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.

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 (PPARgamma), and CCAAT/enhancer binding protein-alpha (C/EBPalpha). 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, PPARgamma, and C/EBPalpha 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/EBPalpha 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 PPARgamma mRNA expression. Leptin, PPARgamma, and C/EBPalpha mRNA levels correlated with each other in the intraperitoneal and extraperitoneal fat of obese subjects, but multivariate analysis revealed that only C/EBPalpha was a predictor of leptin expression in extraperitoneal tissue (partial r=0.6096, P<0.001). Intraperitoneal PPARgamma 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 PPARgamma 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 PPARgamma mRNA expression is inversely associated with cardiovascular risk factors.

Uncoupling protein-3 gene expression: reduced skeletal muscle mRNA in obese humans during pronounced weight loss.

AIMS: Uncoupling protein-3 is a member of a protein family that serves to dissipate energy in the form of heat thereby modulating energy expenditure. Alternative processing of uncoupling protein-3 transcripts results in two mRNA species that encode a large and small protein, perhaps differing in functional activity. Since obesity is associated with disrupted energy homeostasis, we measured muscle mRNA expression in morbidly obese and lean subjects. METHODS: The two uncoupling protein-3 mRNA species were quantified in muscle tissue using an RNase protection assay. Gene locus effects on mRNA expression were studied by quantitative allele-specific primer extension. RESULTS: In both obese and lean subjects, the mRNA species encoding the small protein isoform was twice as abundant as the mRNA species encoding the large protein isoform. Neither the total uncoupling protein-3 mRNA expression nor the molar abundance ratios of the two mRNA species differed between obese and lean male or female subjects. Women who had lost 37+/-22 kg of weight in response to dietary restriction and continued a hypocaloric diet displayed lower mRNA than obese (p<0.005) or lean women (p<0.05). Primer extension assays in lean and obese subjects showed similar allelic mRNA abundance in all but one subject studied. CONCLUSION: Muscle expression of the two uncoupling protein-3 mRNA species is similar in obese and lean people. In obese patients, prolonged hypocaloric diet downregulates uncoupling protein-3 mRNA expression in muscle and can thereby enhance its energy efficiency. Sequence substitutions at the gene locus may only be minor determinants of mRNA expression in muscle tissue.

Plasma leptin levels: interaction of obesity with a common variant of insulin receptor substrate-1.

Obesity is associated with insulin resistance and other major cardiovascular risk factors. A common amino acid polymorphism at codon 972 of the insulin receptor substrate-1 (IRS-1) has been shown to interact with obesity in the expression of insulin resistance. The plasma concentration of the adipocyte-specific hormone leptin is increased in obesity and is correlated with adipose tissue mass. Because in vitro studies demonstrated inhibitory effects of leptin on insulin signaling, leptin may be involved in obesity-associated insulin resistance. To gain insight into the relationship between insulin and leptin in obesity, we studied plasma leptin levels and several cardiovascular risk factors, as well as their modification by the IRS-1 codon 972 genotype, in 156 obese individuals and 131 lean control subjects. In both groups, 10% of the subjects were heterozygous for the IRS-1 codon 972 variant. Obese individuals harboring the IRS-1 variant displayed significantly lower plasma concentrations of leptin than obese subjects without the polymorphism (means, 26.7 versus 37.8 ng/mL, P<0.0293). In a subgroup of obese patients, leptin mRNA abundance was measured in the adipose tissue and was significantly lower in carriers of the IRS-1 variant than in subjects with the wild-type variant (P<0.0291). Our data suggest that insulin signaling influences plasma leptin concentrations at the mRNA expression level and argue against leptin as a major causative factor of insulin resistance.

Uncoupling protein-1 mRNA expression in obese human subjects: the role of sequence variations at the uncoupling protein-1 gene locus.

Uncoupling protein-1 (UCP-1) activity in brown adipose tissue increases thermogenesis, contributes to facultative energy expenditure in humans, and has been implicated in the pathogenesis of rodent obesity. To determine genetic factors controlling UCP-1 expression in humans, we measured intra- and extraperitoneal UCP-1 mRNA abundance levels by a competitive RT-PCR method and compared expression levels with common sequence variations in the beta3-adrenergic receptor gene and the distal UCP-1 gene promoter in obese human subjects. While median and average UCP-1 mRNA levels in both the intra- and extraperitoneal tissue were lower in subjects heterozygous for the Trp64Arg mutation in the beta3-adrenergic receptor gene, this difference was not statistically significant. However, a strong association of intraperitoneal UCP-1 mRNA abundance with the UCP-1 gene polymorphism at -3826 relative to the transcription start site was observed that explained 19.3% of the interindividual variability. The minor allele imparted a dose-dependent reduction on UCP gene expression. The importance of sequence variations at the UCP-1 gene locus as a common source of UCP-1 mRNA abundance variability was supported by allele-specific expression studies utilizing a newly identified polymorphism in exon 2 of the UCP-1 gene that predicts a substitution of alanine by threonine. In four subjects heterozygous for the -3826 polymorphism, the mRNA species transcribed from the wild-type allele accounted for 63+/-6% percent of total intraperitoneal mRNA abundance. In one subject homozygous for the minor promoter allele, wild-type mRNA was also more abundant than variant mRNA. Thus, the UCP-1 polymorphism at -3826 is probably only a marker for a frequent mutation causing reduced mRNA expression.

Human obese gene expression: alternative splicing of mRNA and relation to adipose tissue localization.

BACKGROUND: The adipocyte-specific protein leptin signals the size of the adipose tissue mass to hypothalamic regions, thereby influencing food intake and energy metabolism. Human obesity is often associated with high leptin levels implying leptin resistance or defective leptin function. Two leptin mRNA species differing only by the presence or absence of a CAG codon encoding glutamine at position 49 of the mature protein arise from alternative splicing owing to two splice acceptor sites immediately following each other at the intron 2 - exon 3 junction. Since glutamine 49 is part of a highly conserved region, we studied possible functional implications of alternative splicing for human obesity. METHODS: We determined, in lean and obese individuals, the relative abundance of both mRNA species in intra- and extraperitoneal adipose tissue in relation to ob gene transcript abundance and plasma leptin levels. RESULTS: Leptin mRNA levels in adipose tissue and concentrations of leptin in plasma were significantly higher in obese subjects than in controls. In both obese and control subjects, leptin mRNA levels were higher in extraperitoneal than in intraperitoneal adipose tissue. Furthermore, leptin mRNA abundance correlated with average fat cell size. In all tissue samples, the predominant ob gene transcript contained the codon for glutamine 49 and the molar ratio of the two leptin mRNA species was similar in patients and controls. No correlation was observed between splice site usage and leptin mRNA abundance or leptin concentration in plasma in our study group. CONCLUSIONS: Differences in the primary structure of leptin due to the presence or absence of glutamine 49 are unlikely to contribute to the apparent 'leptin resistance' commonly observed in obese individuals.