA applied and varying exposuretimes had been previously established.As a reference, 4 pg of a stock RNA preparation from rats that had beenexposedto cold for 48 h was blotted on all slot blots. The corresponding laser reading was assignedthe value one. All other values were expressedrelative to this reference(“relative units”). As a negative reference, 4 pg rat brain RNA samplewas blotted on every slot blot [brain has a very low lipoprotein lipase mRNA level (19, 30, 31, 49)]. For Northern blots, the RNA sampleswere denatured with loadingbuffer containing 50% formamide and 6% formaldehyde for 5 min at 65°C before being loaded on the gel. Total RNA samples(10 Kg) were applied to a 1.0% agarosegel containing 6% formaldehyde and run in a buffer consisting of 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0), 8 mM sodium acetate, and 1 mM EDTA (pH 8.0). After electrophoresis,the RNA was blotted onto Zetaprobe filter paper. The filter paper wasprehybridized for several hours at 42°C in a solution containing 50% formamide, ~5 SSC, ~5 Denhardt’s solution, 50 mM sodium phosphate (pH 6.5), 0.5% sodium dodecyl sulfate (SDS), and 100 pg/ml degradedherring sperm DNA. The hybridization wascarried out overnight with the lipoprotein lipase

probe labeled with [a-32P]dCTP with a random priming kit (Boehringer Mannheim). After hybridization, the filter papers were washed four times for 15 min with ~2 SSC and 0.1% SDS at room temperature, followed by two washes with X0.1 SSC and 0.1% SDS at 55°C. The filter papers were then exposed to Kodak X-Omat films with an intensifying screen. RESULTS

Effect of cold exposure on expression of the lipoprotein lipase gene. The level of lipoprotein lipase mRNA in the brown adipose tissue of rats was low (Fig. 1) and remained low under normal conditions at room temperature (Fig. 1). However, exposure of rats to cold led to both a rapid and marked increase in the level of lipoprotein lipase mRNA (Fig. 1). Within 1 h, the amount (per pg RNA) had increased to three times the control levels, and it remained at this high level for at least 2 days. The RNA levels then slowly decreased toward control levels, although control levels were not fully reached, even after 1 wk. Because the total amount of RNA in the tissue remains stable for the first days in cold but then increases severalfold (52), the apparent decrease in the specific lipoprotein lipase mRNA level (expressed per pg RNA) seen after 1 wk may at least partly be due to the dilution caused by this increase in total RNA; thus, despite the decrease in the specific level, it is likely that the total amount of lipoprotein lipase mRNA in the tissue remained elevated during the cold exposure period. These observations on specific lipoprotein lipase

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RNA

Lipoprotein lipase mRNA

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Fig. 1. Effect of cold exposure on level of lipoprotein lipase mRNA in rat brown adipose tissue. Top: 3 rats were placed in cold for 16 h and 3 remained at 21°C. Rats were killed, and total RNA was extracted from interscapular brown adipose tissue depot. RNA (10 pg) was separated on 1% agarose gel containing 1 ppm ethidium bromide. To ensure RNA integrity and equal loading, RNA was vizualized under ultraviolet light (left); 28 S and 18 S rRNA bands are clearly visible. RNA was then blotted over to Zetaprobe filters, and amount of lipoprotein lipase mRNA was determined (right) as described in MATERIALS AND METHODS. Bottom: rats were placed in cold at 9:00 A.M. (filled circles) or remained at 21°C (open circles) and were killed after indicated time periods (note logarithmic time scale). RNA was extracted, and amount of lipoprotein lipase mRNA was determined by slot blots as described in MATERIALS AND METHODS. Symbols indicate mean of values from 2 examined animals/time point, and bars represent the 2 individual values (this representation is mathematically equivalent to mean + SE). Amounts of lipoprotein lipase mRNA are expressed in relative units (RU)/pg RNA, relative to reference sample arbitrarily set to 1.0.

mRNA levels may be compared with our earlier investigations on specific lipoprotein lipase activity (per mg protein) under identical experimental conditions (5). The first time point was from rats that had been in the cold for 4 h, at which time the activity had already increased to four times the control levels, and the activity remained high for >l wk. Thus, during cold exposure, there was good temporal correlation between the changes in lipoprotein lipase gene expression measured here and those of lipoprotein lipase activity observed earlier. Lipoprotein lipase mRNA stability. To investigate the stability of lipoprotein lipase mRNA, we inhibited the transcription process with actinomycin and followed the decrease in lipoprotein lipase mRNA levels in rats at 21 or at 4°C. The amount of actinomycin used was based on earlier investigations in which we had found that this amount of actinomycin fully blocked cold-induced expression of the gene for the uncoupling protein thermogenin (27) and that the inhibition of transcription

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apparently persisted for - 10 h (29). The half-life of lipoprotein lipase mRNA estimated in this way was remarkably long in both groups (Fig. 2A), and only a small reduction in the mRNA level occurred during the 10-h interval. In rats at 21°C, the calculated half-life was -40 h, and, in rats at 4”C, it was - 20 h, but these values were not statistically different. However, since the values obtained, if anything, would indicate a tendency to a shorter mRNA halflife in the cold than at 2l”C, it is clear that the increase in the level of lipopro-

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Fig. 2. Lipoprotein lipase (LPL) mRNA stability. A: LPL mRNA halflife. Rats were preacclimated for 1 wk to 21°C. For each time point, 2 rats were killed. Points are thus means, and bars represent individual values; where not shown, individual values fell within symbol size. Open circles: rats remaining at 21°C were injected with actinomycin (act) and killed at indicated time points. Initial mean LPL mRNA content was set to lOO%, and other values were expressed in relation to this. Line was obtained by regression analysis and had a correlation coefficient of 0.66. Calculated mRNA half-life was 44 h. Filled circles: rats were placed in the cold, injected after 4 h with actinomycin, and remained in the cold. Experiment was run in 2 series, and, in both series, initial mRNA content at injection time (time 0) was set to 100%; other values were expressed in relation to this. Line was obtained by regression analysis and had a correlation coefficient of 0.50. For rats in the cold, calculated mRNA half-life was 19 h. B: physiological half-life. Rats were placed in cold and were returned to control conditions after 4 h (21 “C). Some rats were injected with actinomycin at same time as retransfer. For each time point and condition, 2 rats were killed. Open squares: untreated rats. Initial mean LPL mRNA value was set to lOO%, and other values were expressed in relation to this. Line was obtained by regression analysis of time points 2-10 h and had a correlation coefficient of 0.87. Calculated half-life was 7 h, with 95% confidence interval of 5-13 h. Filled squares: actinomycin-injected rats. Experiment was run in 2 series, and, in both series, initial mRNA content at injection time (time 0) was set to lOO%, and other values were expressed in relation to this. Line was obtained by regression analysis of time points 2-10 h and had a correlation coefficient of 0.53. mRNA half-life thus calculated was 18 h.

GENE

EXPRESSION

tein lipase mRNA in the cold (Fig. 1) could not be due to an increased stability of the mRNA but must have resulted from an increased rate of gene transcription. Under identical conditions, the half-life of lipoprotein lipase activity after transcription inhibition by actinomytin injection was found earlier to be X4 h (6), with no significant difference between the two physiological conditions. Thus the half-life of the decay of activity after inhibition of gene transcription observed earlier and the half-life of the mRNA observed here were similar, indicating that the disappearance of lipoprotein lipase activity under these conditions follows disappearance of lipoprotein lipase mRNA. This result is compatible with the fact that lipoprotein lipase enzyme activity per se has a comparatively short half-life. When estimated after cycloheximide inhibition of protein synthesis, the activity half-life is only -2 h (6); thus, the kinetics are such that activity would reflect mRNA half-life rather than protein half-life. Physiological half-life of lipoprotein lipase mRNA. As demonstrated above, the half-life of lipoprotein lipase mRNA is very long under stable physiological conditions. If the synthesis of the lipoprotein lipase enzyme is governed solely by the level of lipoprotein lipase mRNA and no posttranslationally regulated activation step is involved, the long mRNA half-life (measured under stable conditions) would predict a very slow transition from high to low activity. Thus, if the stimulus that had induced a high level of lipoprotein lipase mRNA suddenly ceased, activity would only slowly decline. In other words, the animal would be forced to produce active lipoprotein lipase even after the activity was no longer physiologically needed. In Fig. 2B, we show the results of experiments designed to follow the events during such a transition phase: the cessation of a cold stimulus. In these experiments, rats were first exposed to cold for 4 h to induce lipoprotein lipase gene expression and were then transferred to 21“C at time 0. As seen in Fig. 2B (open symbols), there was a rapid breakdown of lipoprotein lipase mRNA, corresponding to a half-life of only 7 h, i.e., much shorter than the half-lives observed under stable conditions (20-40 h; Fig. 2A). It would thus seem that, when the cold stimulus had ceased, a process was initiated that led to a more rapid breakdown of lipoprotein lipase mRNA than that which occurred under stable conditions. Earlier studies of the decrease in lipoprotein lipase activity observed under these identical conditions, i.e., after the cessation of a cold stress (6), demonstrated a half-life of activity of -4 h. Thus this rapid decline in activity is apparently a reflection of a rapid breakdown of lipoprotein lipase mRNA under these conditions. This breakdown would seem to be physiologically controlled and induced by the absence of the stimulatory stimulus. To investigate whether this breakdown process could be due to an induction of a novel RNase activity, we treated the rats with actinomycin immediately before the transfer back to 2lOC. It is seen (Fig. 2B, filled symbols) that this inhibition of transcription had a marked effect on the rate of breakdown of lipoprotein lipase mRNA. The half-life was now prolonged to 18 h, i.e., it was now virtually identical to that observed in rats that were not in a transition situation (Fig. 2A). We therefore suggest

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LIPOPROTEIN

LIPASE

that a specific RNase activity is induced when the stimulus for increased expression of the lipoprotein lipase gene is withdrawn. It would seem that the induction of this RNase activity requires gene transcription. Adrenergic

effects on lipoprotein

lipase gene expression.

Most of the effects of a cold stimulus on brown adipose tissue are believed to be mediated via an increased sympathetic activity in the tissue and to be caused by the neurotransmittor norepinephrine. In the experiment depicted in Fig. 3, we have examined whether norepinephrine in itself was able to increase the lipoprotein lipase mRNA level in the tissue. The experiment was started at 9:00 A.M. in rats living in a 1212-h light-dark cycle, with the light turned on at 8:00 A.M. Thus, in the uninjected rats (open symbols), it was possible to follow a part of the daily rhythm in lipoprotein lipase mRNA levels. The lipoprotein lipase mRNA level declined during the middle of the day, but the activity rose toward the end of the light period (probably in anticipation of food intake). Earlier observations on daily rhythm of lipoprotein lipase activity (20) indicate a similar pattern. In the norepinephrine-injected rats (Fig. 3, filled symbols), there was a rapid increase in the level of lipoprotein lipase mRNA. The level peaked 4 h after the injection when it was more than double the initial value, after which it started to decline. It is noteworthy that the lipoprotein lipase mRNA level continued to increase for 4 h after the injection, despite the fact that it is evident from studies in intact rats that the acute thermogenic effect of a single norepinephrine injection does not persist for much more than 1 h (35). This implies that the initiated gene transcription remains activated for a period after the cessation of the hormonal stimulus. The norepinephrine-induced increase in lipoprotein lipase gene expression is in agreement with our earlier

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observation that lipoprotein lipase activity is increased in this tissue by norepinephrine injection under identical conditions (5). To define the adrenergic subtype involved in the response to norepinephrine injection, we compared the ability of different adrenergic agonists to increase lipoprotein lipase mRNA in the rats. Based on the results shown in Fig. 3, the lipoprotein lipase mRNA level was investigated 4 h after the injections. It is seen in Fig. 4 that a norepinephrine-induced increase in lipoprotein lipase mRNA levels was again observed. Norepinephrine is approximately equipotent in its ability to stimulate CY-and P-adrenergic receptors. The P-selective agonist isoprenaline was almost as effective in its ability to induce an increase in the level of lipoprotein lipase mRNA as was norepinephrine, but the a-selective agonist phenylephrine was without effect on lipoprotein lipase mRNA levels in the tissue. It was therefore concluded that the adrenergic stimulation of the expression of the lipoprotein lipase gene was mediated via P-adrenergic receptors. Principally the same conclusion was reached by Carneheim et al. (6), based on investigations of lipoprotein lipase activity. Thus the present experiments indicate that the increase in lipoprotein lipase activity in ,&adrenergically stimulated brown adipose tissue is due to an increased gene expression. A4ediation of cold stress. If norepinephrine is the agent responsible for the induction of lipoprotein lipase gene expression in cold-exposed rats, it would be assumed that it should be without effect when injected into already cold-exposed rats. The experiments depicted in Fig. 5 confirmed that this was the case. As can be seen, a single norepinephrine injection and exposure to cold for 4 h each caused, in

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Fig. 3. Effect of norepinephrine (NE) injection on level of LPL mRNA in brown adipose tissue. Rats preacclimated to 21 “C were injected at 9:00 A.M. with NE. At each time point, 2 injected (filled circles) and 2 noninjected (control; open circles) animals were killed, and their LPL mRNA level was measured. All results are expressed relative to standard RNA preparation given the value of 1 RU. Results are thus means and individual values of 2 animals/point.

Fig. 4. Adrenergic subtype involved in stimulation of LPL gene expression. Rats preacclimated to 21°C were injected at 9:00 A.M. with NE, isoprenaline (Iso), or phenylephrine (Phe). Controls (0) were not injected. Rats were killed 4 h later, and their LPL mRNA level was measured. Results are expressed relative to standard RNA preparation given the value of 1 RU. Values are means + SE of 3-4 animals/ treatment. *(*) and **Significant effects of injected vs. noninjected rats, P < 0.02 and P < 0.01, respectively (Student’s t test). NS, not significant.

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Fig. 5. Effect of NE injection combined with cold exposure on LPL mRNA levels in brown adipose tissue. At start of experiment, some rats were injected with NE and were either placed in cold (4°C) or remained under control conditions (21°C). Later (4 h), all rats were killed, and RNA was prepared and analyzed as described in MATERIALS AND METHODS. All results are expressed relative to standard RNA preparation given the value of 1 RU. Values are means + SE of 4-6 animals in each point. ** and ***Significant differences from noninjected animals under control conditions, P < 0.01 and P < 0.001, respectively (Student’s t test). In cold-exposed rats, there was no significant effect of NE injection.

themselves, an increase in lipoprotein lipase gene expression (as described above). However, when norepinephrine was injected into rats simultaneously with the initiation of cold exposure, no further effect of norepinephrine injection (above that caused by the cold exposure) was evident. This result is thus compatible with norepinephrine being the agent causing increased lipoprotein lipase gene expression in cold-exposed rats. Effect of insulin. In white adipose tissue, only insulin (and not norepinephrine) has the ability to increase lipoprotein lipase activity (13), and insulin has been demonstrated to increase lipoprotein lipase mRNA in white adipocytes (41). Insulin has also been demonstrated to be able to increase lipoprotein lipase activity in brown adipose tissue, at least in fasted animals (4). We have investigated whether this insulin-induced increase in lipoprotein lipase activity is also caused by an increase in lipoprotein lipase gene expression. Thus rats were restriction fed, as described by Carneheim and Alexson (4). After the food was removed (16 h), some of the rats were injected with insulin, and 4 h later the level of lipoprotein lipase mRNA was measured and compared with that in noninjected rats. As seen in Fig. 6, insulin caused a marked increase in lipoprotein lipase mRNA levels in brown adipose tissue. This indicates that the increase in lipoprotein lipase activity seen after insulin injection is also caused by an increased lipoprotein lipase gene expression. DISCUSSION

Although the site of regulation of lipoprotein lipase activity in brown adipose tissue has been discussed earlier, it is only the isolation of cDNA clones corresponding to lipoprotein lipase mRNA that has allowed a more thorough examination of this question. Changes in lipoprotein lipase mRNA levels in brown adipose tissue were first observed postnatally (37) and have also recently been

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Fig. 6. Effect of insulin on LPL gene brown adipose tissue. Rats were restriction fed as described in MATERIALS AND METHODS and some were injected with insulin 16 h after food was removed. Later (4 h), all rats were killed. All results are expressed relative to standard RNA preparation given the value of 1 RU. Results are means + SE from 4 rats in each group. ***Significant effect of insulin injection, P

Regulation of expression of the lipoprotein lipase gene in brown adipose tissue.

The regulation of lipoprotein lipase gene expression in brown adipose tissue was studied. Rats were preacclimated to 21 degrees C. Exposure to cold (4...
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