Overexpression of leptin and leptin receptors in breast cancer positively correlates with clinicopathological features

Obesity has been reported to increase postmenopausal breast cancer risk by 30% to 50%, and obese breast cancer patients have been shown to present more aggressive breast cancer pathological features As most breast cancers are sex hormone-dependent, adipocytokines derived from adipose tissue, such as leptin, may account for the positive association between obesity and breast cancer. Leptin, a 16-kDa protein product of the obese gene, was initially regarded as a neuroendocrine factor in the hypothalamus to maintain homeostasis of body weight by regulating food intake and energy expenditure.

Serum leptin and soluble leptin receptor in non-alcoholic fatty liver disease

AIM: To determine the role of leptin system in non-al- coholic fatty liver disease (NAFLD) development by deli- neating the changes in serum levels of leptin and soluble leptin receptor (sOB-R). METHODS: Blood samples were collected from 30 consecutive patients with liver-biopsy-proven NAFLD and 30 patients with cholecystolithiasis (stationary phase) as controls. Serum leptin levels were determined by radio- immunoassay and concentration of sOB-R was measured by ELISA. Body mass index (BMI) was calculated for all subjects, and serum insulin, C-peptide, and lipoprotein levels were also detected. RESULTS: Mean serum leptin level and BMI in the NAFLD group were significantly higher than in the con- trols (both P < 0.001), but mean sOB-R level was lower in the NAFLD group when compared to the controls. Both men and women in the NAFLD group had higher mean serum leptin levels and lower sOB-R levels than did the men and women in the control group (all P < 0.001). The- re was a significant negative correlation between serum leptin and sOB-R levels (r = -0.725, P < 0.001). Multiva- riate analysis showed that the percentage of hepatocyte steatosis, sex, BMI, and homeostasis model assessment of insulin resistance (HOMA IR) were independently rela- ted to serum leptin levels. CONCLUSION: Elevated serum leptin seems to be afeature of steatosis, and serum leptin seems to increase as hepatocyte steatosis develops. An enhanced release of leptin is accompanied by an decrease in sOB-R con- centration, which suggests higher resistance of periphe- ral tissues towards the action of leptin.

Possible involvement of leptin and leptin receptor in developing gastric adenocarcinoma

AIM： To investigate the expression of leptin and leptin receptor （ob-R） in intestinal-type gastric cancer and precancerous lesions, and to explore the possible mechanism and role of the leptin system in developing intestinal-type gastric adenocarcinoma.METHODS： Immunohistochemistry was performed to examine the expression of leptin and leptin receptor in archival samples of gastric adenocarcinoma and preneoplastic lesions, including intestinal metaplasia and mild to severe gastric epithelial dysplasia. Positive staining was identified and percentage of positive staining was graded.RESULTS： Dual expression of leptin and leptin receptor were detected in 80% （16/20） intestinal metaplasia, 86.3% （25/30） mild gastric epithelial dysplasia, 86.7% （26/30） moderate gastric epithelial dysplasia, 93.3% （28/30） severe gastric epithelial dysplasia, 91.3% （55/60） intestinal-type gastric adenocarcinoma and 30.0% （9/30） diffuse-type gastric carcinoma. The percentage of dual expression of leptin and leptin receptor in intestinal-type gastric adenocarcinoma was significantly higher than that in diffuse-type gastric adenocarcinoma （χ^2 = 37.022, P〈0.01）.CONCLUSION： Our results indicate the presence of an autocrine loop of leptin system in the development of intestinal-type gastric adenocarcinoma.

Expression of Leptin Long-form Receptor mRNA in Luteinized Granulosa Cells of Obese Women with Polycystic Ovary Syndrome

To investigate the expression of mRNA of leptin long-form receptor （OB-Rb） in luteinized granulosa ceils of obese women with polycystic ovary syndrome （PCOS）, and to determine the role of leptin in the physiopathology of PCOS, luteinized granulosa cells were collected from the follicle fluid of 10 obese women who met the diagnostic criteria for PCOS and their BMI was equal to or greater than 25 kg/m^2, and at the same time, granulosa cells were collected from 10 normal women undergoing IVF-ET who served as the control group. Some luteinized granulosa cells were taken from normal women for in-vitro culture, into which human leptin of different concentrations was added （0, 10, 100 and 1000 ng/mL）. After stimulation with leptin for 48 h, RT-PCR was employed for the detection of the expression of OB-RLInRNA in the luteinized granulosa cells. Our results showed that the level of OB-RLmRNA in luteinized granulosa cells of obese PCOS women was higher than those in the control （P〈0.05）. In luteinized granulosa cells cultured in vitro and stimulated by human leptin for 48 h, the level of OB-RLmRNA was higher than those without leptin stimulation （P〈0.01）, and when leptin concentration was at 100 ng/mL, and the level of OB-RLmRNA reached a peak, It is concluded that in obese PCOS women, the level of serum leptin is increased, which promotes the expression of OB-RL in luteinized granulosa cells and increases the sensitivity of the granulosa cells to leptin. Leptin may contribute to anovulation in obese women with PCOS.

Neuropeptide Y and leptin receptor expression in the hypothalamus of rats with chronic immobilization stress

In this study, Sprague-Dawley rats were immobilized to a frame for 3 hours a day for 21 days to establish a model of chronic immobilization stress. The body weight and food intake of rats subjected to chronic immobilization stress were significantly decreased compared with the control group. Dual-labeling immunofluorescence revealed that the expression of leptin receptor and the co-localization coeffient in these leptic receptor neurons in the arcuate nucleus of the hypothalamus were both upregulated, while the number of neuropeptide Y neurons was decreased. Chronic immobilization stress induced high expression of leptin receptor in the arcuate nucleus and suppressed the synthesis and secretion of neuropeptide Y, thereby disrupting the pathways in the arcuate nucleus that regulate feeding behavior, resulting in diminished food intake and reduced body weight.

Clinical significance of the leptin and leptin receptor expressions in prostate tissues

Aim： To evaluate the expression of leptin and leptin receptor in benign prostatic hyperplasia （BPH） and prostate cancer （PCa）, and to investigate whether they are associated with the development and progression of PCa. Methods： hnmunohistochemical staining was performed to examine the expression of leptin and leptin receptor in BPH and PCa. PCa was divided into three groups： localized PCa, locally advanced PCa and metastatic PCa. The positive staining was identified and the percentage of the positive staining was graded. We also assessed the relationship between both the Gleason score and body mass index （BMI） and PCa. Results： The percentage of the leptin expression in PCa was significantly higher than that in BPH （P 〈 0.01）. For the PCa group, the expressed levels of leptin showed a considerable correlation with localized PCa and metastatic PCa （P 〈 0.05）. Leptin receptor, however, did not reveal a definite difference between BPH and PCa. The expression of leptin indicated a significant difference between well-differentiated PCa （Gleason score ≤6） and poorly differentiated PCa （Gleason score 8-10） （P 〈 0.05）. The relation between the leptin expression level in PCa and the BMI was not remarkable （P = 0.447）. Conclusion： Our results suggest that leptin might have a promoting effect on the carcinogenesis and progression of PCa.

Expression of Long Form Leptin Receptor mRNA in Pituitary of Pigs around Puberty

[ Objective] To study the expression of long form leptin receptor （Ob.Rb） mRNA in pituitary of pigs around puberty and explore the rela- tionship between Ob-Rb mRNA and porcine development around pituitary. [Method] Three Sujiang pigs were randomly selected at the age of 120 d, puberty and 180 d, respectively. A pair of primers was designed according to the Ob-Rb sequence published in the GenBank. Total RNAs were extracted from pituitary. The expression of Ob-Rb mRNA was detected by the real-time quantitative RT-PCR. [ Result] The Ob-Rb mRNA was de- tected in pituitaries of pigs at the age of 120 d, puberty and 180 d, respectively. The expression level of Ob-Rb mRNA was lowest at puberty. It was significantly different from that in the 120-day-old pigs and not significantly different from that in the 180-day-old pigs. [ Conclusion] The low expres- sion level of Ob-Rb mRNA is conductive to the arrival of puberty.

Breeding of Leptin Receptor Gene Knockout Mice and Genotype Identification of the Offspring

The leptin（ LP） receptor gene heterozygous mice were mated in four ways. Genomic DNA was extracted from offspring tails and analyzed by PCR. The result showed that the ratios of the offspring genotypes fit the Mendel＇s laws. The male and female LP^（-/-）mice（ DB / DB mice） were infertile. LP^（-/-）mice could be effectively bred by mating of male and female LP^（＋/-）mice. PCR methods could identify LP^（-/-）mice precisely.

Leptin signaling and leptin resistance

Leptin is secreted into the bloodstream by adipocytes and is required for the maintenance of energy homeostasis and body weight.Leptin deficiency or genetic defects in the components of the leptin signaling pathways cause obesity.Leptin controls energy balance and body weight mainly through leptin receptor b(LEPRb)-expressing neurons in the brain,particularly in the hypothalamus.These LEPRb-expressing neurons function as the first-order neurons that project to the second-order neurons located within and outside the hypothalamus,forming a neural network that controls the energy homeostasis and body weight.Multiple factors,including inflammation and endoplasmic reticulum(ER)stress,contribute to leptin resistance.Leptin resistance is the key risk factor for obesity.This review is focused on recent advance about leptin action,leptin signaling,and leptin resistance.

Influence of catgut implantation at acupoints on leptin and insulin resistance in simple obesity rats

OBJECTIVES:To investigate the influence of catgut implantation at acupoints on leptin resistance(LR) and insulin resistance(IR) in the simple obesity rat.METHODS:Rats were made obese with high-fat diets,after which surgical catgut was implanted at Zusanli(ST 36) and Neiting(ST 44) acupoints once a week for 4 weeks(implantation group).Rats from the implantation group were compared with normal rats and unoperated obese rats(control group).Gene expression of the leptin receptor(OB-R) was evaluated using RT-PCR and northern blot.Serum and hypothalamus leptin and insulin(INS) levels were determined by radioimmunoassay.Body weight,Lee's index,body fat,serum and hypothalamus leptin and INS levels,and hypothalamic OB-R gene expression were determined before and after treatment.RESULTS:Body weights,Lee's index,body fat,and serum leptin and INS levels were significantly higher in obese than in normal rats.Hypothalamic leptin and INS levels and OB-R gene expression were significantly lower in obese rats.Catgut implantation at acupoint promoted weight loss and decreased serum leptin and INS levels.Hypothalamic leptin and INS levels and OB-R gene expression increased significantly.CONCLUSIONS:Catgut implantation at acupoint adjusts central and peripheral leptin and promotes hypothalamic OB-R gene expression.This may be an important method for regulation of LR,IR and abnormal endocrinology and metabolism.

Regulation of leptin on insulin secretion and sulfonulurea receptor 1 transcription level in isolated rats pancreatic islets

Objective To investigate the regulation of leptin on insulin secretion and expression of ATP-sensitive potassium channel subunit sulfonulurea receptor 1 (SUR1) mRNA, and to determine whether the effects of leptin are mediated through known intracellular signaling transduction.Methods Pancreatic islets were isolated by the collagenase method from male SD rats. The purified islets were incubated with different concentrations of leptin for 2 h in the presence of different concentrations of glucose. Insulin release was measured using radioimmunoassay. Expression of SUR1 mRNA was detected by RT-PCR.Results In the presence of leptin 2 nmol/L, insulin release was significantly inhibited at either 11.1 or 16.7 mmol/L glucose concentration (both P<0.05), but insulin release was not altered at glucose of 5. 6 mmol/L physiological concentration. The dose-response experiment showed that the maximal effect of leptin on insulin secretion achieved at 2 nmol/L. Exposure of islets to 2 nmol/L leptin induced a significant increase of SUR1 transcription levels by 71% (P<0. 01) at 11.1 mmol/L glucose and by 56% (P<0. 05) at 16. 7 mmol/L glucose concentration. Selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin significantly prevented the leptin effect on insulin secretion and SUR1 mRNA expression.Conclusions Regulatory effects of leptin on insulin secretion could be biphasic at different concentrations of glucose and leptin. The stimulatory regulation of SUR1 transcription levels may be mediated through activation of PI 3-kinase pathway, which may be a possible mechanism of leptin in regulating insulin secretion.

Assessment of ghrelin and leptin receptor levels in postmenopausal women who received oral or transdermal menopausal hormonal therapy

Objective： In postmenopausal women, an increased leptin concentration and reduced levels of ghrelin and adiponectin were observed. The aim of this study was to evaluate the concentrations of the active form of ghrelin, total ghrelin, leptin receptor, lipoprotein（a） （Lp（a））, and plasminogen activator inhibitor type 1 （PAl-l） in postmenopausal women who received oral or transdermal menopausal hormonal therapy （MHT）. Methods： The study involved 76 healthy women： 46 women aged from 44 to 58 years who received oral （26） or transdermal （20） MHT; the control group consisted of 30 women aged from 44 to 54 years who did not receive MHT. The plasma concentrations of total ghrelin, the active form of ghrelin, Lp（a）, and PAI-I：Ag were measured by enzyme-linked immunosorbent assay （ELISA）. The concentration of the leptin receptor was measured by enzyme immunometric assay （EIA）. Results： We observed a significantly higher concentration o~ total ghrelin and the active form of ghrelin in women who received transdermal MHT in comparison with those who took oral MHT. We also found a significantly lower concentration of total ghrelin in women who received oral MHT compared with the control group. A higher concentration of PAl-1 ：Ag was found in the group of women who took transdermal MHT in comparison with those who took oral MHT and with the control group. The differences were statistically significant. Additionally, we found a significant negative correlation between the concentrations of total ghrelin and PAl-1 ：Ag and a positive correlation between the concentrations of total ghrelin and leptin receptor in women who received transdermal MHT. Conclusions： The study showed that women who used transdermal MHT had higher levels of total ghrelin than women who took oral MHT. This indicates a beneficial effect of the transdermal route of MHT. However, transdermal therapy was associated with adverse effects with regard to the observed higher levels of PAl-1 ：Ag, which in turn, can lead to a reduction in fibrinolytic activity.

Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene

Leptin resistance is a main mechanism of acquired childhood obesity,and the suppression of long form of leptin receptor(OBRb)gene expression in diet-induced obese rats indicates that the down-regulation of OBRb gene expression plays a pivotal role in the mechanism of leptin resistance.The aim of the present study was to construct the lentiviral RNA interference(RNAi)vector of rat OBRb gene and evaluate the effects of siRNA on silencing OBRb gene expression.The target sequence of siRNA-OBRb was designed,and the com-plementary DNA containing both sense and antisense oligonucleotides was synthesized.After phosphorylation and annealing,these double-stranded DNA was cloned to pRNA-lentivector-VGFP to construct pRNA-Lenti-OBRb-VGFP recombinants with U6-containing promoter,target sequence and Poly III terminator.Then,the products were confirmed by electrophoresis and sequencing analy-sis,and the effects of RNAi on reducing gene expression were further confirmed by real-time polymerase chain reaction in transfected rat glioma cells expressing OBRb.The target sequence of siRNA-OBRb was successfully cloned to pRNA-lentivector-VGFP,and the RNAi protocol specifically reduced the expression of OBRb mRNA by approximately 80%compared with controls in transfected rat glioma cells.The successful construction of rat lentivirus vectors expressing OBRb-specific shRNA may be useful for further investigation in vivo.

Lentivirus-mediated RNA interference targeting the ObR gene in human breast cancer MCF-7 cells in a nude mouse xenograft model

Background There is a significant association between obesity and breast cancer, which is possibly due to the expression of leptin. Therefore, it is important to clarify the role of leptin/ObR （leptin receptor） signaling during the progression of human breast cancer. Methods Nude mice with xenografts of MCF-7 human breast cancer cells were administered recombinant human leptin subcutaneous via injection around the tumor site. Mice in the experimental group were intratumorally injected with ObR-RNAi-lentivirus, while negative control group mice were injected with the same dose of negative-lentivirus. Tumor size was blindly measured every other day, and mRNA and protein expression levels of ObR, estrogen receptor a （ERa）, and vascular endothelial growth factor （VEGF） for each group were determined.Results Knockdown of ObR-treated xenografted nude mice with a high leptin microenvironment was successfully established. Local injection of ObR-RNAi-lentivirus significantly suppressed the established tumor growth in nude mice. ObR level was significantly lower in the experimental group than in the negative control group, while the amounts of ERα and VEGF expression were significantly lower in the leptin group than in the control group （P 〈0.01 for all）.Conclusions Inhibition of leptin/ObR signaling is essential to breast cancer proliferation and possible crosstalk between ObR and ERa, and VEGF, and may lead to novel therapeutic treatments aiming at targeting ObR in breast cancers.

Obesity phenotype in relation to gene polymorphism among samples of Egyptian children and their mothers

Obesity is complex heterogeneous disease controlled by genes,environmental factors,and their interaction.Genetic factors account for 40e90%of the body mass index variations.Body mass index(BMI)of children correlates more closely with maternal than paternal BMI.So,this studu was aimed to investigate the role of leptin receptor LEPR Gln223Arg,the uncoupling protein 2(UCP2 G 866 A)and insulin receptor gene(INSR exon 17)polymorphisms in the pathogenesis of obesity.A cross-sectional study executed on 130 children and their obese mothers;classified into 2 groups according to their BMI.The 2 groups were evaluated regarding the anthropometry.Restriction fragment length analysis for LEPR Gln223Arg,UCP2-866 G/A and INSR exon 17 polymorphisms were applied.It was reported that increased risk of obesity was found in LEPR AG t AA genotype and the A allele.Significant statistical difference was detected only in female children.Concerning UCP2,the AG followed by the GG genotype was the most frequent in all groups and the G allele was the mostly present in obese mothers and obese male children but with no statistical significance.There was difference in the INSR genotype and alleles between groups,but this difference was not statistically significant.This study concluded that the LEPR Gln223Arg,UCP2 G 866 A and INSR exon 17 polymorphisms are related to obesity in Egyptian population.Further researches on larger population are recommended to ascertain the implications of LEPR,UCP2 and INSR polymorphisms in obesity.

Expression of Kisspeptin/kiss1r System is Down-regulated in the Hypothalamic Arcuate Nucleus of Pubertal Male Rats with High-fat-diet

Objective To explore the potential function of nutrition on kisspeptin/kisspeptin receptor （kisslr） system in the arcuate nucleus （ARC） of male rats. Methods Pregnant rats were obtained and male pups were used to establish obesity model. Body parameters and blood samples were evaluated. Immunohistochemistry was used to determine the localizations and protein levels of kisspeptin, kisslr, and leptin receptor （LepR） immunoreactivity （IR） in ARC. QRT-PCR was used to determine kissl-, kisslr-, LepR-, and GnRH-mRNA levels. Results The establishment of obesity model was successful as body parameters and hormones levels changed noticeably. Kisspeptin-, kisslr-, and LepR-IR were detected and protein levels decreased significantly in high-fat-diets （HFD） rats than controls. The mRNA levels of kissl, LepR and GnRH significantly decreased in the ARC of HFD-fed rats. No difference was observed in kisslr mRNA levels between the two groups. Conclusion These data suggest that failure to increase GnRH levels with HFD may be associated with pubertal down-regulation of LepR and kisspeptin/kisslr system in the ARC of male rats.