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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 2 828-833
Copyright © 2001 by The Endocrine Society

Original Studies

Age-Related Differences in Messenger Ribonucleic Acid Expression of Key Proteins Involved in Adipose Cell Differentiation and Metabolism1

Pascal Imbeault, Hubert Vidal, Angelo Tremblay, Nathalie Vega, André Nadeau, Jean-Pierre Després and Pascale Mauriège

Physical Activity Sciences Laboratory, Department of Social and Preventive Medicine, Laval University (P.I., A.T., P.M.), Ste-Foy, Québec, Canada G1K 7P4; INSERM U-449, Faculté de Médecine R.T.H. Laënnec (H.V., N.V.), F-69372 Lyon, France; Diabetes Research Unit (A.N.), and Lipid Research Center (J.-P.D., P.M.), Laval Medical Research Center, Québec, Canada G1V 4G2; and Quebec Heart Institute, Laval Hospital (J.-P.D.), Québec, Canada G1V 4G5

Address all correspondence and requests for reprints to: P. Mauriège, Ph.D., Lipid Research Center, Laval University Medical Research Center, 2705 boulevard Laurier, Room TR-93, Ste-Foy, Québec, Canada G1V 4G2. E-mail: mauriege{at}club-internet.fr


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
This study was performed to compare the expression of key proteins [lipoprotein lipase (LPL), hormone-sensitive lipase (HSL), complement 3 (C3), and peroxisome proliferator-stimulated receptor-{gamma} (PPAR{gamma})] involved in sc abdominal adipose tissue (AT) metabolism of young (n = 13) vs. middle-aged (n = 16) men. The sc abdominal AT-LPL activity as well as fat cell lipolysis were also measured in both groups of men. Young and middle-aged men displayed similar body weight and sc abdominal fat accumulation, measured by computed tomography. However, middle-aged men were characterized by a higher percent body fat (28 ± 5% vs. 22 ± 7%; P < 0.05) than young subjects. No difference between groups was observed in sc abdominal adipose tissue LPL activity. On the other hand, maximal lipolytic responses of sc abdominal adipocytes to isoproterenol (ß-adrenergic agonist) or to postadrenoceptor agents such as dibutyryl cAMP, forskolin, and theophylline were lower in middle-aged than in young men (P < 0.05). AT-LPL messenger ribonucleic acid (mRNA) levels were similar regardless of the subject’s age. However, HSL, C3, and PPAR{gamma} mRNA levels were higher in middle-aged than in young individuals (P < 0.01–0.05). After correction for percent body fat, only HSL and C3 mRNA levels remained significantly different between groups (P < 0.05). Taken together, these results suggest that aging has an effect on the up-regulation of HSL and C3 mRNA levels, whereas PPAR{gamma} expression seems to be related mainly to increased adiposity.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
SUBSTANTIAL CHANGES in body fat distribution and function occur with aging (1, 2). Indeed, the peak fat mass is attained by middle age and further declines during senescence (2). This phenomenon is paralleled by a decline in the intrinsic preadipocyte replication potential as well as by an impaired capacity of human preadipocytes to differentiate (3). Studies from animals have also revealed that the preadipocyte differentiation program is impaired with senescence, as messenger ribonucleic acid (mRNA) levels of ß-actin and {alpha}-tubulin (early differentiation markers), lipoprotein lipase (LPL) (midway marker), and glycerol-3-phosphate dehydrogenase (late marker) were decreased in preadipocytes cultured from rats of various age (4).

As opposed to young individuals, middle-aged men commonly present greater body fatness (2). However, little is known about changes occurring in fat cell metabolism at this specific period of human life (40–65 yr). We recently reported that middle-aged vs. young men displayed a selective decrease in the sc adipose tissue lipolytic capacity, which appears to be related to postreceptor alterations rather than to increased adiposity (5).

In the last years, the discovery of proteins expressed and secreted by adipocytes has conferred to adipose tissue a more active role in the control of energy balance (6). In the adipocyte, lipid storage is almost entirely dependent on the uptake of fatty acids released from the hydrolysis of circulating triglyceride-rich lipoproteins by lipoprotein lipase (LPL) (7). Conversely, adipose tissue lipid mobilization is stimulated by hormones such as catecholamines that activate cell surface receptors, thereby increasing the cellular concentration of cAMP that induces adipocyte lipolytic activity via the phosphorylation of hormone-sensitive lipase (HSL) (8). Moreover, adipocyte produces complement 3 (C3), which interacts extracellularly with adipsin and factor B to form C3a, from which terminal arginine is removed by carboxypeptidases, generating the acylation-stimulating protein (ASP), a potent stimulator of glucose transport and triglyceride synthesis in the adipocytes (9). These latter adipose cell enzymes (LPL and HSL) or product (C3) are to some extent responsible for control of the adipose tissue lipolysis/lipogenesis balance. More recently, characterization of transcription factor-binding sites led to the identification of a protein family that plays an important role in the induction of the fully differentiated adipocyte, the peroxisome proliferator-activated receptors (PPAR) (10, 11). Among these, PPAR{gamma} has been identified to play a key role in adipocyte differentiation (12). To the best of our knowledge, few studies have examined the impact of aging on potential targets involved in the control of lipolysis/lipogenesis adipose tissue balance as well as adipocyte differentiation in humans. We therefore quantified the mRNA levels of LPL, HSL, C3, and PPAR{gamma} in the sc abdominal fat depot of young vs. middle-aged men.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Twenty-nine healthy Caucasian men were recruited through the media and gave their written informed consent to participate in this study, which was approved by the Laval University medical ethics committee. Thirteen young (mean ± SD, 31 ± 5 yr) and 16 middle-aged (56 ± 6 yr) men were compared for potential differences in sc adipose tissue mRNA levels of genes involved in the control of adipocyte differentiation and metabolism. All individuals underwent a medical evaluation by a physician, which included a medical history. Subjects with cardiovascular disease, diabetes mellitus, endocrine disorders, or taking medication that could influence triglyceride metabolism (ß-blockers, antihypertensive drugs, etc.) were excluded from the study. All participants were sedentary, nonsmokers, and moderate alcohol consumers. None had recently been on a diet or involved in a weight-reducing program, and their body weights had been stable during the last 6 months before the study.

Total body fatness and regional fat distribution

Body density was determined by the underwater weighing technique, from which percent body fat was derived with the Siri formula (13). Pulmonary residual volume was measured using the helium dilution method (14). Fat mass and fat-free mass were derived from percentage of body fat and total body weight. Waist girth was measured according to procedures recommended at the Airlie Conference (15). Computed tomography was performed with a Siemens Somatom DRH scanner (Erlangen, Germany) according to the methodology previously described by Sjöström et al. (16). Briefly, subjects were examined in the supine position with both arms stretched above the head. Computed tomography scans were performed at the abdominal (between L4 and L5 vertebrae) level, using an abdominal scout radiograph to establish the position of the scans to the nearest millimeter. Total adipose tissue (AT) areas were calculated by delineating the abdomen with a graph pen and then computing AT surfaces with an attenuation range of -190 to -30 Hounsfield units (17). Abdominal visceral AT area was measured by drawing a line within the muscle wall surrounding the abdominal cavity. The abdominal sc AT area was determined by subtracting the visceral AT area from the total abdominal AT area.

Adipocyte isolation and lipolysis

After an overnight fast, participants underwent a biopsy of sc fat in the periumbilical region. A small cutaneous incision (1 cm) was performed in the abdominal site under local anesthesia (1% lidocaine, without epinephrine), and about 400 mg sc adipose tissue were surgically removed from the fat depot.

Samples of 250 mg adipose tissue were used for the measurement of fat cell lipolysis. Adipocytes were isolated according to the method of Rodbell (18) in a Krebs-Ringer bicarbonate buffer (pH 7.4) containing 4% BSA and 5 mmol/L glucose (KRBA) plus 1 mg/mL collagenase, as previously described (19). Digestion took place in a shaking water bath under an air-gas phase of 95% O2 and 5% CO2 for 40 min at 37 C. The suspension was then filtered, and the cellular filtrate obtained was rinsed three times with 5 mL KRBA. Isolated adipocytes were finally resuspended in KRBA to obtain a final concentration of approximately 500 cells/50 µL.

Extracellular glycerol release was used as the indicator of adipocyte lipolysis. Fifty-microliter aliquots of the continuously stirred cell suspension were placed in 1.5-mL conical tubes. Two of these tubes were used for cell counting and sizing, as previously described (20); 2 others containing 10 µL KRB were immediately placed on ice and provided evaluation of the initial concentration of glycerol in the medium. Pharmacological agents stimulating lipolysis were added just before starting the incubation in 10-µL portions. After a 2-h incubation at 37 C in a shaking water bath under a 95% O2 and 5% CO2 gas phase, 50 µL HCl (1 N) were added to all tubes to stop the reaction, then 50 µL NaOH (1 N) were added to neutralize the medium. All tubes were stored at -20 C until glycerol determination. The NADH concentration was measured by bioluminescence with a luciferase solution, using an automated 2250 luminometer (Dynatech Corp., Chantilly, VA). For each concentration of stimulator agent, the amount of glycerol was taken as the average of the quantities obtained from the 2 incubated tubes. Glycerol measurement by bioluminescence is very sensitive and especially well adapted when only small amounts of adipose tissue are available (19, 21). Adipose cell diameters were determined using a Leitz microscope equipped with a graduated ocular (Rockleigh, NJ). Mean fat cell diameter was assessed from the measurement of at least 500 cells, and the density of triolein was used to transform adipose cell volume into fat cell weight, as previously described (20).

The lipolytic activity of isolated fat cells was tested with isoproterenol (nonselective ß-adrenergic receptor agonist) (21). Ascorbic acid (0.1 mmol/L) was included in the medium to prevent catecholamine degradation. Some experiments were conducted with forskolin (direct activator of adenylate cyclase), dibutyryl cAMP (a stimulator of the protein kinase hormone-sensitive lipase complex and phosphodiesterase-resistant cAMP analog), and theophylline (mainly inhibitor of cGMP-inhibited phosphodiesterase) (21). Lipolysis was expressed either per cell number (i.e. micromoles of glycerol per 106 cells x 2 h) or per unit cell surface area (i.e. nanomoles of glycerol per µm2 x 108/2 h); the latter mode of expression was used to correct for variation in fat cell size, which is well known to influence the rate of lipolysis (21, 22). The responsiveness was expressed as the difference between basal glycerol release and the lipolytic rate at the maximum effective concentration of the agents tested (10-5 mol/L isoproterenol or forskolin, 10-3 mol/L dibutyryl cAMP or theophylline).

AT-LPL activity

Samples of approximately 100 mg adipose tissue from the abdominal site were immediately frozen in liquid nitrogen and stored at -80 C. Heparin-releasable LPL activity was measured within 1 month of frozen storage according to the method of Savard et al. (23). AT-LPL activity was expressed as micromoles of free fatty acids (FFA) released per h/106 cells. As AT-LPL activity is associated with fat cell size (7, 23), AT-LPL activity was also expressed per cell surface area (i.e. nanomoles of FFA per h/µm2 x 108).

Total RNA preparation

Total RNA from approximately 50 mg adipose tissue was prepared using the RNeasy total RNA kit from QIAGEN (Courtaboeuf, France) as previously described (24). The total RNA concentration was determined by absorbance measurement at 260 nm. The 260/280 nm absorption of all preparations ranged between 1.8 and 2.0. The average yield of total RNA was 1.6 ± 0.7 µg/100 mg adipose tissue, and no significant difference was observed between groups.

Quantification of mRNA

LPL, HSL, C3, and total PPAR{gamma} adipose tissue mRNA levels were determined by RT reaction followed by competitive PCR (RT-competitive PCR). Briefly, this method relies on the addition of a known amount of a competitor DNA molecule to the PCR to standardize the amplification process. The construction of the competitors, the validation of assays, and the conditions of the RT-PCR reactions have previously been described in detail [LPL and C3 (25), HSL (26), and PPAR{gamma} (27)]. For each mRNA, the specific first strand complementary DNA was synthesized from 0.1 µg total RNA. During the PCR, sense primers with the 5'-end labeled with Cy-5 fluorescent dye (Eurogentec, Seraing, Belgium) were used. The use of these primers allowed the synthesis of fluorescent PCR products that were analyzed with an automated laser fluorescence DNA sequencer (ALFexpress, Pharmacia Biotech, Uppsala, Sweden) in 4% denaturing polyacrylamide gels. The initial concentration of target mRNA was determined at the competition equivalence point as previously described (24).

Drugs and chemicals

Collagenase, BSA, and enzymes for glycerol assays were obtained from Roche Molecular Biochemicals (Laval, Canada). Ascorbic acid, theophylline, forskolin, dibutyryl cAMP, (-)-isoproterenol bitartrate, (-)-epinephrine bitartrate, and cold triolein were purchased from Sigma (St. Louis, MO). [14C]Triolein was obtained from NEN Life Science Products (Mississauga, Canada). All other chemicals and organic solvents were of the highest purity grade commercially available. The same batches of pharmacological agents, collagenase, and albumin were used in all experiments.

Statistical analyses

Student’s t test was used for comparisons between young and middle-aged subjects. An analysis of covariance was used to determine whether there were significant differences between mRNA levels once the effect of body fat percentage or visceral adipose tissue was controlled. Associations between two variables were quantified by using the Pearson’s product-moment correlation coefficients. All analyses were performed using the Jump version 3.2.2 program (SAS Institute, Inc., Cary, NC) adapted for Macintosh computers.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The various primer sequences used for the RT-competitive PCR and the sizes of the target and competitor PCR products generated during the reaction are presented in Table 1Go. The subjects’ physical characteristics are presented in Table 2Go. As expected, a significant difference was observed in age (P < 0.001). Although both groups displayed similar body weight and body mass index, middle-aged men had higher percent body fat, waist girth, and visceral adipose tissue accumulation than young subjects (P < 0.01–0.05). Moreover, sc abdominal adipocytes were larger in middle-aged than in young individuals (P < 0.01).


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  		Table 1. Sequences of the primers used for the RT-competitive PCR

 

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  		Table 2. Physical characteristics of subjects

 
AT-LPL expression and activity were similar in both groups (Fig. 1Go). No significant relationship was observed between the mRNA level and the activity of this enzyme in the whole group (r = 0.34; P = NS). As shown in Fig. 2Go, mRNA levels of HSL were higher in middle-aged than in young men (P < 0.01). However, no difference was observed between groups in basal lipolysis of sc adipocytes, whereas maximal lipolytic responses of sc adipose cells to isoproterenol (ß-agonist) and to postreceptor agents such as dibutyryl cAMP, forskolin, and theophylline were lower in middle-aged than in young men (P < 0.01–0.05). Furthermore, HSL expression was not related to either basal or stimulated lipolysis (-0.10 < r < -0.22; P = NS). Finally, middle-aged men displayed higher mRNA levels of C3 and PPAR{gamma} than young subjects (P < 0.01; Fig. 3Go).



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  		Figure 1. Adipose tissue mRNA levels and activity of LPL from the sc abdominal depot of young (n = 13) and middle-aged (n = 16) men. AT-LPL activity is expressed per adipocyte surface area. Values are the mean ± SE.

 


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  		Figure 2. Adipose tissue mRNA levels of HSL as well as basal and maximal lipolytic response of sc abdominal adipocytes to isoproterenol and postadrenoceptor agents in young (n = 13) and middle-aged (n = 16) men. Fat cells were incubated in the presence of isoproterenol (ISO; 10-5 mol/L), dibutyryl cAMP (DcAMP; 10-3 mol/L), forskolin (FK; 10-5 mol/L), or theophylline (THEO; 10-3 mol/L). Basal glycerol release has been subtracted from maximal lipolytic response. Values are the mean ± SE. *, P < 0.05; {dagger}, P < 0.01 (significant difference between groups).

 


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  		Figure 3. Adipose tissue mRNA levels of C3 and PPAR{gamma} from the sc abdominal depot of young (n = 13) and middle-aged (n = 16) men. Values are the mean ± SE. {dagger}, P < 0.01 (significant difference between groups).

 
As middle-aged men showed a greater percent body fat than young individuals, values of each mRNA target corrected for body fat percentage were compared between groups. As shown in Fig. 4Go, mRNA levels of PPAR{gamma} did not remain different between groups, whereas HSL and C3 mRNA levels were still significantly higher in middle-aged than in young men even after adjustment for variation in body fatness (P < 0.05). Similar results were found when mRNA levels were adjusted for visceral adipose tissue accumulation (not shown).



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  		Figure 4. Adipose tissue mRNA levels of LPL, HSL, C3, and PPAR{gamma} from the sc abdominal depot of young (n = 13) and middle-aged (n = 16) men after correction for variation in body fatness. Values are the mean ± SE. *, P < 0.05 (significant difference between groups).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this study the expression of key proteins involved in adipose tissue metabolism was compared between young and middle-aged men. Our results showed that sc abdominal adipose tissue mRNA levels of LPL were not different regardless of age, whereas HSL, C3, and PPAR{gamma} mRNAs levels were greater in middle-aged than in young individuals. However, only HSL and C3 mRNAs remained higher in middle-aged men after correction for body fat percent variation.

The passage of youth to middle age is associated with the peak in fat mass (2) and numerous studies have documented that fat deposition is principally concentrated in the abdominal region of middle-aged men (28, 29, 30). We recently reported a body fat gain during a follow-up period of 12 yr in men aged 44 ± 5 yr at baseline even if both a decrease in their relative fat intake and an increase in their participation in physically activity occurred (31). These results support the idea that the middle-age period (40–59 yr) is accompanied by some metabolic events playing an important role in the regulation of fat balance. The results of the present study show impaired maximal isoproterenol- and postreceptor-induced lipolysis in middle-aged compared with young men, although the former had larger sc fat cells. A loss of the ability to translocate HSL to lipid droplet has recently been suggested to explain the diminished lipolytic response to catecholamines with age in rats (32). That middle-aged individuals presented higher HSL mRNA levels suggests that the mRNA template’s ability to be translated is reduced with aging, thus leading to a decreased HSL protein level and a reduced adipose cell lipolytic capacity. Because a novel form of human HSL produced by alternative splicing has recently been discovered (33) and as the primers used in our study did not detect the alternate, it is possible that the decreased adipose tissue lipolytic capacity observed in middle-aged men could result from an increase in the proportion of the short, but inactive, transcript HSL compared with the long active HSL form. Further studies will be needed to explore this possibility.

To the best of our knowledge, this study is the first to show that C3 mRNA levels are specifically up-regulated in sc adipose tissue of middle-aged compared with young men. This finding is based on the fact that C3 expression remained significantly higher in middle-aged than in young individuals, even after correction for body fat variation. Although we are aware of the fact that the mRNA content of C3 does not necessarily reflect the functional activity of the ASP protein, it is tempting to speculate that the up-regulation of C3 expression in middle-aged men may indirectly explain their impaired adipose tissue lipolytic capacity. Indeed, ASP has recently been shown to inhibit basal and norepinephrine-stimulated FFA release from adipocytes by its marked increase in the fractional FFA reesterification and, to a lesser extent, its inhibition of FFA produced during lipolysis (34).

As expected, middle-aged men displayed a greater insulin-resistant state (estimated by the area under the plasma insulin curve after an oral glucose load) than young individuals (not shown), probably because of their greater body fat percentage or visceral adipose tissue accumulation. However, noteworthy is the fact that fasting glucose as well as glucose and insulin areas were not further different between the two groups of men after correction for body fatness or visceral adipose tissue accumulation (not shown). Therefore, as HSL and C3 mRNA levels remained significantly higher in middle-aged than in young men after controlling for variation in subjects’ body fatness or visceral adipose tissue levels, we can assume that the expression of these genes is up-regulated with aging regardless of insulin sensitivity.

Neither AT-LPL mRNA content nor enzyme activity was altered by aging in the present study. Recently, LPL mRNA levels were reported to be inversely related to age in rhesus monkeys (35). One possible explanation for this controversy might be related to the wide age range of the animals studied (7–30 yr). Indeed, adipocytes of senescent animals have been shown to have a decline in fat cell lipogenic activity, which may thus explain the decrease in fat mass observed in these animals (1). In addition, Hauner et al. (3) previously reported a reduced potential of differentiation of stromal-vascular cells obtained from old human donors. However, further studies are needed to verify whether individuals older than the subjects of the present study would display decreased AT-LPL expression. On the other hand, the fact that LPL mRNA levels were not related to LPL activity is concordant with the important posttranscriptional and posttranslational mechanisms involved in the regulation of this enzyme activity as previously reported (36, 37, 38). On the basis of the fact that adipose tissue LPL activity may contribute to regional fat distribution (36), it was thus expected to observe a similar sc adipose tissue LPL activity in young and middle-aged men of the present study because of their comparable adipose tissue accumulation in this region.

Although PPAR{gamma} expression was higher in middle-aged than in young men, this difference disappeared after correction for variation in body fatness. This finding suggests that PPAR{gamma} expression is not up-regulated during middle age, but seems to be mostly related to increased obesity, as previously reported (39, 40). It would have been expected that adipose tissue in middle-aged men presents lower PPAR{gamma} mRNA levels than that in young subjects with regard to the impaired capacity of preadipocytes to differentiate with aging (3). Once again, it is likely that the subjects of the present study were not old enough to exhibit such an effect. It is also possible that the use of whole adipose tissue complicates the interpretation of our results, as PPAR{gamma} may have an entirely different role in preadipocytes (i.e. promotion of differentiation) vs. mature adipocytes (regulation of metabolically important genes), as the proportion of these cells changes with aging (41, 42). However, this does not exclude that PPAR{gamma} expression might be associated with the expansion of fat mass observed during middle-age in humans. Further studies are therefore warranted to clarify the role of PPAR{gamma} in the middle-age-related obesity phenomenon.

In conclusion, the present study demonstrates that sc abdominal adipose tissue LPL expression is similar in middle-aged and young men. On the other hand, HSL and C3 mRNA levels are up-regulated by aging, whereas PPAR{gamma} expression seems to be related to increased adiposity.


   Acknowledgments
 
We express our gratitude to the staff of the Physical Activity Sciences Laboratory and the Lipid Research Center for data collection. We thank Marie Tremblay and Rachelle Duchesne of the Diabetes Research Unit for their assistance. The subjects are also gratefully acknowledged.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada and the Fonds FCAR-Québec. Back

Received August 9, 2000.

Revised October 16, 2000.

Accepted October 25, 2000.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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