| Background:With the improvement of people’s living standard and lifestyle changes, the prevalence of type2diabetes mellitus (T2DM) was extraordinary increasing in our country. According to the latest data of2009, the prevalence of T2DM was9.7%and the number of patients was9.24million in our country, which had ranked first in the world. The various kinds of acute and chronic complications caused by diabetes leading to multiple tissue or systems dysfunction and have severe impacts on people’s quality of life and longevity. Diabetes has become a critical public health problem threatening people’s life worldwide.The concept of "incretins" was firstly proposed by La Barre in1932. Incretin hormones are gut-derived peptides, which act to potentiate meal-induced insulin secretion. In man, the two most important incretins are glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1(GLP-1). GLP-1is secreted by enteroendocrine L cells, which can promote insulin secretion glucose-dependently and proliferation of islet β cells, inhibits β cells apoptosis. Now it has been confirmed that beside its insulinotropic function, GLP-1has the advantages on anti-inflammation, anti-oxidative stress and improving the functions of endothelial cells. The discovery of incretins has made breakthrough progress on the pathogenesis and treatment of type2diabetes. However, it is not clear whether the disfunction or absent of incretin in the patients of T2DM is the consequence or the pathogenesis of diabetes itself. The previous study has discovered that after taken glucose orally, the effects of incretin hormones on stimulating insulin secretion were decreased in patients of T2DM compared with health control, mainly reflect the defects of GIP and the diminished secretion of GLP-1. Subsequent study has revealed that the impairment effects of incretin were generally serious in the obese than the leaner patients of T2DM, the underlying mechanisms may be include the hyposecretion, efficacy and metabolic abnormalities of the incretins. The scientists compared the characteristic of impaired GIP and GLP-1in patients of T2DM and discovered the impaired secretion of GLP-1is more serious than that of GIP. No matter the total or the activated level of GLP-1are far behind to the normal control group. The reduced secretion of GLP-1is the main feature of the impaired incretin effects in patients of T2DM.The early damage or apoptosis of intestinal L cells may contribute to the reduced secretion of GLP-1and play an important role in the genesis and development of T2DM. Current researches are focus on the regulate effects of different drugs on the endogenous GLP-1secretion and possible mechanisms of L cells secrete GLP-1, but the functional status of L cells are out of the hotspot.There are more and more evidence indicating that gut microbiota play a considerable role in the host metabolism, immunity and even the genesis and development of metabolic diseases such as obesity and diabetes with the development of genomics, proteome and bacterial culture. Evidence shows that there are obviously changes in gut microbiota ecology in patients of T2DM. The proportion of Firmicutes is extremely decreased while the Bacteroidetes are increasing. Lipopolysaccharide (LPS), as the constituent of the Gram negative bacteria (for example, the Bacteroidetes), is persistent generated in the gut by the constant reproduction and decomposition of bacteria. The human body absorbs the increased LPS from the gut leading to the higher plasma LPS levels. The continuous elevated LPS concentration in the gut exerts an inevitably damage to the function of L cells which scattered distributed the distal intestinal tract. The gene encoding GLP-1is known as the proglucagon gene (gcg gene) was separated from rodent and human in the1980s. It was found that the promoter G1and enhancer G2-G5, which are located in the gcg gene promoter region, have a significant regulate effect on the gcg gene transcription. The key effector to the canonical Wnt signaling pathway is the transcription factor β-catenin, which bind to one of the four members of the T cell transcription factor family (TCF-1, LEF-1, TCF-3and TCF7L2) to form the bipartite transcription factor β-catenin/TCF. TCF7L2being the major partner of P-catenin in the intestinal epithelia. In the absence of Wnt, the cellular concentration of free P-catenin is tightly controlled by a "destructive complex" via the proteasome-mediated degradation process. With the existence of Wnt, the P-catenin accumulates and enters the nucleus to form the bipartite transcription factor β-catenin/TCF, then triggers the downstream target genes. For the past few years, many examinations have shown that the Wnt pathway is involved in the regulation of gcg gene expression of intestinal L cells. The bipartite transcription factor β-catenin/TCF bind with subsequence of gcg gene enhancer G2, thereby regulate gcg gene transcription.Once LPS bind to their ligand, they promote the production of the intracellular reactive oxygen species (ROS) via the NADPH oxidase pathway. Even the little elevation of ROS level can stimulate FOXOs transcription factor family and augment the transcriptional activity. In2005, Essers et al. found that FOXOs proteins can also bind to P-catenin in an evolutionarily conserved way which is up-regulated by the existence of oxidative stress. It is confirmed that FOXO competes with TCF for interaction with P-catenin, thereby inhibiting TCF transcriptional activity in the colon carcinoma cells and Bone mesenchymal stem cells. Our study is aim to investigate whether LPS inhibit the regulational effects of P-catenin/TCF on gcg gene expression in L cells through the interaction of p-catenin with FOXOs via the stimulated increased ROS level.Therefore, this topic focus on the enteroendocrine L cell line GLUTag cells, and discuss from the following aspects:1.We observe the impact of LPS on ROS production and gcg mRNA expression and discuss the possible mechanisms.2. We discuss whether LPS exerts the effects on gcg mRNA expression via FOXO4gene, and whether LPS regulates gcg gene transcription through the Wnt signaling pathway and the underlying mechanisms. In order to further clarify the molecular mechanism of decreased GLP-1secretion in vivo and provide new experimental basis to explore the new therapeutic targets in T2DM.Part I The effects of lipopolysaccharide on gcg mRNA expression of GLUTag cellsObjective:To study the effects of lipopolysaccharide on gcg mRNA expression of GLUTag cells and the possible mechanisms.Method:(1) The culture of GLUTag cells:GLUTag cells were cultured with DMEM (low-glucose) containing10%fetal bovine serum in37℃,5%CO2incubator. Medium was changed every two days.(2) The effects of various concentrations of LPS on ROS levels of GLUTag cells: GLUTag cells were cultured in6-well plates, pretreatment by serum-free DMEM (low-glucose), cultured in37℃,5%CO2incubator for6h, made the cells in a synchronization state. The cells were divided into4groups:the blank control group (to abbreviate:ctrl group), which were cultured in DMEM (low-glucose) and without LPS; the lOng/ml LPS group (to abbreviate:lOng/ml group), cultured in DMEM (low-glucose) with10ng/ml LPS; the100ng/ml LPS group(to abbreviate:100ng/ml group),cultured in DMEM (low-glucose) with100ng/ml LPS; the500ng/ml LPS group (to abbreviate:500ng/ml group),cultured in DMEM (low-glucose) with500ng/ml LPS. The cells were cultured in37℃,5%CO2conditions for24hours, and then collected cells and labeled with DHE probe. Detect ROS levels of the cells by immunofluorescence and flow cytometry, respectively.(3) The effects of various concentrations of LPS on gcg mRNA expression of GLUTag cells:The cells were divided into4groups and cultured as above, detect gcg mRNA expression of the cells by realtime fluorescence quantitative PCR.(4) The effects of NADPH oxidase inhibitor apocynin on ROS levels and gcg mRNA expression of GLUTag cells:The cells were cultured in6-well plates, pretreated by serum-free DMEM (low-glucose), cultured in37℃,5%CO2incubator for6h, made the cells in a synchronization state. The cells were divided into4groups:the blank control group; the apocynin group:the cells treated with600μM apocynin for1hour; the100ng/ml LPS group; the100ng/ml LPS with apocynin group:the cells were pretreated with600μM apocynin for1hour, then treated with LPS for24hours. Detect ROS levels of the cells by flow cytometry and gcg mRNA expression by real-time fluorescence quantitative PCR.Results:(1) The effects of various concentrations of LPS on ROS levels of GLUTag cells:the blank control group and the10ng/ml LPS group both showed a weak red fluorescence intensity, and the100ng/ml LPS group showed an enhanced red fluorescence intensity. While the500ng/ml LPS group showed the strongest red fluorescence intensity among these groups. As for the flow cytometry results, he fluorescence intensity of the control group was quite low (31.00±4.65), the ROS levels of the100ng/ml and500ng/ml groups were obviously higher, getting to52.17±4.09and67.13±4.46respectively (P<0.001and P<0.001). The ROS level of the10ng/ml group was mildly rised (32.47±2.77), but had no significant difference compared with the control group (P=0.670). The ROS levels of GLUTag cells were significantly different among the four groups (F=54.127, P<0.001).(2) The effects of different concentrations of LPS on gcg mRNA expression of GLUTag cells:the2-△△Ct value of the control group was0.99±0.11, and0.98±0.11for the10ng/ml group. There were no significance between the two groups. The2-△△Ct value of the100ng/ml and500ng/ml groups were0.49±0.07and0.24±0.03respectively, much more lower than the control group. There were significantly difference among the four groups (F=113.354, P<0.001).(3) The effects of NADPH oxidase inhibitor apocynin on ROS levels and gcg mRNA expression of GLUTag cells:The ROS level of apocynin pretreated group was decreased from52.17±4.09to37.7±5.8, compared with the LPS group without apocynin.A significant difference was existed (P=0.024). Meanwhile, the expression level of gcg mRNA was increased from0.49±0.07to0.88±0.06, the difference was significant (P<0.001).Conclusions:The ROS levels of GLUTag cells were increased along with the concentration of LPS, while the gcg mRNA expression were homologously decreased. Indicating that the ROS production stimulated by LPS may be related to the decreased gcg mRNA expression of the enteroendocrine L cells.Part II The related mechanisms of LPS inhibit gcg mRNA expression of the GLUTag cellsObjective:To investigate the effects of transcription factor FOXO4on gcg mRNA expression of GLUTag cells and then reveal the related mechanisms of LPS inhibit gcg mRNA expression of the GLUTag cells.Method:(1) The effects of FOXO4gene on gcg mRNA expression of GLUTag cells:the GLUTag cells were cultured in24-well plates, pretreatment by serum-free DMEM (low-glucose), cultured in37℃,5%CO2incubator for6h, made the cells in a synchronization state. The cells were divided into4groups:the blank control group (to abbreviate:the ctrl group), which were cultured in DMEM (low-glucose) and without LPS for24hours; the100ng/ml LPS group(to abbreviate:the LPS group), cultured in DMEM (low-glucose) with100ng/ml LPS for24hours; the shFOXO4overexpression plasmid transfection group (to abbreviate:the shFOXO4group), the cells were transfected with shFOXO4overexpression plasmid for24hours, then cultured in DMEM (low-glucose) without LPS for another24hours; the siRNA-FOXO4small interfering RNA transfection group (to abbreviate:the si-FOXO4group), the cells were transfected with siRNA-FOXO4small interfering RNA for24hours, then cultured in DMEM (low-glucose) with100ng/ml LPS for another24hours. Detect gcg mRNA expression of the GLUTag cells by real-time fluorescence quantitative PCR.(2) The effects of different concentrations of LPS on the activity of the bipartite transcription factor p-catenin/T cell transcription factor:the GLUTag cells were cultured in24-well plates, pretreated by serum-free DMEM (low-glucose), cultured in37℃,5%CO2incubator for6h, made the cells in a synchronization state. The cells were divided into4groups:the blank control group (to abbreviate:the Ctrl group); the100ng/ml LPS group (to abbreviate:100ng/ml group); the50ng/ml of recombinant protein Wnt3a group (to abbreviate:the Wnt3a group); the group containing50ng/ml Wnt3a and100ng/ml LPS(to abbreviate:the100ng/ml group). Detect the activity of the transcription factor P-catenin/TCF by the dual luciferase reporter gene.(3) The effects of FOXO4on the activity of the bipartite transcription factor β-catenin/TCF:The GLUTag cells were cultured in24-well plates, pretreatment by serum-free DMEM (low-glucose), cultured in37℃,5%CO2incubator for6h, made the cells in a synchronization state. The cells were transfected with pTOPFLASH luciferase reporter gene and the shFOXO4overexpression plasmid or si-RNA. The cells were divided into4groups:the blank control group (to abbreviate:the Ctrl group); the group containing100ng/ml LPS (to abbreviate:the LPS group); the shFOXO4overexpression plasmid transfection group (to abbreviate:the shFOXO4group); the siRNA-FOXO4small interfering RNA transfection group (to abbreviate: the si-FOXO4group). These groups were detected the activity of the transcription factor β-catenin/TCF by the dual luciferase reporter gene with or without the recombinant protein Wnt3a, respectively.(4)The effects of LPS on the formation of FOXO4/β-catenin and TCF7L2/β-catenin of the GLUTag cells:The GLUTag cells were divided into4groups:the blank control group (to abbreviate:the ctrl group);the50ng/ml of recombinant protein Wnt3a group(to abbreviate:the Wnt3a group); the100ng/ml LPS group (to abbreviate:100ng/ml group); the group containing50ng/ml Wnt3a and100ng/ml LPS (to abbreviate:the Wnt3a+LPS group). Using the protein immune co-precipitation assay (Co-IP) to detect the level of β-catenin expression from the proteins precipitated by the anti-FOXO4and anti-TCF7L2antibody.Results:(1)The effects of FOXO4gene on gcg mRNA expression of GLUTag cells:the level of gcg mRNA expression of GLUTag cells was0.49±0.07, had a significant difference compared with the control group (0.99±0.11)(P<0.001). The level of gcg mRNA expression was decreased to0.41±0.05after cells transfected with shFOXO4overexpression plasmid (P<0.001).While the si-FOXO4group increased to0.80±0.08and had a significant difference compared with the LPS group (P<0.001). There were significant difference among the four groups (F=72.180, P<0.001).(2) The effects of different concentration of LPS on the activity of the bipartite transcription factor β-catenin/T cell transcription factor (β-catenin/TCF):there were interactions between LPS group and Wnt3a group by two-way factorial ANOVA on relative luciferase unit (RLU) value (F=81.389,P<0.001).The value of RLU of the LPS group (0.17±0.01)was evidently lower than the control group (0.26±0.01)(P<0.001). Treated GLUTag cells with Wnt3a for24h followed by LPS treated for another24h, the RLU value was up to0.47±0.03compared with the Wnt3a group (0.74±0.01)(P<0.001).(3) The effects of FOXO4on the activity of the bipartite transcription factor β-catenin/TCF:The value of RLU were significantly different among the groups (F=47.950, P<0.001). The value of RLU of shFOXO4group and LPS group were both declined to0.13±0.02and0.17±0.01when compared with the control group (0.26±0.01)(P<0.001and P<0.001) respectively. While the value of RLU of the siFOXO4group was increased from0.17±0.01to0.23±0.02when compared with the LPS group (P=0.001).In the existence of recombinant protein Wnt3a, the value of RLU were significantly different among the groups (F=145.072, P<0.001).RLU was decreased from0.74±0.01to0.38±0.03and0.47±0.03respectively by the group of shFOXO4and LPS (P<0.001和P<0.001).After transfection with si-FOXO4,the value of RLU of LPS group was increased to0.57±0.02(P<0.001).(4) The effects of LPS on the formation of FOXO4/β-catenin and TCF7L2/β-catenin of the GLUTag cells:The level of β-catenin expression from the proteins precipitated by the anti-FOXO4antibody using the Co-IP were analyzed by two-way factorial ANOVA. There were interactions between LPS group and Wnt3a group (F=47.365, P<0.001). When compared with the control group (1.00±0.04), the β-catenin binded to FOXO4were increased to1.68±0.07(P<0.001) of the LPS group. The β-catenin binded to FOXO4were raise to2.43±0.05of the Wnt3a+LPS group and had a significant difference compared with the Wnt3a group(1.30±0.06)(P<0.001).The level of β-catenin expression from the proteins precipitated by the anti-TCF7L2antibody using the Co-IP were analyzed by two-way factorial ANOVA. There were no interactions between LPS group and Wnt3a group (F=0.062, P=0.809). When compared with the control group (1.00±0.03), the p-catenin binded to TCF7L2were decreased to0.62±0.08(P=0.002) of the LPS group. The β-catenin binded to TCF7L2were decreased to1.37±0.10of the Wnt3a+LPS group and had a significant difference compared with the Wnt3a group(1.77±0.11)(P=0.009).Conclusion:LPS may interfere in the activity of the bipartite transcription factor β-catenin/TCF by FOXO4. The mechanisms underlying was that FOXO4and TCF7L2compete for a limited pool of P-catenin, thus attenuate the activities of Wnt signaling pathway, ultimately inhibit the expression of gcg mRNA. |
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