Preliminary Research On The Effect Of Estrogen On The Key Enzymes Of Glucose Metabolism In H9c2 Cells And Their Mechanisms

Insulin resistance describes an impaired biological response to insulin in periphery tissues such as skeletal muscle, fat and liver, including muscule. Many factors cause insulin resistance.The reduce of estrogenic level is very prevalent in postmenopausal women. Postmenopausal women develop visceral obesity and insulin resistance and are at increased risk for type 2 diabetes mellitus. Links between insulin resistance and cardiovascular disease (CVD) have long been suggested by an increased incidence of CVD and other vascular diseases in diabetes, particularly in female patients.Many scientific research showed, estrogen plays an important role in curing menopause insulin resistance syndrome and preventing cardiovascular diseases. Estrogen can lower the fasting level of serum glucose and insulin of postmenopausal women, improve insulin sensitivity, relieve insulin resistance syndrome, and make the rate of cardiovascular diseases decrease by 35-50%.In July, 2002, an investigation result of a clinical experiment of the American State Sanitation Academy Women's Health Initiative shows that, in accordance with the clinical observation the rate of suffering from apoplexy and cardiopathy in those women who received HRT was obviously increased and advised to end HRT. However, many other home and broad experts agree estrogen replacement therapy, and believe that the main purpose of WHI is to study the relationship between HRT and the danger of suffering from cardiovascular diseases and breast cancer without summarizing the benefits of HRT overall. Temporarily, the shortcoming and benefits of hormone replacement therapy become a hot issue for present studies again. In 2005, Michael E. etc raised their point in Science that it is necessary to improve the understanding of biology and gender differences in the perimenopause, and elucidate the effect of estrogen on heart at molecular and cellular basisThis paper probes into the effect and mechanism of estrogen on glocuse metabolism of myocardium at cell level and molecular level. The study will offer theoretical evidence for understanding the shortcoming and benefits of hormone replacement therapy.MethodH9c2 cells were cultured in six-well cluster plates in DMEM medium, supplemented with 10%(v/v) fetal bovine serum, at 37℃in a humidified 5% CO₂ atmosphere. The serum was stripped of estrogen by dextran T-70-coated activated charcoal. After confluence, cells were washed once with serum-free DMEM, incubated in serum-free DMEM for 1 h, then cultured in DMEM with 2% stripped fetal bovine serum, finally incubated in as described below: (1) For the short-term effect of estradiol (E2 ), cells were treated with 0 , 1 nM or 100 nM E2 for 10 min before enzymatic assays and the cell treated in 0 E2 served as control. (2) For the long term effect of E2, cells were incubated for 24 hrs with 0 , 0．01 nM, 1 nM, 100 nM, or 1μM E2 before experiments and the cell cultured in 0 E2 served as control. (3) In blocking experiment, cells were cultured for 24 hrs with 1 nM E2, 1 nM E2 plus 10 nM ICI or 1 nM E2 plus 100 nM ICI respectively, and the cells cultured by 1 nM E2 served as control. In the experiment we test (1) the enzymatic activity of PFK (6-Phosphofructo-1-kinase, PFK), GS (Glycogen synthase, GS) and ICDH (NAD⁺-Isocitrate dehydrogenase, ICDH), (2) semiquantitative analysis of gene expression of PFK-M, GS and ICDH-αby RT-PCR on the mRNA level , (3) the protein expression of PFK-M, GS by Western blotting. Result:(1) After treated with E2 for 10 min, the activity of PFK with 1 nM and 100 nM E2 are similar to that of control(p > 0．05, p > 0．05) . The total or active activity of GS with 1 nM and 100 nM E2 are similar to that of control(p > 0．05, p > 0．05, p > 0．05, p > 0．05, respectively). The activity of ICDH with 1 nM and 100 nM E2 are similar to that of control( p > 0．05,p > 0．05) .(2) After treated with E2 for 24hrs, the activity of PFK in the groups of 0．01 nM, 1 nM, 100 nM, or 1μM E2 was increased compared with that of the control ( p < 0．05, p < 0．05, p < 0．05, and p < 0．05 respectively ). The activity of PFK in the cells cultured with 1 nM E2 was higher than those of the cells treated by 0．01 nM E2 (p < 0．05) , 100 nM E2 ( p < 0．05) , or 1μM E2 ( p < 0．05) . There was no significant difference among 0．01 nM, 100 nM and 1μM E2 groups (p> 0．05 ). The total activity and activel activity of GS with 0．01 nM, 1 nM, 100 nM, 1μM E2 increased compared with the control (p< 0．05,p < 0．05,p < 0．05 and/p < 0．05 respectively ). The total activity and active activity of GS in the cells with 1 nM E2 were higher than those in the groups treated by 0．01 nM E2 (p < 0．05,p < 0．05) , 100 nM E2 (p < 0．05,p < 0．05 ) and 1μM E2 ( p < 0．05, p < 0．05, respectively ). However, with the same treated condition of E2, the activity of ICDH in the groups of 0．01 nM, 1nM, 100 nM and 1μM E2 was similar to that of control ( p > 0．05, p > 0．05,p> 0．05, p> 0．05, respectively).(3) The estrogen receptor antagonist, ICI 182,780 could decreased the activity of PFK and total GS activity of the cells treated with E2．Compared with the control, the activity of PFK in the groups of InM E2 plus 10 nM ICI and 1nM E2 plus 100nM ICI had significant decreased about 36% (p <0．05 ) and about 70% (p < 0．05 ). And compared with the control, the total activity of GS in the groups of InM E2 plus 10nM ICI and InM E2 plus 100nM ICI had significant decreased about 38% ( p <0．05 ) and about 68% (p < 0．05 ).(4) In the results of RT-PCR, the mRNA expression of PFK-M of 0．01 nM, 1 nM, 100 nM or 1μM E2 in H9C2 cells were increased for 24 hrs (p < 0．05,p < 0．05,p < 0．05, and p < 0．05 respectively ) compared with the control. And the mRNA expression of 1 nM E2 group were higher than those in the groups treated by 0．01 nM E2 (p < 0．05 ), 100 nM E2 (p < 0．05 ) and 1μM E2 (p < 0．05 ). Compared with control, the mRNA expression of GS in H9C2 cells were increased by incubation with 0．01 nM, 1 nM, 100 nM or 1μM E2 for 24 hrs (p < 0．05,p < 0．05,p< 0．05, and p < 0．05 ). And the mRNA expression of 1 nM E2 group were higher than those in the groups treated with 0．01 nM E2 (p < 0．05 ), 100 nM E2 (p < 0．05 ) and 1μM E2 (p < 0．05 ). The mRNA expression of ICDH-αsubunit of 0．01 nM, 1 nM, 100 nM or 1μM E2 for 24 hrs was no significant difference with that of 0 nM E2 (p > 0．05, p> 0．05, p> 0．05, p > 0．05) .(5) Compared with control, the protein expression of PFK-M of 0．01 nM, 1 nM, 100 nM or 1μM E2 groups were increased ( p < 0．05, p < 0．05,p < 0．05, and p < 0．05 respectively ). And the expression of 1 nM E2 group were higher than those in the groups treated by 0．01 nM E2 ( p< 0．05 ), 100 nM E2 ( p < 0．05 ) and 1μM E2 ( p < 0．05 ). Compared with control, the protein expression of GS were increased by incubation with 0．01 nM, 1 nM, 100 nM or 1μM E2 for 24 hrs (p < 0．05,p < 0．05,p< 0．05, and p < 0．05 respectively ). And these mRNA expression of 1 nM E2 group were higher than those in the groups treated by 0．01 nM E2 (p< 0．05 ), 100 nM E2 (p < 0．05 ) and 1μM E2(p < 0．05 ).(6) The mRNA expression of PFK-M in the cells cultured by InM E2 plus 10nM ICI or InM E2 plus 100nM ICI for 24hrs had significant decreased compared with the control (p <0．05 and p < 0．05 respectively ). The decreased PFK mRNA were about 35% by 10 nM ICI and 63% by 100nM ICI. The mRNA expression of GS in the cells cultured by 1nM E2 plus 10nM ICI or 1nM E2 plus 100nM ICI for 24hrs had significant decreased compared with the control (p <0．05 and p < 0．05 respectively ). The decreased GS mRNA were about 32% by 10nM ICI and 60% by 100nM ICI. The tendency of protein expression changes of PKF and total GS in InM E2 plus 10nM ICI or 1nM E2 plus 100nM ICI groups consisted with the results of their mRNA respectively.Conclusion1．Estrogen could enhance the activity of PFK and GS by up-regulating the expression of PFK-M and GS on mRNA and protein level through ER, which could improve glocuse metabolism.2．It was demostrateed that estrogen regulates the activity of PFK and GS by gene expression, not short-term effect pathway.3．Estrogen could not affect the activity of ICDH and mRNA expression of ICDH-α.4．A dose-dependent effect of E2 on the enzyme activity was observed in this study. The optimal effect appears at 1nM E2．5．It is suggested that the dose of E2 used in hormone replacement therapy (HRT) is important to the menopausal women.