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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:443-449

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:443.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Leptin, Peroxisome Proliferator-Activated Receptor-{gamma}, and CCAAT/Enhancer Binding Protein-{alpha} mRNA Expression in Adipose Tissue of Humans and Their Relation to Cardiovascular Risk Factors

Franz Krempler; David Breban; Hannes Oberkofler; Harald Esterbauer; Emanuel Hell; Bernhard Paulweber; Wolfgang Patsch

From the Departments of Internal Medicine (F.K.) and Surgery (E.H.), Krankenhaus Hallein; and the Departments of Laboratory Medicine (D.B., H.O., H.E., W.P.) and Internal Medicine (B.P.), Landeskrankenanstalten, Salzburg, Austria.

Correspondence to Franz Krempler, MD, Department of Internal Medicine, Krankenhaus Hallein, Bürgermeisterstraße 34, A-5400 Hallein, Austria. E-mail w.patsch{at}lkasbg.gv.at


*    Abstract
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 *Abstract
down arrowIntroduction
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 down arrowResults
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Abstract—Obesity is a prevalent disorder that increases the risk for premature cardiovascular disease. The adipose tissue itself plays an active role in the regulation of fuel metabolism and energy homeostasis by expressing a number of regulatory genes, such as leptin, peroxisome proliferator–activated receptor-{gamma} (PPAR{gamma}), and CCAAT/enhancer binding protein-{alpha} (C/EBP{alpha}). To study the in vivo relationships among these genes and their associations with cardiovascular risk factors, plasma levels of leptin, lipids, apolipoproteins (apo), insulin, and glucose were measured in 216 obese, 165 nonobese, and 36 weight-losing postobese subjects. mRNA expression of leptin, PPAR{gamma}, and C/EBP{alpha} in the extraperitoneal and intraperitoneal adipose tissue was quantified in subsets of subjects. In obese individuals, plasma leptin was associated with apoA-I (r=0.2346, P<0.001) and insulin (r=0.2125, P<0.002). Leptin and C/EBP{alpha} mRNA expression in extraperitoneal and intraperitoneal adipose tissue of obese patients was higher than in the respective tissues of nonobese or postobese subjects. No significant differences among the study groups were found for PPAR{gamma} mRNA expression. Leptin, PPAR{gamma}, and C/EBP{alpha} mRNA levels correlated with each other in the intraperitoneal and extraperitoneal fat of obese subjects, but multivariate analysis revealed that only C/EBP{alpha} was a predictor of leptin expression in extraperitoneal tissue (partial r=0.6096, P<0.001). Intraperitoneal PPAR{gamma} expression was inversely related to fasting insulin (r=-0.2888, P<0.017) and a fasting insulin resistance index (r=-0.2814, P<0.021) in obese subjects. In postobese patients, intraperitoneal PPAR{gamma} expression was associated with plasma HDL cholesterol (r=0.5695, P<0.018) and apoA-I (r=0.6216, P<0.008) but was inversely related to LDL cholesterol (r=-0.5101, P<0.03) and apoB (r=-0.6331, P<0.007). These findings suggest a relationship between plasma leptin and HDL metabolism as well as adipose-tissue site–dependent associations among leptin, C/EBP-{alpha}, and PPAR-{gamma} mRNA expression. Furthermore, our results suggest that C/EBP-{alpha} enhances leptin expression in vivo and that PPAR{gamma} mRNA expression is inversely associated with cardiovascular risk factors.


Key Words: leptin • peroxisome proliferator–activated receptor-{gamma} • CCAAT • enhancer binding protein-{alpha} • obesity • cardiovascular risk


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Obesity is the most prevalent nutritional disorder in western societies and is a major risk factor for premature cardiovascular disease.1 2 The disorder results from an imbalance in food intake and energy expenditure. Adipose tissue itself is actively involved in the regulation of energy homeostasis.3 Among the proteins secreted by adipose tissue, leptin plays a pivotal role in energy metabolism by signaling adipose tissue mass to hypothalamic centers that control food intake and influence energy usage.4 5

The metabolic signals that alter adipocyte function, including leptin synthesis and secretion, are incompletely understood. Peroxisome proliferator–activated receptor-{gamma} (PPAR{gamma}) and CCAAT/enhancer binding protein-{alpha} (C/EBP{alpha}) are principal regulators of adipocyte differentiation and function.6 7 C/EBP{alpha} mediates transactivation of leptin transcription in vitro.8 9 10 Other in vitro studies suggested a functional antagonism of C/EBP{alpha} and PPAR{gamma} in leptin expression,11 but little information is available about the in vivo relationship among leptin, C/EBP{alpha}, and PPAR{gamma} mRNA expression in human subjects.

Disturbances in lipid transport and carbohydrate metabolism contribute substantially to the increased cardiovascular risk in overweight subjects.12 13 C/EBP{alpha} and PPAR{gamma} are integrated in the control of lipid and carbohydrate metabolism.7 14 15 16 17 Obesity-associated risk of cardiovascular disease is modulated by the type of fat distribution in that intra-abdominal obesity is associated with a higher risk than subcutaneous obesity,13 18 and biochemical evidence suggests differences in metabolic properties of intraperitoneal and subcutaneous adipose tissue.19 20 21

In the present study, we quantified mRNA levels of leptin, C/EBP{alpha}, and PPAR{gamma} in intraperitoneal and extraperitoneal adipose tissue of lean, obese, and postobese subjects to gain insight into the in vivo control of leptin gene expression and to determine associations of mRNA expression levels with established cardiovascular risk factors.


*    Methods
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 *Methods
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Study Population
This study included unrelated white subjects; 216 subjects were obese patients referred consecutively for weight reduction surgery who underwent a gastric banding procedure. Thirty-six different individuals referred to as postobese subjects underwent an elective procedure, such as adjustment or removal of the gastric tape or cholecystectomy. This group had lost 32.8±17.7 kg after a gastric banding operation. One hundred sixty-five subjects were lean individuals without a major illness. This control group included 20 lean subjects who underwent an elective surgical procedure, such as cholecystectomy or repair of hernias. Among diabetic subjects, only 3 obese patients received hypoglycemic drugs, and none received insulin or a thiazolidinedione. No subject of the entire study group was on lipid-lowering medication. Study subjects provided informed consent, and the study was approved by the institutional review board. After an overnight fast, general anesthesia was induced, and fat biopsies were taken from the abdominal subcutaneous fat, referred to as extraperitoneal adipose tissue, and/or the omental fat, referred to as intraperitoneal adipose tissue, at the beginning of the surgical procedure. Adipose tissue specimens were submerged in ice-cold saline and immediately transported to the laboratory, where they were divided into aliquots and frozen at -70°C, as described previously.22 23 Body mass index (BMI; kilograms per meter squared) was calculated from measurements of weight and height.

Biochemical Measurements
After an overnight fast, blood was collected into tubes containing EDTA. Plasma glucose was measured by a hexokinase/glucose-6-phosphate dehydrogenase method. Plasma insulin was measured by immunoassay (MEIA, Abbott Laboratories). Fasting insulin resistance index (FIRI) was calculated from fasting glucose and insulin concentrations (mmolxµU/25) as described by Duncan et al.24 Cholesterol and triglyceride were measured by enzymatic procedures (catalogue Nos. 1489437 and 1058550, Boehringer Mannheim Diagnostics). HDL cholesterol was determined in supernatants after precipitation of plasma with phosphotungstic acid/magnesium chloride, and LDL cholesterol was calculated by the formula of Friedewald et al.25 Apolipoprotein (apo) B and A-I levels were determined by nephelometric procedures (Array 360, Beckman). Plasma leptin was measured by radioimmunoassay (Linco Inc).

Isolation of RNA From Adipose Tissues and Northern Blot Analysis
Total RNA was isolated from 2 g of adipose tissue according to the method of Chomczynski and Sacchi.26 The integrity of RNA was ascertained by electrophoresis in formaldehyde gels. RNA concentrations were determined by absorbance measurements at 260 nm. Total adipose tissue RNA (30 µg) was denatured with 1 mol/L glyoxal, 50% DMSO and separated by electrophoresis in 1.2% agarose. The RNA was transferred to S&S Nytran membranes (Schleicher & Schüll) by capillary blotting and hybridized to a nearly full-length 32P-labeled leptin cDNA probe labeled by the random priming method using [32P]dCTP (3000 Ci/mmol, Amersham). The relative abundance of leptin mRNA was determined from the intensities of bands that were quantified on the autoradiographs by densitometry with Molecular Analyst software (Bio-Rad). After the leptin probe had been stripped according to the manufacturer’s recommendations, membranes were subsequently hybridized with human 32P-labeled PPAR{gamma} and C/EBP{alpha} cDNA probes. The PPAR{gamma} cDNA probe was obtained by reverse transcription–polymerase chain reaction amplification of adipose tissue RNA using 5'-CCAGCATTTCTACTCCACATT-3' (+300 to +320) and 5'-GCGGTTGTCGAAGAGGAAGAG-3' (+757 to +737) as upstream and downstream primers, respectively (GenBank accession No. L40904). The nearly full-length C/EBP{alpha} cDNA probe was kindly provided by Per Antonson, Stockholm, Sweden. Relative abundance levels of leptin, PPAR{gamma}, and C/EBP{alpha} mRNA were corrected by use of the relative abundance of GAPDH (GenBank Accession No. M33197) and ß-actin mRNA (GenBank Accession No. M10278) abundance levels as described previously.22 23 mRNA signals were normalized by use of overlapping RNA samples in Northern blots.

Statistical Analyses
Two-way ANOVA27 was used to test the equality of such continuous variables as age and biochemical measurements among obese, nonobese, and postobese subjects. A transformation was made on the original variable if the equal variance and normality assumptions of the 2-way ANOVA were rejected. For some analyses, mRNA abundance levels were adjusted, by multiple regression, for sex and/or age. To compare categorical variables, a contingency {chi}2 test was used. Stepwise multiple linear regression models were used to identify independent predictor variables for plasma leptin, leptin mRNA expression, and PPAR{gamma} mRNA expression. Independent variables with a value of P<0.05 (2-sided) were considered statistically significant.


*    Results
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 *Results
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Approximately 4 times more female than male patients were admitted for bariatric surgery. Therefore, more female than male patients were recruited for the control group. According to the selection criteria for surgery, average BMI and leptin levels were considerably higher in obese than in nonobese subjects (Table 1Down). Although the majority of postobese subjects were still losing weight according to their own weight measurements, accurate dietary and weight data were not available in many postobese subjects. We therefore estimated the actual calorie intake status of postobese subjects by comparing their plasma leptin values with those predicted from multivariate analysis using BMI and sex as predictor variables in the population of obese and nonobese subjects. According to these calculations, the average leptin level in postobese subjects was 62.24% (CI 50.51% to 73.98%) of the level predicted. Hence, postobese subjects were most likely on a hypocaloric diet at the time of the study.


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  		Table 1. Characteristics of Study Subjects

Compared with nonobese control subjects, average values for plasma insulin, leptin, triglyceride, and apoB were significantly higher in obese subjects, whereas average values for HDL cholesterol and apoA-I were significantly lower. Blood pressure readings, as well as the prevalence of non–insulin-dependent diabetes, were also higher in obese subjects than in control subjects. Systolic blood pressure readings, plasma insulin, leptin, and apoB levels were significantly lower in postobese than in obese patients. Exclusion of diabetic subjects reduced plasma glucose in the obese study group to 85±18 mg/dL (P<0.024) but had no considerable effect on the average values of the other variables shown in Table 1Up.

Relation of Plasma Leptin With Sex, BMI, and Cardiovascular Risk Factors
In all 3 study groups, plasma leptin was significantly higher in women than in men (obese: 40.9 versus 27.6 ng/mL, P<0.001; nonobese: 10.5 versus 3.8 ng/mL, P<0.001; postobese: 14.2 versus 4.4 ng/mL, P<0.04). Sex-adjusted plasma leptin correlated with BMI in obese (r=0.3297), nonobese (r=0.5809), and postobese subjects (r=0.6548) (all P<0.001). In obese patients, sex-adjusted leptin correlated with apoA-I (r=0.2346, P<0.001) and insulin (r=0.2125, P<0.002). No such associations were found in nonobese or postobese subjects. Exclusion of diabetic patients, whether they were receiving hypoglycemic drugs or not, did not alter the associations found in obese subjects.

Multivariate analysis in obese subjects demonstrated that BMI, sex, apoA-I, and insulin were independent predictors of plasma leptin (Table 2Down, obese subjects). This model explained 26% of the variance in plasma leptin. In multivariate analyses of nonobese or postobese subjects, only sex and BMI were retained as independent variables, and these models explained 50% or 48% of the variance in plasma leptin (Table 2Down, nonobese and postobese subjects). Again, exclusion of diabetic subjects did not alter the relationships in the obese and control groups.


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  		Table 2. Partial Correlations of Plasma Leptin With Sex, BMI, ApoA-I, and Fasting Insulin in Obese, Nonobese, and Postobese Study Subjects

Leptin, PPAR{gamma}, and C/EBP{alpha} mRNA Expression
Relative mRNA abundance levels of leptin, PPAR{gamma}, and C/EBP{alpha} in intraperitoneal and extraperitoneal adipose tissues of obese, nonobese, and postobese subjects are shown in Table 3Down. No sex-associated differences were found in the mRNA expression levels of these genes (not shown). In obese subjects, leptin mRNA expression in extraperitoneal adipose tissue correlated with plasma leptin (r=0.3411, P<0.002). Adjustment for sex did not substantially alter the association in obese patients (P<0.003). Moreover, in obese and nonobese subjects, Spearman rank order correlations showed associations of intraperitoneal leptin mRNA abundance with BMI (R=0.1988, P<0.037 and R=0.6294, P<0.009).


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  		Table 3. Relative mRNA Abundance Levels in the Adipose Tissue

In obese patients, leptin and C/EBP{alpha} but not PPAR{gamma} mRNA abundance levels were significantly higher in the extraperitoneal than in the intraperitoneal adipose tissue, whereas no such differences were observed in nonobese and postobese subjects (Table 3Up). Compared with nonobese and postobese subjects, obese patients exhibited higher extraperitoneal and intraperitoneal expression levels of leptin and C/EBP{alpha} mRNA. No significant differences among the 3 study groups were found in PPAR{gamma} mRNA levels. Results were not substantially altered when diabetic patients and/or subjects receiving any type of medication were excluded from the analyses.

The relationships among leptin, PPAR{gamma}, and C/EBP{alpha} mRNA expression in adipose tissue deposits of obese patients are shown in Table 4Down. Significant associations of intraperitoneal with extraperitoneal tissue expression were observed for C/EBP{alpha} and PPAR{gamma} but not leptin mRNA in univariate analyses. Multivariate analysis, adjusted for age, sex, BMI, and insulin (all forced into the model) demonstrated that C/EBP{alpha} but not PPAR{gamma} mRNA expression was a predictor of leptin mRNA abundance in the extraperitoneal adipose tissue (partial r=0.6096, P<0.001). This model accounted for 40% of the variance of leptin mRNA expression. In intraperitoneal adipose tissue, neither C/EBP{alpha} nor PPAR{gamma} mRNA abundance was maintained as an independent predictor of leptin mRNA expression on multivariate analysis. These associations remained similar after exclusion of diabetic subjects.


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  		Table 4. Spearman Correlation Coefficients Among Leptin, PPAR{gamma}, and C/EBP{alpha} mRNA Abundance in Intraperitoneal and Extraperitoneal Adipose Tissue of Obese Patients

In nonobese and postobese patients, only C/EBP{alpha} mRNA abundance exhibited significant correlations between the 2 tissue sites (P<0.01 for both study groups). Furthermore, no associations among the expression levels of the 3 mRNA species were noted in any tissue.

PPAR{gamma} mRNA Expression and Cardiovascular Risk Factors
In obese nondiabetic patients, intraperitoneal PPAR{gamma} mRNA was inversely related with fasting insulin and FIRI in univariate analyses (r=-0.2888, P<0.017 and r=-0.2814, P<0.021, respectively) and in multivariate analyses after adjustment for age and sex (r=-0.2926, P<0.017 and r=-0.2998, P<0.015, respectively).

Striking relationships were observed in postobese subjects. Univariate analyses showed correlations of intraperitoneal PPAR{gamma} mRNA with HDL cholesterol (r=0.5695, P<0.018) and apoA-I (r=0.6216, P<0.008) and inverse relationships with LDL cholesterol (r=-0.5101, P<0.031) and apoB (r=-0.6331, P<0.007). In multivariate models with intraperitoneal PPAR{gamma} mRNA as the dependent variable, both HDL cholesterol and LDL cholesterol were retained as independent variables after adjustment for age and sex (Table 5Down, HDL and LDL Cholesterol). HDL cholesterol and LDL cholesterol could be replaced by apoA-I and apoB without diminishing the predictive value of the model (Table 5Down, ApoA-I and ApoB). The proportion of the PPAR{gamma} mRNA variance explained by lipoprotein lipids or apolipoproteins was 57% and 61%, respectively, providing internal consistency for the relationship of lipid transport with intraperitoneal PPAR{gamma} mRNA expression in postobese subjects.


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  		Table 5. Partial Correlations of Intraperitoneal Adipose Tissue PPAR{gamma} mRNA Abundance With HDL Cholesterol and LDL Cholesterol or ApoA-I and ApoB in Postobese Subjects


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The main findings of our study are that (1) plasma leptin correlated with apoA-I and insulin in obese subjects; (2) associations among leptin, C/EBP{alpha}, and PPAR{gamma} mRNA expression were adipose-tissue site–dependent; (3) C/EBP{alpha} but not PPAR{gamma} mRNA abundance was an independent predictor of leptin mRNA expression in extraperitoneal adipose tissue of obese subjects; and (4) PPAR{gamma} mRNA expression was inversely associated with CAD risk factors in obese and postobese patients.

As has been reported in numerous previous studies,28 29 plasma leptin levels correlated with BMI and were higher in women than in men in all 3 study groups. Sex and BMI explained {approx}50% of the variance of plasma leptin in nonobese and postobese subjects. In obese patients, the predictive value of sex and BMI for plasma leptin was considerably lower, suggesting that other factors gain importance in the regulation of leptin or that the control of plasma leptin levels is disturbed in obese subjects. Both univariate and multivariate analyses revealed associations of leptin with apoA-I and insulin in obese patients. A relationship between leptin and apoA-I was reported recently in overweight children.30 Although leptin synthesis, which depends on fat cell size and total number of adipocytes, is the main determinant of circulating leptin levels,31 different rates of adipocyte leptin secretion may also influence plasma leptin levels.32 We previously reported that a variable fraction of leptin is associated with HDL after ultracentrifugation.30 One may therefore speculate that apoA-I, analogous to its function in reverse cholesterol transport, is involved in the transport or release of leptin from adipocytes. Conversely, leptin itself may modulate apoA-I synthesis and/or metabolism, since it has been shown to stimulate lipolysis and oxidation of fatty acids.33

In obese patients, plasma leptin was associated with insulin, a relationship well known from previous studies.23 34 35 Although such an association may be explained, at least in part, by the relation of insulin resistance with BMI,36 growing evidence suggests that insulin stimulates leptin synthesis not only in rodents but also in humans. We have recently shown that obese subjects with impaired insulin signaling due to a variation in the IRS-1 gene displayed lower leptin mRNA and plasma levels than obese wild-type subjects.23 Consistent with a stimulatory role of insulin on leptin gene expression are increases of leptin mRNA and plasma levels in patients with insulin-secreting tumors.37 The result of the present study showing that insulin is a predictor of plasma leptin independent of BMI and sex lends further support to a role of insulin in the long-term regulation of leptin gene expression.

To fulfill its central role in energy homeostasis, adipose tissue deposits must undergo continuous remodeling, which includes differentiation of precursor cells into adipocytes.38 PPAR{gamma} and C/EBP{alpha} regulate adipocyte differentiation6 7 but must also play a role in mature adipose tissue function. In our study, C/EBP{alpha} expression was associated with obesity and was highest in the extraperitoneal adipose tissue of obese subjects, which is known to harbor larger and more fat-laden adipocytes.22 39 Hence, increased C/EBP{alpha} synthesis may be required for maintaining adipocyte function when fat cell size increases and obesity advances. In contrast, PPAR{gamma} expression was not related to BMI. This finding must be qualified, however, because mRNA expression levels do not necessarily predict protein concentrations or functional activities. Moreover, several isoforms of PPAR{gamma} have been identified.40 41 PPAR{gamma}1 mRNA was much more abundant in human adipose tissue than PPAR{gamma}2 mRNA, which accounted for {approx}13% of total PPAR{gamma} mRNA.42 Conflicting results regarding the association of the 2 isoforms with obesity were reported. PPAR{gamma}2, but not PPAR{gamma}1, was increased in subcutaneous adipose tissue of obese humans in 1 study,43 whereas the expression ratios of the 2 mRNA species were similar in various adipose tissue depots and independent of the degree of obesity in another study.42 PPAR{gamma}3, a third recently identified isoform, is also expressed in adipose tissue,41 but data on a possible relation of PPAR{gamma}3 with obesity are not yet available. Because we determined the abundance of total PPAR{gamma} mRNA, possible differences among adipose tissue sites and in relation to obesity status of mRNA species encoding the PPAR-{gamma} isoforms could not be assessed.

PPAR{gamma} and C/EBP{alpha} mRNA levels were significantly correlated in the extraperitoneal and intraperitoneal adipose tissue of obese subjects. This result is consistent with a synergistic function of the 2 genes in maintaining adipocyte function,44 but the greater variance of C/EBP{alpha} mRNA abundance implicates differences in the regulation of C/EBP{alpha} and PPAR{gamma} mRNA expression.

Signaling pathways operating through PPAR{gamma} and C/EBP{alpha} are central to adipocyte function, because a number of adipocyte-specific genes possess binding sites for PPAR{gamma} and C/EBP{alpha}, and overexpression of these factors can induce adipocyte differentiation.6 7 C/EBP{alpha} expression precedes the expression of leptin during adipocyte differentiation.9 Studies of the leptin promoter identified a C/EBP{alpha} motif that mediated transactivation of leptin transcription.8 9 In contrast, thiazolidinedione treatment or PPAR{gamma}/retinoid X receptor-{alpha} cotransfection did not alter transcription from the leptin promoter in transient transfection studies.10 However, a putative binding site for PPAR{gamma} was localized in the leptin promoter, and transfection studies in primary rat adipocytes and CV-1 cells suggested a functional antagonism of C/EBP{alpha} and PPAR{gamma} in leptin expression. Interestingly, this antagonism was observed with constructs containing the C/EBP{alpha} but devoid of the PPAR{gamma} binding site.11 Moreover, thiazolidinediones induced expression of both C/EBP{alpha} mRNA and protein in fully differentiated 3T3-L1 adipocytes,45 and C/EBP{alpha} expression vectors were shown to activate transcription from the PPAR{gamma} promoter in UMR 106 cells.46 Thus, the regulation of leptin expression by C/EBP{alpha} and PPAR{gamma} appears to be complex in vitro. In our in vivo study, leptin mRNA abundance levels in the intraperitoneal and extraperitoneal adipose tissue correlated with the expression of PPAR{gamma} and C/EBP{alpha} in obese patients and tended to correlate in nonobese and postobese subjects. In a multivariate model, C/EBP{alpha}, but not PPAR{gamma}, was an independent predictor of leptin mRNA abundance in the extraperitoneal adipose tissue. Moreover, the decrease in C/EBP{alpha} expression in going from the obese to the postobese state was paralleled by a decrease in leptin mRNA abundance (Table 3Up). These results support the proposed role of C/EBP{alpha} as a transcriptional activator of the leptin gene. Reduced insulin sensitivity of the omental compared with the subcutaneous adipose tissue has been implicated in the differences of leptin mRNA expression between the 2 tissue sites.47 Our results strongly suggest that C/EBP-{alpha}–mediated upregulation represents an additional mechanism that contributes to the higher leptin expression in the extraperitoneal adipose tissue. The repressor effect of PPAR{gamma} on the leptin gene that has been demonstrated in cell culture experiments11 was not apparent at the mRNA level measured in the present study. Because PPAR{gamma} total mRNA expression does not reflect the integrated activities of PPAR{gamma} protein isoforms, an inhibitory effect of PPAR{gamma} on leptin expression may have been concealed in our in vivo study. Supportive evidence for a role of PPAR{gamma} in leptin expression in vivo comes from a recent report demonstrating that carriers of a PPAR{gamma}2 polymorphism had higher plasma leptin levels than wild-type subjects.48 Studies in rats suggested that leptin itself may regulate PPAR{gamma}, because leptin injection increased the expression of PPAR{gamma} protein in adipocytes.49 Our data showed no correlations of plasma leptin with PPAR{gamma} or C/EBP{alpha} mRNA expression.

C/EBP{alpha} mRNA abundance levels in the extraperitoneal and intraperitoneal adipose tissue deposits correlated in all 3 study groups, indicating that common signaling mechanisms regulate the expression of this gene in both tissue sites. PPAR{gamma} mRNA expression correlated only in the obese group. Leptin mRNA expression levels in intraperitoneal and extraperitoneal tissue sites exhibited no correlation in any of the study groups. Hence, tissue site–specific factors contributed significantly to leptin gene expression.

In obese subjects, intraperitoneal PPAR{gamma} expression was inversely related to fasting insulin and FIRI, both indicators of insulin resistance. PPAR{gamma} is a receptor for thiazolidinediones, a new class of antidiabetic compounds.14 15 Activation of PPAR{gamma} is believed to be central to the insulin-sensitizing effect of these drugs.3 Our findings are thus consistent with a role of PPAR{gamma} in glucose homeostasis and suggest that PPAR{gamma} may be a key factor in maintaining insulin sensitivity as obesity develops.

In postobese subjects, intraperitoneal PPAR{gamma} expression correlated with HDL cholesterol and apoA-I but was inversely related to LDL cholesterol and apoB. No such relationships were observed in obese or nonobese subjects. Postobese subjects had lost a considerable amount of weight and were still losing weight, judged by the relation of actual to predicted plasma leptin values. After prolonged hypocaloric nutrition, energy stores are reduced and several metabolic processes, including VLDL synthesis by the liver as well as cellular lipid metabolism, are altered. The associations of PPAR{gamma} expression with HDL and LDL components may relate to the sterol content of adipocytes, because LDL and HDL would be expected to have opposing effects on cellular cholesterol homeostasis. Sterol depletion of cells induces a proteolytic release of sterol regulatory element binding proteins (SREBPs) from the endoplasmic reticulum. After translocation into the nucleus, SREBPs activate a number of genes, thereby augmenting cellular cholesterol synthesis and uptake.50 A SREBP motif has been identified in the PPAR{gamma}3 promoter,44 and transactivation by SREBPs might contribute to the relationship of PPAR{gamma} mRNA with HDL and LDL in postobese subjects. Such a scenario would be consistent with the interrelationship of lipid and glucose metabolism originally proposed by Randle et al.51 Because PPAR{gamma} is involved in the remodeling of adipose tissue,52 it is also possible that changes in adipose tissue metabolism are the primary event that secondarily affects plasma levels of HDL and LDL. Irrespective of the mechanism, our findings further underline the central role of the intraperitoneal adipose tissue for metabolic processes that affect cardiovascular risk.


*    Acknowledgments
 
This study was supported by a grant from the Medizinische Forschungsgesellschaft Salzburg (to W.P.) and by the Jubilaeumsfondsprojekt Nr. 7228 of the Oesterreichische Nationalbank (to W.P.). The technical assistance of C. Winkler and the assistance of Dr C. Breban in the management of study patients are gratefully acknowledged.

Received January 19, 1999; accepted September 1, 1999.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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