Gene Expression And Function Study Of Melanin-Concentrating Hormone Receptor 2

Obesity, a chronic disease characterized by excessive accumulation of body fat, has become a global health problem and is a significant risk factor for developing many serious diseases, including cardio- and cerebro- vascular diseases, diabetes, hypertension, dyslipidemias and some cancers. In recent years, it has been well established that obesity is associated with the neuroendocrine control of the hypothalamus. The identification of the hypothalamic orexigenic neuropeptide melanin-concentrating hormone (MCH) and its two receptors (MCHR1 and MCHR2) provide new targets for treatment of obesity. Major advances have been made in identifying the relevance of MCH and MCHR1 to obesity and energy homeostasis by transgenic animal and gene knockout animal. However, the function of MCHR2 and its relevance to obesity is still indistinct. In this study, we first construct the eukaryotic expression vector of human MCHR2 by DNA recombination technique. Then, we stably transfect mammalian cell line with the vector and analyse the expression, localization and biologic activity of MCHR2. Finally, we investigate the physiological function of MCHR2 in 3T3-L1 preadipocyte to evaluate whether it is involved in mediating the effects of MCH on energy balance and its relevance to obesity. This study includes three parts:1. Construction and identification of eukaryotic expression vector of human MCHR2. Methods: The full-length MCHR2 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and was oriently inserted into eukaryotic expression vector pcDNA3.1(+). The positive clone was obtained after transformation and antibiotic screening and was identified by restriction endonuclease digestion, PCR, and DNA sequencing. Results: The 1023bp MCHR2 cDNA fragment was obtained and cloned into the downstream of CMV promoter of pcDNA3.1(+). The recombinant plasmid was identified by restriction endonuclease digestion and PCR. DNA sequencing confirmed that the sequence of the inserted element was correct. Conclusion: The eukaryotic expression vector pcDNA3.1-MCHR2 was successfully constructed.2. Stably expression and biological activity of MCHR2 in CHO cells. Methods: CHO cells were transfected with the recombinant plasmids using lipofectamine. Stable cell line was established by G418 selection. Expression of MCHR2 in transcriptional level and protein level was detected by RT-PCR and western blot. Localization of MCHR2 in CHO cells was assayed by immunofluorence technique. Radioligand binding assays were performed to survey receptor density and affinity of MCHR2. Signal transduction pathways mediated by MCHR2 were analyzed by measurement of intracellular cAMP and calcium. Results: CHO cell line stably transfected with pcDNA3.1-MCHR2 plasmids was generated after liposome transfection and G418 selection. MCHR2 gene was efficiently transcribed and translated, and its protein exactly localized on the cytomembrane. MCHR2 could specific bind with ¹²⁵I-MCH with high-affinity and saturability (Bmax=309.97±1.14fmol/mg protein, Kd=0.170±0.0006nmol/L). MCH stimulation had no effect on the production of cAMP. MCH induced a clear and transient increase of intracellular calcium, and this procedure was completed within 30⁵0s. The effect of MCH on intracellular calcium showed dose-dependent response with an effective half-maximal concentration (EC50) of 2.32±0.01nmol/L. It is suggested that MCHR2 is only coupled to Gq protein. Conclusion: Stably transfected CHO cell line was established successfully. It is confirmed that MCHR2 expressed in mammalian cells is a functional receptor for the MCH by the analysis of its expression, localization, receptor characteristics, and signal pathways. This present study provides a reliable experimental foundation for further studies on the function of MCHR2 in vitro.3. Influence of MCHR2 on the biological characteristics of adipocyte. Methods: 3T3-L1 cell line stably transfected with pcDNA3.1-MCHR2 plasminds was generated by transfection and G418 selection and named as 3T3-L1-MCHR2 while the control cell line transfected with pcDNA3.1(+) was named as 3T3-L1-mock. The expression and biological activity of MCHR2 were identified by RT-PCR, western blot, immunofluorence, radioligand binding and intracellular calcium assay. Cell proliferation was determined by MTT assay. Cell cycle was detected by flow cytometry. Cell differentiation was induced by hormone cocktail (MIX+DEX+insulin) and examined by Oil red O staining. The marker genes of adipocyte differentiation were examined by RT-PCR. The production of triglyceride and secretions of FFA and leptin were measured. Results: Stably transfected cell line named 3T3-L1-MCHR2 was established and passed the identification of expression and biological activity of MCHR2. MCH had no effect on the proliferation of 3T3-L1-MCHR2 and 3T3-L1-mock cells. Both the two cell lines were induced to differentiate to mature adipocytes by MCH. The speed and degree of differentiation in 3T3-L1-MCHR2 cells were higher than those in 3T3-L1-mock cells. Following was the detailed display of differentiation in 3T3-L1-MCHR2 cells: accumulation of lipid increased at day 9 (in 3T3-L1-mock it increased at day 12); marker genes of mature adipocyte including PPARγ2,C/EBPαand aP2 increased at day 3, leptin increased at day 6, while in 3T3-L1-mock cells, up-regulation of PPARγ2 and leptin appeared at day 6, up-regulation of C/EBPαand aP2 appeared at day 9, and the increasing levels were lower. There was no difference of the FFA secretion between MCH-treated group and control group. The triglyceride content in 3T3-L1-MCHR2 and 3T3-L1-mock mature adipocytes obviously increased by MCH, and the rising rate were 22.61% and 12.17%, compared with corresponding untreated cells. MCH stimulated leptin releases from 3T3-L1-MCHR2 mature adipocytes at 6h and 24h, the rising rate were 28.17% and 18.25% compared with the untreated cells. In contrast, secretion of leptin from 3T3-L1-mock mature adipocytes only increased by 14.57% at 24h. Conclusion: MCHR2 mediates the positive control of MCH on preadipocyte differentiation, triglyceride synthesis and leptin secretion without the influence on preadipocyte proliferation and lipolysis. The present study tentatively confirms that the physiological function of MCHR2 involvs in the regulation of energy balance.