Regulation Of Leptin On POMC And Hypothalamus-pituitary Axis

In 1994, Zhang et al. had successfully cloned the little mouse's obese gene and human homology sequence using positional cloning technology. The protein of ob gene code is known as leptin. The discovery of leptin confirmed the hypothesis about forty years ago: energy stores represented by the adipose tissue might produce some signaling molecules able to modulate both energy balance and reproduction to CNS sites. Ob/ob mouse and db/db (diabetic gene, db) mouse have similar physical features because of the gene mutation of leptin and gene mutation of leptin receptor separately: the serious early obesity, the serious insulin resistance, hyperphagia, reduced energy expenditure and infertility in both sex. However, after injecting leptin to the abdominal cavity of ob/ob mouse, not only their weights and diets back to the normal level, but also retrieved reproduction ability. In female ob/ob mouse, ovary and uterine weight increased, serum LH raised, primordial follicles and vesicular follicles increased. weight gained in male ob/ob mouse's testicle and seminal vesicle gland, sperm's quantity increased. The observation suggested that leptin controlled reproduction as modulating energy expenditure and food intake.Though there are a large number of evidence supporting the role of leptin to regulate the reproduction function, whether leptin regulates the hypothalamus-pituitary-gonadal axis directly or indirectly, still needs more studies to clarity. Therefore, this experiment study on leptin's probable function on the hypothalamus-pituitary axis, and expect to tentatively probe into leptin's mechanism of regulating reproduction, offer certain experimental data for clinical practice, via observing leptin's influence on the expression of POMC (Proopiomelanocortin) ih hypothalamus and the regulating function on the hypothalamus-pituitary axis hormone. Research contents and results are as follows:1 Regulation of leptin on POMC gene and its protein Objective: To observe the influence after injection of leptin into the cerebral ventricle on the female OEP(ovariectomized, estrogen primed) rat's POMC mRNA and its protein.Methods: Select adult female Wistar rat to make OEP model , from the 8th day after the operation, hypodermic injection of Estradiol Benzoate about 25μg everyday for each rat, after 5 days injection, located the cerebral ventricle according to"The Rat Brain in stereotaxic coordinates"written by George Paxinos and Charles Watson. 5μl leptin was injected into the cerebral ventricle in the experimental group, the control group was injected with saline 5μl. After injection, the brain tissue was cut out between optic chiasma and mamillary body rapidly at different time then weigh and grind under the low temperature, centrifuged for 15 min at 4℃3000rpm, the supernatants were collected and stored at -80℃. According to the Trizol interpretation, hypothalamus tissues were stored at liquid nitrogen and RNA was extracted strictly. After detection and quantitation, expression of POMC mRNA after microinjection was measured by RT-PCR. According to the experimental step of Western blot, observing the change of POMC protein. All the results were analyzed by statistical analysis.Results: 1 After microinjection 1.0h later POMC mRNA expression in rats'hypothalamus increased slightly, but there was no significant difference in leptin-injected group when compared with control group(P>0.05). POMC mRNA expression in hypothalamus increased in leptin-injected rats both 2.0h later and 4.0h later, and there was significant difference between control group and leptin-injected group(P<0.05). And 6.0h later, there was no significant difference between control group and leptin-injected group(P>0.05). 2 Effects on POMC expression by lateral cerebral ventricle injection of leptin: 1.0h later after injection, POMC expression in rats'hypothalamus no significantly increased compared with control group, there was no significant difference in leptin-injected group(P > 0.05). Both 2.0h later and 4.0h later after microinjection, POMC expression in hypothalamus begun to increase and then reach its highest level, there was significant difference between leptin-injected group and control group(P<0.05). 6.0h later, expression of POMC in hypothalamus decreased in leptin-injected group, but there was also significant difference between the groups(P<0.05).Conclusions: 1 After the lateral cerebral ventricle injection of leptin, the expression of POMC mRNA in hypothalamus increased, leptin injection show a positive regulation to POMC mRNA. 2 After injection of leptin, POMC protein expression in hypothalamus also increased, leptin injection show a positive regulation to POMC protein expression. 3 Leptin could play a regulating role to energy balance and nutrition state through POMC neuron.2 Effects on the hormone secretion of hypothalamus-pituitary axis by lateral cerebral ventricle injection of leptin andα-MSHObjectives: To observe the influence on rats'plasma GnRH, LH, FSH by the lateral cerebral ventricle microinjection of leptin andα-MSH. Explore the effects of POMC neuron on hypothalamus-pituitary- reproduction axis.Methods: Adult female Wistar rats were divided into three groups randomly: control group, Leptin group andα-MSH group. All the rats were ovariectomized in sterile condition. Since the 8th day after operation, rats were injected s.c. with 25μg of estradiol benzoate everyday and were used for 5 days. Then according to the《The Rat Brain in Stereotaxic Coordinates》written by George Paxinos and Charles Watson, lateral cerebral ventricle was located: posterior to bregma point 1.0mm, lateral 1.5mm, deep 4.0mm. Injecting respectively leptin 5μl,α-MSH 5μl, saline 5μl into lateral cerebral ventricle according to the groups. 1h, 2h, 4h and 6h later after microinjection, open the thorax of the rat under anesthesia, blood samples were collected from the right atrium and stored at -20℃after centrifugation until measurements of GnRH,LH and FSH by ELISA kits.Results: 1 Effects on rats'serum GnRH by lateral ventricle microinjection of leptin: the concentration of GnRH increased significantly. after microinjection 1.0h later, After microinjection 2.0h later, the concentration of GnRH reached its highest level. Microinjection 4.0h later, the concentration of GnRH decreased significantly. Concentration of GnRH after microinjection 1.0h,2.0h,4.0h later, there was significant difference compared with control group(P<0.05). After microinjection 6.0h later, there was no significant difference between treated group and control group (P>0.05). 2 Results of rats serum LH level by lateral ventricle injection of leptin: measurements of ELISA were as follows: after microinjection 1.0h later, serum LH level increased significantly, and there were significant changes between treated group and control group(P<0.05). 2.0h later, compared with saline group, serum LH level increased continuously in leptin-injected group. After microinjection 4.0h later, serum LH level decreased significantly, and there was also significant difference between the two groups(P<0.05). However 6.0h later, rats serum LH level decreased to normal level in leptin-injected group, and there was no significant difference when compared with control group(P>0.05). 3 Effects on rats serum FSH by lateral ventricle injection of leptin: after microinjection 1.0h later, there was no significant changes on serum FSH level in treated group and control group(P>0.05). But 2.0h later, compared with control group, serum FSH level increased quickly in leptin-injected animals, and there was significant difference between the two groups(P<0.05). FSH level decreased significantly after microinjection 4.0h later, but there was still significant difference between control group and leptin-injected group(P<0.05). There was no significant difference between treated group and control group 6.0h later(P>0.05). 4 Results after lateral ventricle injection ofα-MSH were as follows: 1.0h later, rats'plasma GnRH concentration was decreased, compared with the control group, and there was significant difference between the two groups(P<0.05). After injection 2.0h, the plasma GnRH concentration continues reducing, compared with the control group, and there was also significant difference between the two groups(P<0.05). 4.0h and 6.0h later, GnRH concentration compared with the control group, there is no significant difference(P>0.05). 5 After the lateral ventricle injection ofα-MSH for 1.0h later, LH plasma concentration reduced obviously. 2.0h later, LH concentration decreased continuously and 4.0h later, the concentration of LH increased significantly. 1.0h,2.0h and 4.0h later, LH concentration compared with control group, there was significant difference. LH concentration returned to normal at the 6h, and there was no significant difference betweenα-MSH group and control group. 6 The resultes of FSH concentration after microinjection ofα-MSH were as follows: after microinjection ofα-MSH 1.0h later, the rats'plasma FSH concentration was slightly decreased, compared with the control group, but there was no significant difference between the tow groups(P>0.05). FSH concentration remarkably decreased after microinjection ofα-MSH 2.0h later. FSH concentration increased after 4.0h later, but also lower than the normal. FSH concentration returned to normal at 6.0h after microinjection ofα-MSH. FSH concentration after injection ofα-MSH 2.0h and 4.0h later, there was significant difference between experiment groups and control groups(P<0.05). 6.0h later, FSH concentration there was no significant difference compared with the control group.Conclusions: 1 There was a positive regulation effect on OEP rats'plasma GnRH,LH and FSH concentration after microinjection of leptin. 2 There was a negative regulation effect on OEP rats'plasma GnRH,LH and FSH concentration after microinjection ofα-MSH.3 Effect of leptin andα-MSH on the secretion of LH,FSH from cultured pituitary cells in vitroObjectives: To study the effect of leptin andα-MSH on reproductive at pituitary level through researching the influence of leptin andα-MSH on the secretion of LH,FSH from cultured anterior pituitary cells in vitro.Methods: Adult female Wistar rats, abdominal cavity anesthesia, skin was sterilized by alcohol, cut the skin to expose the skull, strip the skull and expose the brain, then took out the pituitary quickly. Put it into the balanced salt solution of D-Hanks. Separating posterior pituitary and anterior pituitary. Cut the anterior pituitary into small pieces( <1mm3), through digesting, centrifugalization, filtering, living cell counting, and observe cells growing. On the 4th day of cells cultured, added leptin andα-MSH of different concentration separately into culture medium. After continuing cells culture for 24h, collecting the culture liquid of every group, stored at -20℃, measuring LH,FSH concentration in the culture liquid with ELISA kits.Results: 1)The shape of cells was round at the beginning of culture and stick to the wall of the glass dish in 6h, some cells began to grow and protrude in 24h, after 48h cell stick to the wall well, then the cells began to get together. The shape of anterior pituitary cells appeared to be oval, triangle or the polygon, like epithelioid cell. In 7d, the shape of gland cell was not changed obviously except growing. From 5th day on, another cell began to grow day by day, and the quantity increased gradually, then developing into the fibroblast. From 7th day on, the fibroblast grew rapidly, and the gland cell began to lose the original shape and characteristic, from 10th day on, the gland cell was almost replaced by the fibroblast completely. 2 The releasing amount of LH increased with the increasing of leptin concentration. When leptin concentration was 10-9 mol/L, LH releasing amount reached its highest level, then the releasing amount of LH decreased with increasing of leptin concentration. Except that when leptin concentration was 10-12 mol/L, the releasing amount of LH was significantly difference compared with the control group(P<0.05). Between different groups of leptin, the releasing amount of LH was also significantly difference(P<0.05). 3 Under the stimulation of leptin, the releasing amount of FSH also increased with the increasing concentration of leptin. When leptin concentration was 10-9 mol/L, FSH releasing amount reached its highest level, then the releasing amount of FSH decreased with increasing concentration of leptin. Except that when leptin concentration were 10-12 mol/L and 10-6 mol/L, the releasing amount of FSH was significantly difference compared with the control group(P<0.05). Between different groups of leptin, except the two groups of 10-12 mol/L and 10-6 mol/L, the releasing amount of FSH was also significantly difference(P<0.05). 4 Under the stimulation ofα-MSH, the releasing amount of LH, FSH there was no significant difference compared between experiment groups and control groups. the releasing amount of LH, FSH there was also no significant difference compared between different experiment groups. Conclusions: 1 The cultured anterior pituitary cells in vitro stick to the wall of the glass dish in 6h, some cells began to grow and protrude in 24h, after 48h cell stick to the wall well. From 5th day on, another group cells began to grow and developing into the fibroblast. In 7d, the shape of gland cell was not changed obviously except growing, then the fibroblast grew rapidly, and the gland cell began to lose the original shape and characteristic. The shape of anterior pituitary cells appeared to be oval, triangle or the polygon. From 10th day on, the gland cell was almost replaced by the fibroblast completely. 2 There was obviously influence on the secretion of LH,FSH from cultured pituitary cells in vitro by leptin stimulation. When leptin concentration was 10-9 mol/L, LH,FSH releasing amount reached its highest level. The effect would be depressed when the concentration of leptin was higher or lower. 3α-MSH did not influence the secretion of LH,FSH from cultured pituitary cells in vitro.