Functional analysis of the C(–188)A polymorphism of the human leptin promoter

Mutational analysis of the promoter region of the leptin gene in morbidly obese Finnish subjects had revealed a previously unidentified C(–188)A polymorphism in the proximal promoter that showed a weak association with elevated serum leptin levels in obese male carriers of the variant (–188A) allele. In this study we demonstrated that neither expression of reporter gene constructs driven by wild-type (–188C) or variant (–188A) proximal promoter regions, nor assay of binding of cellular proteins reveal a genotype-related difference in promoter activity. Received: 16 April 1998 / Accepted: 8 July 1998     

Similarities and differences in the transcriptional regulation of the leptin gene promoter in gastric and adipose cells

The stomach was reported to synthesize and secrete leptin mainly in the gastric lumen. Gastric leptin release is markedly increased after food intake, by vagal cholinergic stimulation and by cholecystokinin and secretin. Here we show that human gastric MKN-74 cells produce leptin that increases upon challenge with cholecystokinin, insulin, glucocorticoids and all-trans retinoic acid through activation of the leptin gene promoter. In addition, we demonstrate that forskolin and BRL37344 which increased cAMP levels, fail to affect the activity of leptin gene promoter in MKN74 expressing beta(3)-adrenoceptor cells but, induce a 2-fold decrease in this activity in adipose 3T3-L1 cells. These data described for the first time, similarities and more interestingly, differences in the regulation of the leptin gene promoter in gastric cells as compared to adipocytes.     

The Alternative Epac/cAMP Pathway and the MAPK Pathway Mediate hCG Induction of Leptin in Placental Cells

Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)₂cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway.     

Leptin promoter variant G2548A is associated with serum leptin and HDL-C levels in a case control observational study in association with obesity in a Pakistani cohort

Leptin is a protein hormone synthesized by adipocytes and is involved in the regulation of food intake and energy expenditure. We hypothesized that any change in the promoter sequence can affect the expression of the gene and hence leptin protein levels in the serum. The aim of the current study was to investigate the relationship of such a promoter variant of the leptin gene, G-2548A polymorphism, with obesity and its effect on various anthropometric and metabolic parameters in a Pakistani cohort consisting of 250 obese and 225 non-obese control subjects. Body weight, height, waist circumference (WC), hip circumference (HC) and blood pressure (BP) were measured by standard methods and levels of fasting blood glucose (FBG), total cholesterol, triglycerides, HDLC, LDLC, and leptin were determined. Genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results showed that the LEP G-2548A polymorphism showed significant association with obesity in Pakistan. In addition, the polymorphism showed association with weight, height, BMI, WC, HDLC and serum leptin levels. The findings suggest that the leptin promoter G-2548A variant may play its part in the progression to obesity by not only affecting the body’s fat distribution but also by changing the serum leptin and HDLC levels.     

G-2548A polymorphism of the leptin gene is correlated with extreme obesity in Taiwanese aborigines

We examined the genetic associations of the G-2548A polymorphism in the promoter of the leptin (LEP) gene and the Gln223Arg (Q223R) polymorphism of the leptin receptor (LEPR) gene with obesity. Two hundred twenty-six obese aboriginal subjects (BMI > or = 27 kg/m2) and 182 aboriginal subjects with normal weight (BMI < 25 kg/m2) participated in this study. The polymorphisms of LEP G-2548A and LEPR Q223R were genotyped by polymerase chain reaction/restriction fragment length polymorphism, and their anthropometric characteristics were measured. Levels of leptin, triglycerides, and cholesterol were measured after overnight fasting. We found that the frequencies of the LEP G/G homozygote (22.6%) with Mendelian recessive (chi2 = 7.89, p = 0.005) and codominant (chi2 = 7.93, p = 0.02) models to be higher in the extremely obese subjects (BMI > or = 35 kg/m2) than in normal weight subjects (6.9%) but not in moderately obese subjects (35 > BMI > or = 27 kg/m2). There was no difference in genotypic frequency of the LEPR Q223R polymorphism between the extreme obese and control groups. We suggest that the LEP -2548 G/G homozygote plays a genetic recessive role in the development of extreme obesity in Taiwanese aborigines.     

Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli

High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.