MiR-425-5p Regulates Glucagon-like Peptide-1 Production and Affects Blood Glucose Levels in High-fat Diet-fed Mice

doi:10.21203/rs.3.rs-948391/v1

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MiR-425-5p Regulates Glucagon-like Peptide-1 Production and Affects Blood Glucose Levels in High-fat Diet-fed Mice

https://doi.org/10.21203/rs.3.rs-948391/v1

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Background

In modern society, obesity has become a global problem with resulting in metabolic disorders and poses high risk for type 2 diabetes mellitus (T2DM). The glucagon-like peptide-1 (GLP-1) has been taken as an effective drug for the therapy of T2DM and obesity. In the present study, the regulatory roles and molecular mechanisms of miR-425-5p in GLP-1 secretion in high-fat diet (HFD)-induced diabetic mice were explored.

Methods

Oral glucose tolerance test and insulin tolerance test were performed to assess glucose metabolism and GLP-1 and LPS levels. Quantitative real time polymerase chain reaction (qRT-PCR) was employed to detect the expression of LPS, GLP-1, GLP-1 receptors, miR-425-5p, phosphatase and tensin homology (PTEN), proglucagon, p65 and β-catenin. Western blot was performed to determine the expression of proglucagon, p65, β-catenin and PTEN.

Results

The results showed that plasma GLP-1 level was negatively correlated with plasma LPS level in HFD-fed mice, and miR-425-5p expression and LPS level were up-regulated in the ileal fluid compared with control groups. LPS injection boosted miR-425-5p expression in ileum. MiR-425-5p ameliorated glucose intolerance and insulin resistance in HFD-fed mice by increasing GLP-1 secretion. Furthermore, p65 protein level in the cytoplasmic and nuclear in the ileum of HFD-fed mice was increased compared with the control group. MiR-425-5p agomir elevated nuclear β-catenin protein level, but reduced PTEN protein level in HFD-fed mice compared with HFD-fed mice treated with the miR-425-5p antagomir.

Conclusions

Our results suggest that miR-425-5p promotes GLP-1 secretion and improves glucose tolerance and insulin resistance in high-fat diet-fed mice.

Nutrition & Dietetics

Endocrinology & Metabolism

high-fat diet

LPS

glucagon-like peptide-1

miR-425-5p

NF-κB

PTEN/β-catenin

In modern society, obesity has become a global problem with resulting in hyperinsulinemia and impairing glucose tolerance, and posed high risk for type 2 diabetes mellitus (T2DM)[1, 2]. After a long-term of treatment for obesity, it seems to be indispensable to use pharmaceutical, except for the diet control and life style modification. Glucagon-like peptide-1 (GLP-1) is secreted by the intestinal L cells and devotes to curbing appetite and reducing insulin resistance, and thus controls blood sugar levels and body mass[3]. Therefore, GLP-1 has been taken as an effective drug for the treatment of T2DM and obesity[4]. GLP-1 is encoded by proglucagon gene, and proglucagon has been proven to promote the secretion of GLP-1 and ameliorate metabolic disorders in the high-fat diet (HFD)-induced diabetic mice[5]. Hence, researching the mechanism of endogenous GLP-1 secretion may provide a novel insight for the prevention and treatment of diabetes and other metabolic diseases caused by HFD.

Lipopolysaccharide (LPS), as a component of the cell wall of gram-negative bacteria, is produced by gut microbiota[6]. In HFD-fed mice, the LPS secretion, the number of intestinal gram-negative bacteria, and serum LPS levels were also increased[7], suggesting that the imbalance of gut microbiota was closely linked to HFD-induced LPS production. LPS is an inducer for the apoptosis of intestinal L cells, which has been demonstrated in our previous study [8], and LPS is also indicated to regulate the secretion of GLP-1[9]. In the present study, our data showed that LPS level was up-regulated and GLP-1 level was down-regulated in diabetic mice. In addition, gut microbiota alterations increased the level of GLP-1, but decreased the level of LPS [10], implying that GLP-1 was related to LPS.

MicroRNAs (miRNAs) are a novel class of non-coding RNAs containing 21-23 nucleotides and effectively regulate eukaryotic gene expression at the post-transcriptional level[11]. Kwon et al.[12] demonstrated that miR-425-5p was closely associated with the dysregulation of insulin/phosphatidylinositol-3-kinase (PI3K)-AKT signaling in HFD-fed mice. Ma et al.[13] reported that IL-1β-induced NF-κB activation enhanced miR-425-5p gene transcription and miR-425-5p contributed to gastric cancer progression by targeting phosphatase and tensin homology (PTEN) gene. We observed that miR-425-5p expression was up-regulated in the ileum of HFD-fed mice in our preliminary study. Therefore, our study explored the regulatory roles of miR-425-5p in GLP-1 secretion in HFD-fed mice, and assessed its potential as a promising therapeutic target for diabetes.

Materials

Male C57BL/6 mice (6-8 weeks) were purchased from the Experimental Animal Centre of Hubei Province and were housed in a pathogen-free room [14-17]. All the mice were randomly divided into two groups (control group and HFD groups). Mice in control group were fed with a rodent normal diet (10 % kcal from fat, D12450B, Research Diets) and in HFD group was fed with HFD containing 60% fat (D12492, Research Diets) for 12 weeks, respectively. The weight of mice fed with HFD was significantly increased and subsequent analysis was performed. 15 mice were included in control group and HFD group, respectively. The experiments were performed at 4, 8 and 12 weeks after high fat feeding. Five mice from each group were sacrificed at each point. All animal studies were approved by the ethical committee of the First Affiliated Hospital of Zhengzhou University, and performed in accordance with the guidelines for the care and use of laboratory animals.

Oral glucose tolerance and insulin tolerance test

Oral glucose tolerance test (OGTT) was performed after the mice was fasted for 12 h with mice were injected glucose at a dose of 3 g/kg. Insulin tolerance test (ITT) was performed by fasting mice for 4 h, followed intraperitoneally injecting insulin at a dose of 0.75 U/kg. Blood was collected after 0, 15, 30, 60, 90, and 120 mins, and plasma glucose levels were measured using a glucometer (Glucotrend, Roche Diagnostics, Germany).

Plasma LPS and GLP-1 measurement

C57/BL6 mice were anaesthetized by intraperitoneal injection of pentobarbital (35mg/kg) after fasting for 12 h. Blood samples were obtained from orbital plexus and were collected in EDTA and DPP-IV inhibitor tubes. Blood samples were centrifugated at 3000 g for 15 minutes at 4℃ to collect plasma. Plasma GLP-1 levels were measured by using the GLP-1 ELISA kit (Solarbio, SEKM-0148). According to the manufacturers’ instructions, the diazo-coupled limulus amebocyte lysate (LAL) chromogenic endotoxin quantification kit (Thermo Scientific, USA) was used to quantify the plasma LPS. Blood samples for OGTT and ITT test were collected from tail vein. Get rid of the first drop of blood and put one drop of blood on the test strip of the blood glucose meter to measure blood glucose levels. The fat tissues were cut into 4-μm standard sections, and stained with hematoxylin and eosin (H&E). Under an optical microscope, the adipocyte area was analyzed by ImageJ software (National Institutes of Health, USA).

LPS determination in colonic fluid

In all, 100 mg colonic fluid was collected from each mouse, diluted using 50 mL PBS, sonicated for 1 h on ice, and then centrifuged at 400 g for 10 mins. Supernatant was collected, filtrated using 0.45- and 0.22-μm filters, diluted in distilled deionized water, and heated at 70°C for 10 mins. Fecal LPS concentration was assayed using the LAL chromogenic assay kit, according to the manufacturers’ instructions.

Gut microbiota analysis

Approximate 100 mg caecal content was used for determining gut microbiota by qRT-PCR. Briefly, DNA was isolated from the caecal content with the QIAmp^® DNA stool mini kit (Qiagen, USA). The obtained DNA was quantified using Infinite^® M200 Pro NanoQuant (Tecan Systems Inc., USA), and its integrity was evaluated by electrophoresis with a 0.8% agarose gel. qRT-PCR was conducted for quantifying the abundance of Firmicutes, Bacteroidetes, Enterobacteriaceae, Lactobacillus, and Bifidobacteria, with total bacteria used as an endogenous control.

RNA extraction and qRT-PCR analysis

Total RNA was extracted from the ileum using the Trizol reagent (Invitrogen, USA) and was reverse-transcribed to cDNA with a MiRcute miRNA first-strand cDNA synthesis kit (Tiangen Biotech, China). Total RNA was reverse-transcribed into cDNA by PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China). Next, real-time PCR was performed on 20 μL SYBR Green PCR Master Mix (TaKaRa). Relative expression of genes was determined using MiRcute miRNA qPCR detection kit (Tiangen Biotech) with an ABI 7500 real-time PCR system (Applied Biosystems, USA). Data was analyzed with 2−ΔΔCt method.

Western blot analysis

Total proteins were obtained by lysing ileum with radio immunoprecipitation assay (RIPA) buffer (Beyotime, China). Nuclear and cytoplasmic proteins were extracted by using NE-PER™ (Thermo Scientific™) according to the instruction. The isolated nuclear protein and cytoplasmic proteins were used to detect the expression of p56 and β-catenin by Western blot. And western blot analysis was performed as previously described [18]. Membranes were incubated with primary antibodies against proglucagon (1:500; Santa Cruz Biotechnology), PTEN (1:500; Santa Cruz Biotechnology), NF-κB p65 subunit (1:500; Santa Cruz Biotechnology), β-catenin (1:1000; BD Biosciences), β-actin (1:500; Sigma-Aldrich), and lamin B1 (1:1000; Abcam). After incubation with the horseradish peroxidase-conjugated secondary antibodies, proteins were visualized using enhanced chemiluminescence reagents (Thermo Scientific) and Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, USA).

The effect of LPS on miR-425-5p expression

HFD-fed mice were allocated into 3 groups (n=3, respectively): control, acute LPS injection, and continuous low dose of LPS injection. In the acute LPS injection group, mice were given intraperitoneal injections of LPS (1 mg/kg), and sacrificed 6 h later[19]. For the continuous injection of low dose of LPS, a mini-osmotic pump was subcutaneously implanted and linked to the peritoneal cavity by a catheter, which had infused LPS at dose of 300 ug/kg per day for 4 weeks in mice [20].

The biological function of miR-425-5p

C57BL/6 male mice were divided into 4 groups (n=5, respectively), control, HFD, HFD+miR-425-5p antagomir, and HFD+miR-425-5p agomir. Briefly, 8 weeks following HFD, HFD-fed mice were intracolonicaly treated with an empty vehicle or miR-425-5p antagomir/agomir (10 mg/kg) each week for consecutive 4 weeks by using a 3.5-cm long polyethylene cannula (Intramedic PE-20 tubing; Becton Dickinson, USA) attached to an insulin syringe that was introduced into the mouse colon up to ~3 cm from the anus. All the mice were sacrificed after 4 weeks. The fat, pancreas tissues and blood samples were collected for further analysis. The adipocyte area and islet area were measured. Moreover, the mice ileum was taken for determining the protein levels of proglucagon, PTEN, nuclear NF-κB p65, and nuclear β-catenin.

Statistical analysis

Data were analyzed using SPSS 22.0 and image J software, and were presented as mean ± standard deviation (SD). Data in two groups or more groups were compared using Student’s t-test or one-way ANOVA. Correlation analysis was performed with using the Pearson correlation analysis. P < 0.05 was considered statistically significant.

LPS level is negatively correlated with GLP-1 level in the plasma of HFD-fed mice

In this study, we first established a HFD-fed mouse model and some parameters were firstly detected. Compared with control group (n=15), the body weight of HFD-fed mice (n=15) was significantly increased compared with control group after 4 weeks (P < 0.01; Figure 1A), and adipocyte area in the HFD-fed mice was also notably larger than that in control group (P < 0.01; Figure 1B). Moreover, the plasma levels of triacylglycerol (TG), total cholesterol (TC), and glucose in HFD-fed mice were markedly up-regulated after 12 weeks as compared to control groups (P < 0.01; Figure 1C). All the above data claimed HFD-induced mice model was successfully established. Importantly, the HFD-fed mice showed the decrease of plasma GLP-1 levels and the increase of plasma LPS levels when compared with control group (P < 0.01 Figure 1D). Subsequently, a significantly negatively correlation was revealed between the plasma GLP-1 and LPS level in HFD-fed mice (r = -0.557, P < 0.05; Figure 1E). Meanwhile, the mRNA and protein expression levels of proglucagon in the ileum of HFD-fed mice were decreased compared with control group (P < 0.01; Figure 1F, Figure 1G). The mRNA levels of the LPS receptors including TLR2 and TLR4 in the ileum of HFD-fed mice was also increased (P < 0.01; Figure 1F). A clear inverse correlation between the expression of proglucagon and LPS (r = -0.651, P < 0.05; Figure 1H) was found in the ileum of HFD-fed mice.

LPS may upregulates miR-425-5p expression through NF-κB activation in HFD-fed mice

Elevated LPS levels are suggested to be mainly derived from intestinal gram-negative bacteria [7]. We next examined the fecal LPS levels and caecal microbiota. Fecal LPS levels were obviously higher in HFD-fed mice (n=15) than that in control group (n=15) (P < 0.01; Figure 2A). As expected, the relative amounts of Enterobacteriaceae (gram-negative) bacteria in HFD-fed mice was significantly more than that in the control groups (P < 0.01; Figure 2B). Subsequently, we demonstrated that the expression of miR-425-5p was noticeably upregulated in the ileum of HFD-fed mice (P < 0.01; Figure 2C).

We further examined the effect of LPS on the expression of miR-425-5p, HFD-fed mice (n=3 in each group) were injected with LPS with an acute or continuous method. As shown in Figure 3E (n=3 in each group), both acute LPS injection and continuous low dose of LPS injection boosted miR-425-5p expression in the ileum of HFD-fed mice (P < 0.05; Figure 2D), and acute LPS injection showed higher efficacy. It implied that LPS-induced miR-425-5p expression in vivo might be associated with injection dose and treatment time course.

A previous study showed that LPS induced NF-κB p65 phosphorylation and thereby activating NF-κB pathway [21]. Therefore, we tested the expression of NF-κB- p65. p65 protein level in the ileum of HFD-fed mice was increased when compared with control group in cytoplasm and nucleus (Figure 2E). These results indicated that LPS may increase miR-425-5p expression through activating NF-κB.

MiR-425-5p may up-regulate β-catenin by targeting PTEN to promote proglucagon production

The targeting effect of miR-425-5p on PTEN gene has been identified in human gastric cancer cells [13]. And the activation of Wnt/β-catenin pathway and subsequent accumulation of β-catenin in nucleus are common features of intestinal L cells[22]. Therefore, we tested the protein levels of proglucagon, PTEN and nuclear β-catenin in the ileum of mice, and the mice were divided into 4 groups (n=5, respectively): control, HFD, HFD+miR-425-5p agomir, and HFD+miR-425-5p antagomir. The expression of miR-425-5p in HFD mice was largely upregulated by miR-425-5p agomir but was reduced by miR-425-5p antagomir (P < 0.01; Figure 3A). Compared with HFD-fed mice treated with the miR-425-5p antagomir, MiR-425-5p agomir elevated proglucagon and nuclear β-catenin protein level, but reduced PTEN protein level in HFD-fed mice (P < 0.05; Figure 3B, Figure 3C). These results indicated that miR-425-5p may promote proglucagon expression by up-regulating β-catenin through PTEN.

MiR-425-5p may ameliorate glucose intolerance and insulin resistance in HFD-fed mice by increasing GLP-1 secretion

To verify the effect of miR-425-5p on GLP-1 level and glucose metabolism, the mice were also divided into 4 groups (n=5, respectively): control, HFD, HFD+miR-425-5p agomir and HFD+miR-425-5p antagomir. MiR-425-5p agomir decreased plasma glucose levels by detecting total AUC (P < 0.01; Figure 4A) and ITT (P < 0.05; Figure 4A) in HFD-fed mice, increased plasma GLP-1 level (P < 0.05; Figure 4B), reduced body weight (P < 0.05; Figure 4B), reduced adipocyte and islet area (P < 0.05 for all; Figure 4C) when compared with that in HFD+miR-425-5p antagomir. The levels of plasma TG, TC, and glucose were clearly reduced by miR-425-5p agomir (p < 0.05; Figure 5A), and LPS expression was also was dramatically decreased in feces and plasma, indicating miR-425-5p agomir relieved metabolic disorder in HFD-fed mice. These results indicated that miR-425-5p ameliorated glucose intolerance and insulin resistance in HFD-fed mice which might attribute to increase GLP-1 secretion.

Our previous study showed that LPS had been indicated to induce the apoptosis of intestinal L cells[8], and LPS regulates the secretion of GLP-1[9]. In the present study, we explored the regulatory mechanism of miR-425-5p in HFD-fed mice, elucidated its action in regulating the secretion of GLP-1 and emphasized its crucial role in affecting glucose metabolic disorders. We found that LPS may increase the level of miR-425-5p through activating NF-κB in HFD-fed mice, and miR-425-5p may further promote proglucagon transcription through PTEN/β-catenin axis and subsequently increase GLP-1 secretion, which contributed to the amelioration of the glucose metabolic disorders caused by HFD. In addition, miR-425-5p can improve insulin resistance and reduce fat mass, which may be related to increase GLP-1 secretion.

We observed that LPS injection in both acute and continuous low dose manner boosted miR-425-5p expression in HFD-fed mice, and acute LPS injection showed higher efficacy (Figure 2D). The results implied that LPS treatment increased miR-425-5p expression though LPS injection in different ways (i.p. dose and treatment duration). However, no significant difference in fecal LPS (P > 0.05; Figure 5B) and plasma LPS level (P > 0.05; Figure 5C) was found between miR-425-5p agomir and miR-425-5p antagomir group.

Emerging evidence indicated that LPS stimulation activated NF-κB by phosphorylating and degrading IκB [23], and the activation of NF-κB enhanced miR-425-5p gene transcription has been previously identified in human [13]. In the current study, NF-κB p65 protein level in both cytoplasma and nucleus were increased in the ilum of HFD-fed mice, and miR-425-5p expression was upregulated. However, nuclear NF-κB p65 ptotein level was significantly changed (P > 0.05; Figure 4B, Figure 4C) in miR-425-5p agomir and miR-425-5p antagomir groups. The transcriptional activation effect of NF-κB on miR-425-5p gene was firstly revealed in mice.

Subsequently, the regulatory mechanism of miR-425-5p on GLP-1 secretion was highlighted. Previously, the targeting effect of miR-425-5p on PTEN gene has been identified in human gastric cancer cells [13]. PTEN is a tumor suppressor gene and is often lost in various human diseases [24]. Loss of PTEN induced the nuclear localization and transcriptional activation of β-catenin [25], activation of the Wnt/β-catenin pathway and subsequent nuclear accumulation of β-catenin are common features of intestinal L cells [22]. In this study, we confirmed that PTEN protein levels were significantly decreased, but proglucagon and nuclear β-catenin protein levels were significantly increased in HFD-fed mice treated with the miR-425-5p agomir when compared with HFD-fed mice. We identified a novel signaling pathway that miR-425-5p negatively regulated PTEN expression in HFD-fed mice, which promoted the translocation of β-catenin into the nucleus. Moreover, the transcriptional activation of the proglucagon gene by β-catenin has been proven in GLUTag cells [26], which is pivotal for the biosynthesis and secretion of GLP-1. More significantly, we proposed a novel and critical pathway in GLP-1 secretion: LPS/miR-425-5p/proglucagon, which not only underlined the roles of miR-425-5p in modulating glucose metabolic disorders, but also focused on the influence of LPS on GLP-1 secretion in HFD-fed mice. Balakumar et al. [27] reported that male C57/BL6 mice fed with HFD (57% fat) for 6 months showed the increase of plasma LPS levels and the decrease of plasma GLP-1 levels. And Singh et al. [28] reported that Male Swiss albino mice fed with HFD (58% fat) for 12 weeks also showed the remarkable upregulation of plasma LPS levels and the decrease of ileum GLP-1 levels. Consistent with above studies, the male C57/BL6 mice in this study fed with HFD (D12492, Research Diets, 60% fat) for 12 weeks showed the significant increase of plasma LPS levels and the downregulation of plasma GLP-1 levels.

More significant, we proposed a possible pathway implicated in the GLP-1 secretion: LPS/miR-425-5p/proglucagon, which not only underlined the roles of miR-425-5p in modulating glucose metabolic disorders, but also focused on the influence of LPS on GLP-1 secretion in HFD-fed mice. It has been revealed that acute injection or continuous infusion of low dose LPS triggers GLP-1-mediated insulin secretion inC57/BL6 mice[19]. Furthermore, a previous study has reported that GLP-1 secretion is raidly increased after 90min of LPS (200ng/ml) stimulation in GLUTag cells[29], which is completely contradictory to our findings on LPS stimulation and GLP-1 secretion. However, in another study, stimulation with LPS (100ng/mL) for 15min did not increase GLP-1 secretion in GLUTag cells[30]. Moreover, Zhu et al. found that low dose of LPS (2 mg/kg) increased plasma GLP-1 levels in LDL receptor knockout mice after 6h [31]. Therefore, we attribute the completely opposite effect of LPS on GLP-1 secretion to the different doses and treatment durations.

In the present study, we observed that LPS regulated miR-525-5p expression, and miR-425-5p regulated β-catenin signaling, which was at least partially mediated by PTEN decline-induced phosphorylation of β-catenin which was the major effector of Wnt signaling that binds to the proglucagon gene promoter. Given the limitation of funding and the consideration of the welfare of the experimental animals, our major in vivo experiment were designed according to the general requirements (n>=5), further studies with large size or vitro models are needed to carry out on the contribution of miR-425-5p in maintaining metabolic homeostasis and the relationship between LPS and GLP-1 secretion. Results were showed in Figure 6.

In conclusion, LPS may increase the level of miR-425-5p by activating NF-κB. miR-425-5p may promote the translocation of β-catenin into the nucleus through negative regulation of PTEN expression. The nuclear localization of β-catenin can promote proglucagon transcription and further increase GLP-1 secretion, thereby improving glucose intolerance and insulin resistance in HFD-fed mice.

T2DM: Type 2 diabetes mellitus; GLP-1: Glucagon-like peptide-1; HFD: High-fat diet; qRT-PCR: Quantitative real time polymerase chain reaction; PTEN: Phosphatase and tensin homology; LPS: Lipopolysaccharide; miRNAs: MicroRNAs; PI3K: Phosphatidylinositol-3-kinase; OGTT: Oral glucose tolerance test; ITT: Insulin tolerance test; LAL: limulus amebocyte lysate; H&E: Hematoxylin and eosin; SD: standard deviation; TG: triacylglycerol; TC: total cholesterol;

Ethics approval and consent to participate

All animal studies were approved by the ethical committee of the First Affiliated Hospital of Zhengzhou University, and performed in accordance with the guidelines for the care and use of laboratory animals.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by grants from the Science and Technology Project of Henan Province (grant no. 192102310064), and the National Natural Science Foundation of China (grant no. 81500646).

Authors’ contributions

JW, LRW and XNZ designed the experiment; FG, FJH, YYZ, LNW and LYL contributed to the experiment and data; XJM, LLC, JL, LLJ and XY contributed to the analysis of the data. JW drafted the manuscript. LRW, XNZ and CZD revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank all the participants of this study. We also thank the First Affiliated Hospital of Zhengzhou University for its support.

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MiR-425-5p Regulates Glucagon-like Peptide-1 Production and Affects Blood Glucose Levels in High-fat Diet-fed Mice

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