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Am J Physiol Endocrinol Metab 294: E600-E606, 2008. First published December 18, 2007; doi:10.1152/ajpendo.00551.2007
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Studies of UCP2 transgenic and knockout mice reveal that liver UCP2 is not essential for the antiobesity effects of fish oil

Nobuyo Tsuboyama-Kasaoka,1 Kayo Sano,1 Chikako Shozawa,1 Toshimasa Osaka,2 and Osamu Ezaki1

1Nutritional Science Program and 2Health Promotion and Exercise Program, National Institute of Health and Nutrition, Tokyo, Japan

Submitted 24 August 2007 ; accepted in final form 13 December 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Uncoupling protein 2 (UCP2) is a possible target molecule for energy dissipation. Many dietary fats, including safflower oil and lard, induce obesity in C57BL/6 mice, whereas fish oil does not. Fish oil increases UCP2 expression in hepatocytes and may enhance UCP2 activity by activating the UCP2 molecule or altering the lipid bilayer environment. To examine the role of liver UCP2 in obesity, we created transgenic mice that overexpressed human UCP2 in hepatocytes and examined whether UCP2 transgenic mice showed less obesity when fed a high-fat diet (safflower oil or lard). In addition, we examined whether fish oil had antiobesity effects in UCP2 knockout mice. UCP2 transgenic and wild-type mice fed a high-fat diet (safflower oil or lard) developed obesity to a similar degree. UCP2 knockout and wild-type mice fed fish oil had lower rates of obesity than mice fed safflower oil. Remarkably, safflower oil did not induce obesity in female UCP2 knockout mice, an unexpected phenotype for which we presently have no explanation. However, this unexpected effect was not observed in male UCP2 knockout mice or in UCP2 knockout mice fed a high-lard diet. These data indicate that liver UCP2 is not essential for fish oil-induced decreases in body fat.

dietary fat; uncoupling protein; high-fat diet; mitochondria

UNCOUPLING PROTEINS (UCPs) are embedded in the inner mitochondrial membrane and belong to the superfamily of mitochondrial ion transporters (20). UCP1, which is preferentially expressed in brown adipose tissue (BAT), causes heat production by inducing proton leaks that uncouple oxidative phosphorylation. In contrast, the physiological functions of UCP2 and UCP3, which are expressed in many tissues, are still unknown. UCP2 and UCP3 knockout mice have normal responses to cold exposure and normal body weight, suggesting that the primary functions of UCP2 and UCP3 are not to promote gross thermogenesis or energetic inefficiency (1, 11, 25, 26). However, several studies have indicated that UCP2 and UCP3 may transport protons and increase the net proton conductance of mitochondria in the presence of activators such as fatty acids and free radical-derived alkenals (4–6). Recently, it was suggested that the lipid environment also affects UCP2 activity; polyunsaturated fatty acids activated human UCP2 in planar lipid bilayers (3). Therefore, activation of UCP2 and UCP3 in vivo by physiological activators or pharmacological intervention might have the capacity to be significantly thermogenic (4, 7).

In liver, under normal metabolic conditions, UCP2 levels are very low in hepatocytes, whereas levels are high in nonparenchymal cells (15). Subsequent studies showed that, under conditions associated with increased hepatic UCP2 expression (e.g., fatty liver and endotoxin-mediated liver injury), cell-specific expression would change with UCP2 being expressed primarily in hepatocytes. UCP2 mRNA and protein levels were increased in hepatocytes from ob/ob mice (8, 10). Bacterial lipopolysaccharide-injected rats showed increased expression of UCP2 mRNA in hepatocytes, whereas UCP2 mRNA levels were decreased in macrophages (9).

In mice fed fish oil or fenofibrate [peroxisome proliferator-activated receptor (PPAR)-{alpha} activator], expression of UCP2 mRNA was increased 8- or 18-fold, respectively, in isolated hepatocytes, whereas these treatments increased UCP2 mRNA expression by only 2- or 2.5-fold, respectively, in nonparenchymal cells (17). Because fish oil appears to have antiobesity effects relative to ingestion of other oils (12, 18, 24), it is possible that the increase in hepatocyte UCP2 expression and/or activation of the UCP2 molecule by fatty acids may underlie the antiobesity effect of fish oil. To clarify this possibility, we generated mice overexpressing UCP2 in hepatocytes and then examined whether overexpression of UCP2 in hepatocytes could prevent obesity caused by a high-fat diet. In addition, we examined whether fish oil has antiobesity effects in UCP2 knockout mice.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic mice and their diet. The cDNA encoding human UCP2 (23) was inserted in the EcoRI site between the serum amyloid component promoter (SAP; a kind gift from Dr. K. Yamamura at Kumamoto University) and SV40 polyadenylation sequence (Fig. 1A). This promoter contains all the necessary elements for selective expression in liver (28). This transgene fragment was microinjected in BDF1 mouse eggs at Japan SLC (Hamamatsu, Japan). The founder chimeras harboring the UCP2 transgene were mated with C57BL/6J mice to produce F1 offspring. C57BL/6J mice (8 wk of age) were obtained from Tokyo Animals Science (Tokyo, Japan). The heterozygous F1 offspring from this breeding were then backcrossed with C57BL/6J mice to obtain F2 offspring. This step was repeated one to three times, and we used F3 to F5 offspring in the experiments. The heterozygous mice were maintained at a constant temperature of 22°C with a fixed artificial light cycle (12:12-h light-dark cycle) and used in the experiments. All animal procedures were reviewed and approved by the National Institute of Health and Nutrition.


Figure 1
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  		Fig. 1. A: map of the uncoupling protein (UCP) 2 transgene construct used for microinjection of fertilized eggs. The cDNA encoding human UCP2 was inserted in the EcoRI site between the serum amyloid component promoter (SAP) to drive UCP2 expression to hepatocytes. B: expression of UCP2 mRNA in liver. Northern blot analysis of UCP2 mRNA in liver from female wild-type (WT) and UCP2 transgenic mice (Tg-L, low UCP2 expression; TG-H, high UCP2 expression). Endogenous mouse UCP2 mRNA appears as a 1.7-kb band, whereas the human UCP2 transgene mRNA appears as a 1.6-kb band. C: Western blot analysis of UCP2 protein. UCP2 protein was 32 kDa.

 
UCP2 transgenic mice and control wild-type (WT) littermate mice were fed a laboratory chow diet (CE2; CLEA) before initiation of dietary experiments. Between ages 3 and 7 mo, the mice were fed either a high-carbohydrate (HC) diet or high-fat (HF) diet (safflower oil or lard) for 2–7 mo. The composition of the diets and protocols was described previously (13). Briefly, in the HC diet, 63% of calories were from carbohydrate, 11% from fat, and 26% from protein. In the HC diet, safflower oil was used as the source of fat. In the HF diet, 14% of calories were from carbohydrate, 60% from dietary oil (safflower oil or lard), and 26% from protein. Safflower oil (high-oleic type) contained 46% oleic acid (18:1n-9) and 45% linoleic acid (18:2n-6) from total fatty acids. Lard contained 24% palmitic acid (16:0), 14% stearic acid (18:0), and 44% oleic acid.

UCP2 knockout mice and their diets. UCP2 knockout mice and their WT mice that have the same genetic background (129S4/SvJae/C57BL6) were kindly supplied by Dr. Bradford B. Lowell at Harvard Medical School (26). Six-month-old UCP2-knockout mice (–/–) and their WT mice (+/+) were fed an HC diet (10% of total calories from fat), high-safflower oil diet (60% of calories from fat), or high-fish oil diet (60% of calories from fat) for 16 wk. Fish oil from tuna contained 7% cis-5,8,11,14,17-eicosapentaenoic acid (20:5n-3) and 24% cis-4,7,10,13,16,19-docashexaenoic acid (22:6n-3). Fish oil was provided by NOF (Tokyo, Japan).

Energy intake measurements. Food consumption was measured daily. The mean food intake per day was estimated by subtracting the food weight of that day from the initial food weight of the previous day and dividing by the number of mice housed in the cage. Considering the evaporation of water in food during presentation to mice, the amount of water evaporation was measured as the change in food weight over 1 day of exposure in the animal chamber.

Northern blotting. cDNA clones containing the coding sequence of human UCP2 were obtained by PCR as described previously (23). Northern blot analysis was performed as described previously (23). A portion of RNA (15 µg/lane) was denatured with glyoxal and dimethyl sulfoxide and analyzed by electrophoresis in 1% agarose gels. After transfer to nylon membranes (NEN) and ultraviolet crosslinking, RNA blots were stained with methylene blue to visualize the 28S and 18D rRNAs and to ascertain the amount of loaded RNA (21). Human UCP2 cDNA was labeled with [{alpha}-32P]dCTP (NEN) with a random prime labeling kit (Multiprime DNA Labeling System; Amersham Biosciences, Piscataway, NJ). The amount of UCP2 mRNA was quantified with an image analyzer (BAS-1800II; Fuji Film, Tokyo, Japan) and expressed as the intensity of phosphostimulated luminescence in 3 h. Collagenase methods for isolation of hepatocytes and nonparenchymal cells were described previously (17).

Immunoblotting. Mitochondria-rich fractions from liver were prepared by centrifugation of tissue homogenates as described previously (26). Mitochondrial proteins (40 µg) separated by SDS-12% PAGE were transferred electrophoretically to Hybond-P (Amersham Life Science, Buckinghamshire, UK). Immunoblotting was performed with goat anti-UCP2 IgG (1:1,000, C-20; Santa Cruz Biotechnology, Santa Cruz, CA) as the first antibody and anti-goat IgG (1:1,000, Santa Cruz) as the secondary antibody. Bands were visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Body composition. For body composition analysis, mice were anesthetized with pentobarbital sodium (0.08 mg/g body wt Nembutal; Abbot, North Chicago, IL) and scanned with a Lunar PIXI mus2 densitometer (Lunar, Madison, WI) (16).

Statistical analysis. All values are means ± SE. Between-group differences were analyzed by unpaired Student's t-test. Data from the four groups were compared by two-way ANOVA of each group (Statview 5.0; Abacus Concepts, Berkeley, CA). When they were significant, each group was compared with the others by Fisher's protected least-significant difference test (Statview 5.0; Abacus Concepts). Statistical significance was defined as P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Human UCP2 mRNA is expressed in hepatocytes from UCP2 transgenic mice. The human SAP was used to drive liver-specific expression of human UCP2 in BDF1 mice (Fig. 1A). The sizes of the transcripts from the human UCP2 transgene and endogenous UCP2 gene were 1.6 and 1.7 kb, respectively. A number of founder mice were generated, and two independent lines, Tg-H, which expressed high levels of UCP2 (~10-fold higher than endogenous liver UCP2), and Tg-L, which expressed low levels of UCP2 (~2-fold higher than endogenous), were bred and used as heterozygotes (Fig. 1B). In parallel with UCP2 mRNAs, UCP2 protein levels in Tg-H mice were larger than those in Tg-L mice (Fig. 1C).

Expression of UCP2 mRNA in transgenic mice was further characterized. As expected from the function of the SAP promoter, the UCP2 transgene was expressed in hepatocytes but not in nonparenchymal cells, whereas endogenous liver UCP2 was expressed primarily in nonparenchymal cells (Fig. 2A). UCP2 mRNA derived from the transgene was present in liver but absent in other tissues, including white adipose tissues (WAT), BAT, gastrocnemius, kidney, heart, lung, and brain (Fig. 2B). To determine whether the level of expression of UCP2 mRNA in liver of UCP2 transgenic mice was within the normal range of physiological variations, UCP2 mRNA levels in transgenic mice fed an HC diet were compared by Northern blotting with those in WT mice fed an HC, high-safflower oil, or high-fish oil diet (Fig. 2C). Levels of liver UCP2 mRNA in Tg-H mice and Tg-L mice were higher and lower, respectively, than the level in C57BL/6 mice fed a high-fish oil diet for 1 wk, suggesting that the level of expression of the UCP2 transgene in UCP2 transgenic mice was within the limits of normal physiological variation. Because higher expression of UCP2 in Tg-H mice was well preserved and an increase of UCP2 mRNA in Tg-H mice was larger than that in mice fed a high-fish oil diet, we used this strain for later experiments.


Figure 2
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  		Fig. 2. A: expression of UCP2 mRNA in hepatocytes and nonparenchymal cells from male WT and Tg-H mice. Perfused liver tissues from WT and Tg-H mice were digested with collagenase and then separated into hepatocyte and nonparenchymal cells by a brief centrifugation. Endogenous and transgene-derived UCP2 mRNAs in each fraction were measured. B: Northern blot analysis of UCP2 mRNA in several tissues from female WT and Tg-H mice. Gastro, gastrocnemius. Endogenous mouse UCP2 mRNA appears as a 1.7-kb band, whereas the human UCP2 mRNA transgene appears as a 1.6-kb band. C: comparison of UCP2 expression in transgenic mice and in mice fed fish oil. Left: expression of UCP2 mRNA in livers of female control (WT) or transgenic Tg-L or Tg-H mice. WT, Tg-L, and Tg-H mice were fed a high-carbohydrate (HC) diet for 1 wk. Right: effects of diet on the expression of endogenous UCP2 mRNA in control female mice. C57BL/6J mice were fed an HC, high-safflower oil, or high-fish oil diet for 1 wk. Northern blot analysis of UCP2 mRNA in liver on the same membrane sheet is shown. On the autoradiogram, each line represents a sample from an individual mouse. The mean phosphostimulated luminescence (PSL) level of each band (endogenous plus transgene derived transcripts) for 3 h is presented. Each point is the mean ± SE. *P < 0.05 and **P < 0.01.

 
Obesity in response to high-safflower oil or high-lard feeding was observed in UCP2-transgenic mice and WT mice. To examine the effect of overexpression of UCP2 in hepatocytes on HF diet-induced obesity, considering that different fatty acids may activate UCP2 differently, we tested two types of dietary fat: linoleic acid-rich safflower oil and saturated fat-rich lard. In the first experiment, 3-mo-old female Tg-H mice and WT littermates were fed a high-safflower oil diet for 27 wk (Fig. 3A). Tg-H and WT mice fed the high-safflower oil diet showed similar increases in body weight. After 27 wk, total UCP2 mRNA (endogenous + transgene UCP2 mRNA) levels in Tg-H mice did not differ between Tg-H mice fed the HC and high-safflower oil diet (data not shown). This result is expected, since the transgene is not expected to respond to diet and since safflower oil does not induce expression of endogenous UCP2 gene markedly. Energy intake, body composition, and tissue weights in this first experiment are shown in Table 1. Energy intake in WT mice fed an HC diet was slightly lower than that in WT mice fed a high-safflower oil diet or transgenic mice fed an HC diet. However, body weights of WT and Tg-H mice fed the HC diet did not differ, suggesting that a small difference in energy intake did not affect body weight. Tg-H and WT mice fed a high-safflower oil diet showed similar increases in body fat, several WAT tissue weights, and liver weights (relative to those of mice fed an HC diet). The lack of a significant difference in the interaction of genotype and diet in WAT tissue weights suggested that overexpression of liver UCP2 did not influence increases in fat mass in response to a high-safflower oil diet.


Figure 3
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  		Fig. 3. A: body weight changes in female Tg-H mice (filled symbols) and WT littermates (open symbols) fed a HC (circles) or high-safflower oil; diet (HF; triangles). The experimental diets were started at 3 mo of age and continued for 27 wk. Each point is the mean ± SE; n = 5–6 mice. Tissue weights are shown in Table 1. B: body weight changes in female Tg-H mice (filled symbols) and WT littermates (open symbols) fed an HC diet (squares) or high-lard diet (HF, triangles). The experimental diets were started at 7 mo of age and continued for 8 wk. Each point is the mean ± SE; n = 4–5 mice. C: body weight changes in male Tg-H mice (filled symbols) and their WT littermates (open symbols) fed an HC diet (squares) or high-lard diet (HF, triangles). The experimental diets were started at 7 mo of age and continued for 8 wk. Each point is the mean ± SE; n = 4–5 mice.

 

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  		Table 1. Energy intake, body fat, and tissue weight in female UCP2 transgenic and WT mice fed a high-carbohydrate or a high-safflower oil diet for 27 wk

 
In the second experiment, lard was used as the HF diet (Fig. 3B). Seven-month-old female Tg-H mice and WT littermates were fed a high-lard diet for 8 wk. There were no significant difference in the change in body weight in response to the high-lard diet between Tg-H and WT mice.

In the third experiment, male mice were examined (Fig. 3C). Seven-month-old male Tg-H mice and WT littermates were fed a high-lard diet for 8 wk. Although the initial body weight of WT mice was marginally larger than that of mice with high levels of UCP2, there was no difference in the increase in body weight in response to the high-lard diet. Similar changes in tissue weights in male and female mice fed a high-lard diet were observed in WT and Tg-H mice without significant changes in energy intake (data not shown).

Antiobesity effects of fish oil in UCP2 knockout and WT mice. Ingestion of fish oil by mice increased UCP2 expression in hepatocytes and had an antiobesity effect relative to ingestion of other oils (12, 17, 18, 24). Although overexpression of UCP2 in hepatocytes did not lead to a reduction in body weight, it is possible that increased production plus activation of hepatocyte UCP2 in response to fish oils may underlie the antiobesity effects. To examine this possibility, we investigated the effects of fish oil on UCP2 knockout mice. UCP2 knockout mice and WT mice were fed an HC diet, high-safflower oil diet, or high-fish oil diet for 16 wk. As observed previously (24), after 16 wk, liver UCP2 mRNA levels were markedly higher in WT mice fed a high-fish oil diet than in WT mice fed an HC diet or a high-safflower oil diet (Fig. 4A). Body weight of female WT mice fed a high-safflower oil diet was higher than that of mice fed the HC diet; however, this increase in body weight was not observed in mice fed the fish oil diet (Fig. 4B, top). Among female UCP2 knockout mice, although the increase in body weight in mice fed the high-safflower oil diet was much smaller than the increase observed in WT mice, mice fed a high-fish oil diet did not show an increase in body weight relative to those fed an HC diet. The smaller change in body weight in female UCP2 knockout mice fed a high-safflower oil diet was specific to safflower oil; female UCP2 knockout and WT mice fed a high-lard diet showed similar, marked increases in body weight (data not shown). In male mice, there was no difference in safflower oil-induced weight gain between UCP2 knockout and WT mice (Fig. 4B, bottom). A similar increase in body weight in response to a high-fish oil diet was observed in both male UCP2 knockout and WT mice (relative to HC diet); however, the increase in mice fed a high-fish oil diet was smaller than that in mice fed a high-safflower oil diet.


Figure 4
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  		Fig. 4. A: Northern blotting of UCP2. WT and UCP2 knockout (KO) mice were fed a HC, high-safflower oil diet (Safflower), or high-fish oil diet (Fish) for 16 wk, and their livers were used for Northern blots. On the autoradiogram, each lane represents a sample from an individual mouse. B: body weight changes in UCP2 KO mice and WT mice fed a HC ({circ}), high-safflower oil ({triangleup}), or high-fish oil ({square}) diet. Body weights of female and male mice are shown. Each point is the mean ± SE; n = 6 mice. Tissue weights are shown in Tables 2 and 3.

 
Energy intake, body compositions, and tissue weights for female and male UCP2 knockout mice are shown in Tables 2 and 3. Because the two HF diets are hypercaloric and contain the same amounts of other nutrients, we limited our comparisons to the effects of the two HF diets, rather than comparing them with the effects of an HC diet. The difference in body weight between groups was because of the difference in body fat weight. There was no difference in lean body mass. Female WT mice fed a high-fish oil diet had lower mesenteric, parametric, retroperitoneal, and subcutaneous WAT weights compared with mice fed a high-safflower oil diet (Table 2). In male WT mice fed a high-fish oil diet, only the mesenteric WAT weight was lower (Table 3). The lower fish oil-induced body weight decrease observed in male mice was because of the absence of reductions in epididymal, retroperitoneal, and subcutaneous WAT. The reasons for this sex-specific difference in fat weights are not clear.


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  		Table 2. Energy intake, body fat, and tissue weight in female UCP2 knockout and WT mice fed a high-safflower oil or high-fish oil diet for 16 wk

 

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  		Table 3. Energy intake, body fat, and tissue weight in male UCP2 knockout and WT mice fed a high-safflower or a high-fish oil diet for 16 wk

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Among HF diets, both safflower oil and lard induce obesity, whereas fish oil does not. In parallel with the change in body weight, fish oil markedly increases UCP2 levels in hepatocyes (17). Fish oil may increase UCP2 expression and activity in hepatocytes to increase energy expenditure (24). In the present study, overexpression of UCP2 in hepatocytes did not prevent obesity induced by a high-safflower oil or high-lard diet, indicating that an increase in UCP2 in hepatocytes does not prevent HF diet-induced obesity. UCP2 knockout mice fed a high-fish oil diet also showed a reduction in body weight (relative to that of mice fed a high-safflower oil diet) similar to that observed in WT mice, indicating that increased UCP2 protein levels or activity in hepatocytes may not contribute to the antiobesity effects of fish oil.

It has been reported that mitochondria isolated from pancreatic β-cells and thymocytes from UCP2 knockout mice show higher ATP levels, possibly because of a decrease in proton leak than those in control WT mice (14, 26). In β-cells, a deficiency of UCP2 or pharmacological inhibition of UCP2 activity increases glucose-stimulated insulin secretion (26, 27). In addition, it was reported recently that, in glucose-excited neurons [proopiomelanocortin (POMC) neurons] in brain, deficiency of UCP2 or pharmacological inhibition of UCP2 activity protects against HF diet-induced loss of glucose sensing (i.e., glucose-stimulated release of {alpha}-melanocyte stimulation hormone from hypothalamus) (19). In contrast, UCP2 ablation in liver does not affect steatohepatitis observed in genetically obese ob/ob mice or mice fed a HF diet, suggesting that the contribution of UCP2 in hepatocytes to uncoupling of oxidative phosphorylation is limited (2). Furthermore, it has been reported recently that the membrane potentials of mitochondria isolated from liver of UCP2 knockout mice and WT mice are similar, suggesting that enhanced uncoupling may not occur in liver from UCP2 knockout mice (22), although this outcome is expected, since UCP2 is not expressed in hepatocytes (Fig. 2A). Taken together, these data suggest that the role of UCP2 in energy dissipation differs among tissues and that liver UCP2 may not be a major physiological uncoupler.

One unexpected finding of the present study was that a high-safflower oil diet caused less obesity in female UCP2 knockout mice than in WT mice (Fig. 4B). We had thought that a high-safflower oil diet might exacerbate obesity in UCP2 knockout mice. However, this unexpected effect was not observed in male UCP2 knockout mice or in UCP2 knockout mice fed a high-lard diet. Although the mechanism is not clear, it is conceivable that dietary safflower oil may affect other UCP2-sensitive tissues, such as thymocytes, β-cells, and POMC neurons, which may prevent an increase in body fat in a sex- or fatty acid-dependent manner.

In conclusion, liver UCP2 is not essential for the antiobesity effects of fish oil. Fish oil showed antiobesity effects, possibly through activation of PPAR{alpha} (18); however, the target genes that may lead to increased energy expenditure are not known.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by a grant-in-aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (Tokyo, Japan), by research grants from the Japanese Ministry of Health, Labour, and Welfare, and by a grant for the Promotion of Fundamental Studies in Health Sciences from the Organization for Pharmaceutical Safety and Research (Osaka, Japan).


   ACKNOWLEDGMENTS
 
We thank Dr. Chen-Yu Zhang and Dr. Bradford B. Lowell at Beth Israel Deaconess Medical Center and Harvard Medical School and Dr. György Baffy at Rhode Island Hospital and Brown Medical School for advice on the UCP2-knockout mice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. Ezaki, Nutritional Science Program, National Institute of Health and Nutrition, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8636, Japan (e-mail: ezaki{at}nih.go.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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