[Construction of Zinc-alpha2-glycoprotein expression plasmid and its expression in 3t3-l1 preadipocytes].

OBJECTIVE: To construct mouse Zinc-alpha2-glycoprotein (mZAG) eucaryotic expression plasmid and identify its expression in 3T3-L1 preadipocytes. METHODS: The total RNA from mouse liver tissue was extracted. The reverse-transcript(RT)-PCR method was used to amplify the complete domain sequence of mZAG, and the confirmed PCR products was inserted into expression plasmid by DNA ligation. The mZAG expression plasmids with various concentrations (0, 0.4, 0.8, and 1.6 microg) were transfected into 3T3-L1 preadipocytes, and ZAG expression in mRNA and protein level was determined by real-time fluorescence quantitative PCR and Western blot, respectively. RESULTS: DNA sequencing confirmed the right sequence of mZAG expression plasmid pcDNA3.1(-)-mZAG. After the mZAG expression plasmid with different concentrations were transfected into 3T3-L1 preadipocytes, mZAG mRNA level significantly increased and reached 2.58 folds (P=0.002), 3.67 folds (P=0.000 and 5.19 folds (P=0.001) of that in the control group (no mZAG transfection). mZAG protein level also significantly increased and reached 2.75 folds of that in the control group (P=0.017). Treating 3T3-L1 cells with small interfering RNA (siRNA) sequence siRNA 1 and siRNA 4 resulted in a decrease of mZAG mRNA to 49% and 41% of those in the control group(no siRNA sequence transfection) (P=0.002P=0.000)and a decrease of mZAG protein to 55% and 62% of that in the control group (P=0.004,P=0.025). CONCLUSIONS: mZAG expression plasmid pcDNA3.1(-)-mZAG was successfully established in this study. This plasmid can be well expressed in 3T3-L1 preadipocytes. siRNA 1 and siRNA 4 can effectively inhibit the expression of mZAG in these cells.

[Effect of bodyweight on the onset of puberty of female children and adolescents].

OBJECTIVE: To investigate the possible effects and roles of bodyweight on the puberty onset in adolescent girls. METHODS: Totally 288 Chinese female children and adolescent girls aged 5 to 16 were followed up yearly for four consecutive years. The height, bodyweight, fat percentage, sexual characteristics, and the serum levels of leptin and insulin-like growth factor-1 (IGF-1) were studied to analyze the influential factors of puberty onset and age of menarche. RESULTS: The serum level of leptin elevated significantly from age 13 [(9.23 +/- 1.25) microg/L] and reached peak at age 16 [(13.19 +/- 1.45) microg/L]. IGF-1 significantly correlated with the timing of puberty onset (r = 0.292, P = 0.016). BMI and fat percentage had no significant effects on the onset of puberty, but were negatively correlated with the age of menarche (r = -0.323, P = 0.037, r = -0.298, P = 0.038 respectively). CONCLUSION: Bodyweight may have effect on puberty onset in female adolescents.

The Changes of Serum Leptin and Kisspeptin Levels in Chinese Children and Adolescents in Different Pubertal Stages.

The aim of the study is to investigate the changes of serum leptin and kisspeptin levels in children and adolescents with different pubertal stages and nutritional states. A total of 647 Chinese children and adolescents were recruited, and serum estradiol, testosterone, pituitary gonadotropins, leptin, and kisspeptin levels were measured. The results showed that serum leptin levels of boys in T2 stage were the highest among the five stages, while they showed a gradual increase from T1 to T5 stage in girls and reached the highest in T5 stage (P < 0.05). Conversely, serum kisspeptin levels of boys were higher in T4 and T5 stages than those in T1 stage, while its levels of girls were the highest in T2 stage, 21.4% higher than those in T1 stage (P < 0.05). Both leptin and kisspeptin levels were positively correlated with BMI, WC, and weight in all boys and girls (all P < 0.05). In conclusion, kisspeptin levels were firstly found to be notably changed in pubertal stages and nutritional status in Chinese children and adolescents with a significant sexual dimorphism. Obese/overweight girls had higher kisspeptin levels, and there was a positive correlation between kisspeptin and FSH and LH and obesity-related parameters in all boys and girls.

[Puberty development of the healthy adolescent girls of Daqing city, Heilongjiang province].

OBJECTIVE: To investigate the process of puberty development of healthy adolescent girls in Northern China. METHODS: 288 adolescent girls of Daqing city, Heilongjiang province, aged 5 to 16, were studied and followed up yearly for four years. The height, weight, fat percentage, second sex characteristics, and the blood levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E(2)) were examined. RESULTS: The mean age of puberty onset of these healthy adolescent girls was 8.5 years +/- 1.1 years. The blood levels of FSH, LH and E(2) were 0.2 mIU/L, 1.1 mIU/L and 0.06 nmol/L respectively (the 95 percentiles were 2.5 mIU/L, 2.3 mIU/L and 0.12 nmol/L respectively). Their mean age of menarche was 12.4 years +/- 1.2 years. The mean age of breast development was 8.8 years +/- 1.1 years. CONCLUSION: The girls in Northern China begin their puberty development at younger ages than reported before.

[The Effect of Cytokines on Expression of Growth Hormone Gene in Rat Pituitary GH(3) Cells].

To study the effect of interleukin-11(IL-11), ciliary neurotropic factor (CNTF) and transforming growth factor-beta (TGF-beta) on the hGH gene promoter activity in rat pituitary GH(3) cells and the interaction with pituitary-specific transcription factor Pit-1, firstly the stable transformed GH(3) cell line which contained hGH gene promoter 484-30 bp and luciferase reporter gene was established, then the concentration of GH in the medium and lysate of GH(3) cells and luciferase activities in GH(3) cells were measured, after treating these cells with the above cytokines, the effects of cytokines on secretion and synthesis of GH, and the promoter activity of the hGH gene were observed. The results of our experiments showed that IL-11(20 nmol/L), CNTF(10 nmol/L) and TGF-beta(5 nmol/L) regulated secretion and synthesis of GH, and the luciferase expression in stable-transformed GH(3) cells. IL-11 and CNTF had a stimulatory effect, whereas TGF-beta had an inhibitory one. Neither overexpression of Pit-1 nor inhibition of Pit-1 expression could affect the regulatory role of these cytokines. In conclusion, IL-11, CNTF and TGF-beta regulated the GH production in pituitary GH(3) cell line by regulating the hGH gene promoter activity, while Pit-1 might not be involved in the roles.

[Mechanism of interleukin-1beta increasing growth hormone expression in rat pituitary GH3 cells].

OBJECTIVE: To investigate the effect(s) of interleukin-1beta (IL-1beta) on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the molecular mechanism. METHODS: The method of luciferase reporter gene was used. We firstly established stable GH3 cell line which contains hGH gene promoter -484-30 bp and luciferase reporter gene. After treating these cells with IL-1beta or IL-1beta plus various signaling transduction inhibitors, the concentration of GH in the medium and lysate of GH3 cells and luciferase activities in GH3 cells were measured to reflect the effect of IL-1beta on secretion and synthesis of GH and the promoter activity of the hGH gene and the molecular mechanism. Results IL-1beta (10-10(4)U/ml) increased secretion and synthesis of GH. IL-1beta at levels of 10(2)-10(4) U/ml promoted the luciferase expression in stable GH3 cells, and the maximal action was 1.61 times of the control (P < 0.001). Among the inhibitors of intracellular signaling transduction pathways, mitogen-activated protein kinases (MAPK) inhibitor PD98059 (40 micromol/L) and p38 MAPK inhibitor SB203580 (5 micromol/L) completely blocked the stimulatory effect of IL-1beta, and phosphoinositide 3-kinase (PI3-K) inhibitor LY294002 (10 micromol/L) partly blocked the induction of IL-1beta. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IL-1beta induction of hGH promoter activity. The stimulatory effect of IL-1beta was abolished following deletion of the -196 to -132 bp fragment. CONCLUSIONS: IL-1beta increases the activity of hGH gene promoter in rat pituitary GH3 cells. This stimulatory effect of IL-1beta appears to require the intracellular MAPK, p38 MAPK, and PI3-K dependent signaling pathways. The effect of IL-1beta requires the promoter sequence that spans the -196 to -132 bp fragment of the gene, but it is unrelated to Pit-1 protein.

Serum Levels of the Adipokine Zinc- α 2-glycoprotein Are Decreased in Patients with Hypertension.

Objective. Zinc-α2-glycoprotein (ZAG) has recently been proposed as a new adipokine involved in body weight regulation. The purpose of this study is to investigate serum levels of ZAG in patients with hypertension and its association with related characteristics. Methods. 32 hypertension patients and 42 normal controls were recruited and the relationship between serum ZAG, total and high molecular weight (HMW) adiponectin, and tumor necrosis factor-α (TNFα) determined by enzyme-linked immunosorbent assay (ELISA) and metabolic-related parameters was investigated. Results. Serum ZAG concentrations were significantly lowered in patients with hypertension compared with healthy controls (61.4 ± 32 versus 78.3 ± 42 µg/mL, P < 0.05). The further statistical analysis demonstrated that serum ZAG levels were negatively correlated with waist-to-hip ratio (WHR) (r = -0.241, P < 0.05) and alanine aminotransferase (ALT) (r = -0.243, P < 0.05). Additionally, serum HMW adiponectin significantly decreased, while TNFα greatly increased in hypertension patients as compared with healthy controls (2.32 ± 0.41 versus 5.24 ± 1.02 µg/mL, 3.30 ± 1.56 versus 2.34 ± 0.99 pg/mL, P < 0.05). Conclusions. Serum ZAG levels are significantly lowered in hypertension patients and negatively correlated with obesity-related item WHR, suggesting ZAG is a factor associated with hypertension.