Effects Of Leptin On Long And Short Leptin Receptor MRNA Expression

ObstractChildhood obesity is becoming increasingly apparent with the changes in children's life - style and eating environment, which is risk factors for adult diseases such as type 2 diabetes and cardiovascular disease. Leptin, the product of ob gene, is an adipocyte - derived hormone that plays a key role in the regulation of food intake, energy expenditure, and whole - body energy balance in rodents and humans. Leptin acts through the leptin receptor (OB - R) , two major isoforms of which are long form ( OB - Rb) and short form ( OB - Ra). Because most of human obesity are not associated with deficiency of leptin due to a mutation in its gene in ob/ob mice, or OB - Rb defect caused by a mutation within the leptin receptor gene in db/db mice, acquired leptin resistance or insensitivi-ty may be a main cause of human obesity. We have found that the levels of leptin were high in obese children suggesting an inhibitory regulation at the level of the leptin receptor or downstream signaling pathway. It, is likely that leptin itself acts as an inhibitory regulator. The present study was designed to investigate the possible regulation effect on leptin receptors by leptin. We observe serum leptin levels as well as expressions of long and short leptin receptor isoforms ( OB -Rb, OB -Ra) mRNA in central (hypothalamus) and peripheral (liver) tissue of fatty SD rats fed with high - fat diet. Since human hepatocellular carcinoma cell line ( HepG2 ) , derived from liver where the OB - Ra was abundant and OB - Rb was also detectable, has been known to express functional OB - Ra, we determined whether HepG2 cells also express OB - Rb, and utilized HepG2 cells to assess the regulatory features of leptin receptor, which possibly contributes to an understanding of the mechanism for leptin resistance in vivo and sug-gests that leptin - induced receptor down - regulation may be relevant to leptin resistance.Materials and MethodsThirty four - week - old healthy male SD rats were divided into two groups randomly. One (20) acting as experimental group fed with high -fat diet containing 20% lard to set up the animal model of dietary obesity; the other (10) serving as control group fed with ordinary diet. Body weights were monitored weekly, At the end of 8 weeks, obese group was obtained according to the standard that body weight go beyond 20% of average body weight of the control. For both obese and control rats, serum leptin concentrations were measured with enzyme immunoassay; expression of OB - Ra, OB - Rb mRNA in hypothalamus was detected with semi - quantitative RT - PCR ( usingjj - actin as an internal standard ) , and the expression of OB - Ra and OB - Rb mRNA in liver was detected with in situ hybridization.The human hepatocellular carcinoma cell line ( HepG2) was cultured in six -well plate, 0. 5 x 105 cells/well, using DMEM containing 10% FBS. The culture medium was changed every other day, on the sixth day of culture, HepG2 cells were incubated for 24 h in serum - free medium containing 0, 10~9, 10~8, 10~7 or 10~ (mol/L) recombinant human leptin or insulin to analyse the effects of leptin and insulin on the expression of OB - Ra and OB - Rb. Total RNA was extracted from the treated cells using ISOGEN according to the manufacturer s suggested procedure. OD levels of RNA A260/A 280 were measured and RNA concentrations were calculated and Dnase treatment. 1 juug RNA used to synthesize cDNA with an Advantage RT - for - PCR kit and the oligo ( dT) 18 primer. To amplify fragments of leptin receptor cDNA, PCR primers were designed on the basis of established GenBank sequences for leptin receptors. Using a semi -quantitative RT - PCR technique ( GAPDH expression was used as an internal standard) , we measured the levels of long (OB - Rb ) and short (OB - Ra) leptin receptor mRNA expression at 24 h incubation with various concentrations of human leptin or insulin. Ten microliter aliquots of PCR products were electro-phoresed in 2% agarose gels and stained with ethidium bromide. Video images of the ethidium bromide - stained gels were quantified by densitometry using the NIH Image 1.61 software. A linear relationship between PCR products and amplification cycles (from 20 to 45 ) was observed.Results1. Rats fed with high - fat diet were observed significant increases in body weight from 4 weeks of high - fat die administration compared with control group, at the end of 8 - weeks feeding, according to the standard above, 14 rats (70% ) were diet -induced -obesity rats and treated as obese group, the body weight of obese group was (370. 07 ± 33. 03) g, increased significantly compared with control group (270. 80 ± 18.61) g,and there were significant differences in body weights between obese and control groups ( p < 0.01). There were higher levels of glucose, triglyceride and insulin in obese group.2. The concentrations of serum leptin in rats of obese group were (13. 84 ± 3. 70) ng/ml, elevated obviously compared with control groups (10.30 ±3.55) ng/ml (p<0.05).3. OB - Ra and OB - Rb existed in hypothalamus both in obese group and control group. The expression (relative gray value)of OB - Ra and OB - Rb in obese group were 5. 23 ± 1. 60 and 5. 97 ± 2. 19 respectively, reduced remarkably as compared with the control group (OB - Ra : 15. 05 ± 6. 22, p < 0. 01; OB - Rb: 30. 24 ± 10. 99, p<0.01), and there was significant negative relationship between OB - Ra or OB - Rb mRNA expression in hypothalamus and serum leptin concentrations in obese group respectively (r = 0.774, p <0.01 or r2= 0.751, p<0.01).4. OB - Ra and OB - Rb existed in liver both in obese group and control group. The expression of OB - Ra was 119.67 ± 15.42, reduced significantly as compared with the control group (211. 50 ±20.99, p <0.01) ; and the expression of OB - Rb in obese group was 78.56 ± 8.63, reduced remarkably as compared with the control group ( 137.41 ± 17. 57, p < 0. 01). There was significant negative relationship between OB - Ra or OB - Kb mRNA expression in liv-er and serum leptin concentrations in obese group respectively ( r2 = 0. 678 or 0.684, p<0.05).5. By RT - PCR and DNA sequencing, we demonstrated that both OB - Ra and OB - Rb mRNA was expressed in this cell line which provides a useful model allowing direct studies of leptin receptor regulation.6. Leptin at 10" -10" (mol/L) produced a significant inhibition of OB- Rb mRNA expression, with maximum decrease (43% of control ) at 10"6 mol/L. Similarly the expression of OB - R219. 1 and OB - R219. 3, two short isoforms of leptin receptor, was also markedly reduced in cells treated with leptin at 10 "8 - 10 "6( mol/L) , with maximum inhibition ( 44% of control at 10 ~7 mol/L for OB - R219.1; 49% of control at 10 "6 mol/L for OB - R219.3 ).7. There is no effect of insulin on OB - Rb and OB - Ra mRNAs expression in HepG2 cells following a 24 - hours incubation with insulin at 10 ~9 ~ 10 ~6 (mol/L).Conclusions1. Obese rat models can be set up through feeding with high - fat diet, the levels of leptin were high in obese rats suggesting leptin resistance.2. OB - Ra and OB - Rb existed in hypothalamus in SD rats. The expression of OB - Ra and OB - Rb mRNA in obese group was reduced significantly as compared with the control group, and there was significant negative relationship between OB - Ra or OB - Rb mRNA expression in hypothalamus and serum leptin concentrations in obese group respectively, suggesting the down - regulation of OB - R in central maybe act as one of the factors that result in leptin resistance in fatty rats.3. OB - Ra and OB - Rb existed in liver in SD rats. The expression of OB- Ra and OB - Rb mRNA in obese group was reduced remarkably as compared with the control group, and there was marked negative relationship between OB- Ra or OB - Rb mRNA expression in liver and serum leptin concentrations in obese group respectively, implying that such down - regulation of leptin receptor may also occurs in peripheral site.