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Correspondence to:
Philip A. Kern.
Elevated plasma leptin levels are found in obese humans, suggesting a defect in the function of leptin in regulating body weight and adiposity. In 53 subjects covering a broad range of adiposity, we examined the relationships between plasma leptin, adipose tissue ob mRNA levels, and adipose tissue TNF mRNA. There was a highly significant correlation between plasma leptin levels and every index of adiposity. In contrast, the relationship between ob mRNA levels and adiposity was weak. Adipose tissue from obese subjects demonstrated higher ob mRNA levels than adipose tissue from lean subjects (lean: 0.49 ± 0.05; obese 0.87 ± 0.09 arbitrary units, P < 0.05). However, there was no significant correlation between body fat and ob mRNA level. In addition, there was no significant relationship between ob mRNA levels and plasma leptin levels, which were measured in the same subjects. In addition to the measure of ob mRNA levels, adipose TNF mRNA levels were measured in 18 subjects. TNF mRNA levels varied with ob mRNA levels (r = 0.44, P = 0.06).
These data show that plasma leptin levels are not directly related to adipose tissue ob mRNA levels, suggesting posttranscriptional regulation of leptin expression, either at the level of the adipocyte, or by alteration of plasma leptin degradation or clearance. In addition, the parallel changes in ob and TNF mRNA in adipose tissue suggest that these two important factors in the defense against obesity may be regulated similarly.—Ranganathan, S., M. Maffei, and P. A. Kern. Adipose tissue ob mRNA expression in humans: discordance with plasma leptin and relationship with adipose TNF
Supplementary key words:
leptin, obese gene, tumor necrosis factor, obesity, adipose tissue
The defective gene in the ob/ob mouse has been described, and the gene product, leptin, is an 167 amino acid protein that is expressed in adipose tissue (1) and placenta (2) (3) (4). When ob/ob mice are injected with recombinant leptin, weight loss ensues (5) (6) (7). The recent description of a receptor for leptin in the hypothalamus (8) (9) supports other studies suggesting that leptin plays an important role in the control of appetite and represents a homeostatic signal intended to limit the expansion of adipose mass.
Several studies have demonstrated that adipose tissue ob mRNA and plasma leptin levels are elevated in obese rodents (10) (11) (12) (13) (14) (15). In humans, several studies have examined plasma leptin levels in patients with varying levels of adiposity (10) (16) (17) (18) (19) (20). Obese subjects generally have higher levels of plasma leptin, suggesting that obese humans are not defective in leptin expression, but rather manifest leptin resistance. Several studies have noted increased ob mRNA expression in obese humans (16) (21). Because of the consistent finding of elevated plasma leptin, there is a tendency to assume that plasma leptin levels are directly related to adipose tissue ob gene expression.
Another important product of the adipocyte is TNF
As described herein, we examined both adipose tissue ob and TNF mRNA levels and plasma leptin levels in the same patients. We found a remarkable lack of correlation between adipose tissue expression and plasma leptin, suggesting that other factors are involved in the regulation of plasma leptin levels.
Subjects and patient recruitment
RNA extraction and mRNA analysis
TNF mRNA was measured using competitive RT-PCR, as described previously (22). In brief, 0.4 µg of total RNA from adipose tissue was added to increasing quantities of a competing RNA construct (31), which contained primer sites for human TNF, but which contained a 49 nt internal deletion. After the reverse transcriptase reaction, PCR was carried out for 35 cycles at 60°C, and the resulting PCR products were resolved on a 2% agarose gel and the ethidium bromide-stained gel was quantitated. The ratio of TNF product/cRNA standard was plotted against the number of copies of cRNA added, to yield the equivalence point between cRNA and TNF mRNA. Data are expressed as the "number of copies"/µg of total RNA, were "number of copies" refers to the number of copies of the cRNA added.
Statistics
As described by us and others, plasma leptin correlated significantly with indicators of adiposity. Such was the case with the subjects in this study. There was a significant relationship between plasma leptin and body mass index (BMI) (r = 0.711, P < 0.0001), and between plasma leptin and total body fat (r = 0.761, P < 0.0001).
In previous studies, a significant relationship was observed between ob mRNA levels and adiposity (16) (21). The ob mRNA levels from our subjects were analyzed with regard to a number of factors. When patients were divided into lean and obese (lean was defined as <20% fat in men, and <30% fat in women), ob mRNA levels were significantly higher in obese subjects ( Figure 1). However, ob mRNA levels did not increase consistently with increased adiposity. As shown in Figure 2, a regression of ob mRNA levels with percent body fat showed no significant relationship. Similarly, there was no significant relationship between other indices of adiposity and ob mRNA levels. The correlation coefficient between % body fat and ob mRNA was 0.18, and between total fat mass and ob mRNA level, the correlation coefficient was 0.05. Of the 36 subjects who underwent fat biopsies, 27 were women. However, the analysis of women and men separately did not improve the correlation between adiposity and ob mRNA levels nor did the relationship improve by excluding the very obese (BMI > 40) subjects. Both plasma leptin and ob mRNA were measured in subjects, and the relationship between these measurements is shown in Figure 3. There was no relationship between the mRNA expression of ob, and the plasma level of the leptin protein. In Figure 4, representative Northern blots are shown to clearly illustrate these points. As shown by the blots and the corresponding plasma leptin levels below each lane, patients with the highest plasma leptin levels did not necessarily have the highest ob expression. Even within the same subjects, the changes in ob mRNA and leptin were poor. As described by us previously (10), weight loss resulted in a consistent decrease in plasma leptin, and a variable decrease in ob mRNA levels. In 8 subjects who had measurements of leptin and ob mRNA both before and after weight loss, the correlation between the percent changes in leptin and ob mRNA was not significant (r = 0.08).
Obese rodents and humans express high levels of TNF in adipose tissue, which may play a role in the development of insulin resistance. In the adipose tissues of 18 subjects, we measured TNF mRNA using competitive RT-PCR. As shown in Figure 5, adipose tissue TNF expression varied with adipose tissue ob mRNA levels (r = 0.44, n = 18, P = 0.06). There was no relationship between plasma leptin levels and adipose TNF mRNA expression (r = 0.01, P = NS).
Since the original description of the ob gene and the corresponding plasma protein (1), numerous studies have analyzed ob regulation and plasma leptin levels. Plasma leptin levels are strongly associated with total adipose tissue mass. Not only is plasma leptin increased in obese subjects (10) (16) (18) (19) (20), and decreased in reduced-obese subjects (10) (32), but leptin is decreased adipose tissue mass induced by exercise (33) (34) and anorexia nervosa (35). Fewer studies have examined the relationship between adipose tissue ob mRNA levels and adiposity. Using a PCR method, Considine et al. (16) measured ob mRNA levels in 54 subjects and found a highly significant correlation with BMI and % body fat. Several studies also found an increase in ob mRNA levels in obese patients (21) (32) (36). However, no previous study has directly compared ob mRNA levels with plasma leptin levels in the same subjects.
In this study, we found that obese subjects as a group demonstrated higher levels of ob mRNA than lean subjects; however, there was no significant correlation between ob mRNA levels and adiposity using either BMI, percent body fat, or total body fat. In addition, we found no significant relationship between adipose tissue ob mRNA level and plasma leptin level. However, as with numerous other studies, we found the same significant relationship between adiposity and plasma leptin levels.
Previous studies on ob mRNA differed from this one in several ways. Isolated adipocytes were used after a collagenase digestion in some studies (16), whereas we used whole adipose tissue. As the ob mRNA is only present at very low levels in preadipocytes (37), either method should yield similar results unless there was selective loss of an adipocyte population during the collagenase digestion. Some studies used PCR for the RNA measurement (16) (32), although without an internal standard to control for the reverse transcriptase reaction and variations in the thermocycling.
In this and most other studies, subcutaneous abdominal adipose tissue was sampled. It is possible that this adipose depot is not representative of plasma leptin and hence the RNA and protein data appear unrelated. Based on other studies, however, this is unlikely. Depending on the study, 74 –93% of total body adipose tissue is found in subcutaneous adipose tissue using MRI or CT with careful quantitation (38) (39) (40). Although the proportion of visceral adipose tissue is a little higher in men, all studies show that the large majority of total body adipose mass is located subcutaneously. Therefore, because subcutaneous adipose tissue makes up the bulk of adipose mass, it is highly unlikely that some other adipose tissue depot could contribute significantly to leptin expression.
The lack of correlation between adipose tissue ob mRNA and plasma leptin suggests that the regulation of ob gene expression may be complex, and plasma leptin levels may not be dependent solely on adipose tissue ob mRNA expression. The high levels of plasma leptin in obese subjects could be due to posttranscriptional mechanisms, such as leptin translation or secretion from the adipocyte. A recent study provided evidence for such posttranscriptional mechanisms. Kirchgessner et al. (41) found that plasma leptin was decreased, with no change in leptin mRNA levels, in TNF knockout mice. In addition, 3T3-L1 cells demonstrated a discordance between leptin secretion and mRNA levels (41). An additional potential site for leptin regulation is the degradation of the plasma protein. Previous studies have suggested that leptin has a short plasma half-life (42) (43) of less than 24 min, and is bound to other proteins, thus raising the possibility for regulation of leptin degradation or availability (44). However, no changes were found in leptin half-life in obese subjects (42). In addition, when adipose tissue leptin production was measured by comparing abdominal venous leptin levels with peripheral leptin levels, a direct correlation between abdominal adipose leptin production and obesity was found (42). Another possible explanation for the apparent differences between studies is patient heterogeneity. Although several studies have observed a decrease in ob mRNA after weight loss (10) (16), this finding was not consistent, suggesting that some subjects decrease plasma leptin through posttranscriptional mechanisms.
We also observed a correlation between adipose tissue ob mRNA and adipose tissue TNF expression. Adipose tissue TNF expression increases with increasing adiposity in humans (22) (24) and has been implicated in the insulin resistance of obesity (23) (25). Other studies have previously noted a relationship between TNF and leptin expression. When TNF was injected into mice and hamsters, the anorexia was accompanied by an increase in plasma leptin and adipose leptin mRNA levels within 8 h (45) (46). In a recent study, leptin secretion by 3T3-L1 cells was stimulated by the addition of TNF (41). In addition, TNF knockout mice had lower levels of plasma leptin than controls (41). Although the relationship between TNF and leptin expression in humans was of borderline statistical significance, these data tend to corroborate the findings noted above in rodents. One possible explanation for the parallel increase in TNF and ob mRNA in obese humans relates to the overall defense of the animal against the deleterious effects of obesity. Thus, it is possible that both adipose TNF expression and ob mRNA expression are different parts of the defense against obesity: one working to control appetite, and the other (TNF) making the organism more insulin resistant.
We wish to thank Rosa Simsolo and Ricardo Bosch for assistance with patient recruitment, and Rami Kaakaji for technical assistance. We also wish to acknowledge the assistance of Dr. Jeffrey M. Friedman. Grant support: a Merit Review Grant from the Veterans Administration, DK 39176 for the National Institutes of Health, and a Grant-in-Aid from the American Heart Association.
Manuscript received November 3, 1997; and in revised form December 1, 1997.
Abbreviations:
TNF, tumor necrosis factor; GAPD, glyceraldehyde phosphate dehydrogenase; BMI, body mass index
Copyright © 1998 by Lipid Research, Inc.
Original Article
Adipose tissue ob mRNA expression in humans: discordance with plasma leptin and relationship with adipose TNF
Subramanian Ranganathana,
Margherita Maffeib, and
Philip A. Kerna
expression
a Department of Medicine, Division of Endocrinology, University of Arkansas for Medical Sciences and the John L. McClellan VA Medical Center, Little Rock, AR 72205
b Rockefeller University, 1230 York Avenue, New York, NY 10021
ABSTRACT
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expression. J. Lipid Res. 1998. 39: 724–730.
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, which is elevated in the adipose tissue of obese rodents and humans (22) (23) (24), and which may play an important role in obesity-related insulin resistance (25). Although adipose tissue TNF expression is related to obesity, there is much interindividual variation in humans (22), suggesting that other factors control TNF expression.
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Fifty-three subjects were recruited and 36 underwent an incisional fat biopsy from the lower abdominal wall under local anesthesia. All subjects were weight stable, were taking no medications likely to affect adipocyte metabolism, were non-diabetic, and did not exercise for at least 2 weeks prior to the biopsies. All subjects were placed on a standardized diet consisting of 35% fat, 20% protein, and 45% carbohydrate, calorically adjusted for weight maintenance, for 2 days prior to the study. Body composition was measured using bioelectrical impedance (26). Plasma leptin was measured using an enzyme-linked immunosorbent assay, as described previously (10). Data on some of these subjects have been reported previously (10) (27) (28).
Ob mRNA was measured by Northern blotting, as described previously (10). RNA was extracted using the method of Chomczynski and Sacchi (29) with minor modifications (30). Equal amounts of total RNA were resolved on a 2.2 M formaldehyde–1% agarose gel, transferred to nylon membrane, and blotted with the [32P-labeled cDNA probes for human ob (1), and glyceraldehyde phosphate dehydrogenase (GAPD). To further demonstrate that equal quantities of total RNA were loaded, samples of the RNA were resolved by electrophoresis and stained with ethidium bromide for quantitation of the 28S and 18S rRNA bands. To quantitate differences between Northern blots, autoradiographic images were quantitated by densitometry on a laser image analyzer.
Data are expressed as the mean ± SEM. Data were analyzed non-parametrically, using the Mann-Whitney test for non-paired data. Linear regressions were performed using the Pearson product correlation coefficient.
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Figure 1.
Ob mRNA levels in lean and obese subjects. Subjects were divided into lean and obese based on % of body fat (see methods). *P < 0.01.
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Figure 2.
Relationship between ob mRNA levels and BMI. Ob mRNA levels are shown in relation to each subject's BMI.
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Figure 3.
Relationship between ob mRNA level and plasma leptin. Ob mRNA/GAPD ratio is shown in relation to the plasma leptin level for each subject.
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Figure 4.
Representative Northern blots. Two representative Northern blots are shown, with each subject's BMI and plasma leptin shown below the lanes.
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Figure 5.
Relationship between ob mRNA and TNF mRNA levels in 18 subjects covering a wide range of adiposity.
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ACKNOWLEDGMENTS
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