Mol Cell Biochem DOI 10.1007/s11010-014-2170-8
MiR-199a is overexpressed in plasma of type 2 diabetes patients which contributes to type 2 diabetes by targeting GLUT4 Shuang-Tong Yan • Chun-Lin Li • Hui Tian Jian Li • Yu Pei • Yu Liu • Yan-Ping Gong • Fu-Sheng Fang • Ban-Ruo Sun
•
Received: 15 February 2014 / Accepted: 24 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Decreased GLUT4 expression and impaired GLUT4 cell membrane translocation are involved in type 2 diabetes mellitus (T2DM) pathogenesis so the factors impacting GLUT4 expression may be associated with T2DM. In this study, we identified four miRNAs: miR-31, miR-93, miR-146a, and miR-199a which suppress GLUT4 expression in HEK293T cells. Subsequently, we determined expression of these four miRNAs in plasma samples of T2DM patients, T2DM susceptible individuals, and healthy controls and found miR-199a was overexpressed in patients’ plasma compared with healthy control. Because the miR-199a binding site in GLUT4 30 UTR is highly conserved among vertebrates, we detected the glucose uptake in rat L6 myoblast cells through gain- and loss-offunction of miR-199a. We found that miR-199a can repress glucose uptake in L6 cells, which was rescued by GLUT4 overexpression. These results indicate that T2DM patients may have a high level miR-199a that reduce GLUT4 expression and contribute to the insulin resistance. Hence, miR-199a may be a novel biomarker for risk estimation and classification in T2DM patients. Keywords Type 2 diabetes mellitus GLUT4 MiRNA Glucose uptake Plasma
S.-T. Yan C.-L. Li (&) H. Tian J. Li Y. Pei Y. Liu Y.-P. Gong F.-S. Fang B.-R. Sun Department of Geriatric Endocrinology, PLA General Hospital, Beijing 100853, China e-mail: [email protected]
Introduction GLUT4 is an insulin-responsive facilitative glucose transporter, which is found mostly expressed in adipose, skeletal muscle, and cardiac muscle cells. Under conditions of low insulin, GLUT4 is sequestered in intracellular vesicles in muscle and fat cells. Insulin induces a rapid increase in the uptake of glucose by inducing the translocation of GLUT4 from these vesicles to the plasma membrane. As the vesicles fuse with the plasma membrane, GLUT4 transporters are inserted and become available for transporting glucose, and glucose absorption increases. GLUT4 expression levels are correlated with whole-body insulin-mediated glucose homeostasis, and it is has been shown that the dysregulation of GLUT4 expression is related to diabetes and obesity. GLUT4 heterozygous mutant mice (GLUT4±) have increased serum glucose and insulin, reduced muscle glucose uptake, hypertension, and diabetic histopathologies in the heart and liver [1]. Overexpression of GLUT4 can partially improve the in vivo glucose tolerance in diabetes mice indicated the importance of GLUT4 in the process of diabetes [2]. MicroRNAs (miRNAs) are short non-coding RNAs which modulate gene expression by binding to complementary segments present in the 30 UTR of the mRNAs of protein coding genes. MiRNAs play very important roles in maintaining normal human body physiology conditions, and abnormal miRNA expressions have been found related to many human diseases spanning from psychiatric disorders to malignant cancers [3–5]. Following the discovery that miRNAs were involved in many relevant biological processes, including energy and fat metabolism, these molecules have received growing attention in the fields of diabetes and obesity research. Recently, two research groups highlighted the presence of miRNAs in plasma [6, 7]. These plasma miRNAs are not
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cell associated, but packaged in microvesicles or bound by AGO proteins that protect them from endogenous RNase activity. Interestingly, plasma miRNAs can display unique expression profiles: specific tumor miRNAs were identified in cancer patients, whereas tissue derived miRNAs constitute a marker for injury [8–10]. Anomalous miRNAs expression was also was found in the plasma of patients with diabetes which was highly associated with diabetes and diabetic complications [11]. Hence, the objective of the current study was to determine if miRNA regulate GLUT4 expression and whether such regulation contributes to the disease pathogenesis in type 2 diabetes mellitus (T2DM) patients.
Materials and methods Dual-luciferase assay Full length GLUT4 30 UTR (1,419 bp) was cloned downstream of firefly luciferase coding region in pGL3 vector (Promega, Madison, WI, USA) to generate luciferase reporter vector. For luciferase reporter assays, HEK293T cells were seeded in 48-well plates. MiRNA mimics and luciferase reporter vectors were co-transfected using Lipofectamine 2000 (Life Technologies, Carlsbad, CA USA). pRL-TK vector containing Renilla luciferase was co-transfected for data normalization. Two days later, cells were harvested and assayed with the Dual-Luciferase Assay kit (Promega, Madison, WI USA). Each treatment was performed in triplicate in three independent experiments. The results were expressed as relative luciferase activity (Firefly LUC/Renilla LUC). Western blotting Protein extracts were boiled in SDS/b-mercaptoethanol sample buffer, and 20 lg of samples was loaded into each lane of 10 % polyacrylamide gels. The proteins were separated by electrophoresis, and the proteins in the gels were blotted onto PVDF membranes (Amersham Pharmacia Biotech, St. Albans, Herts, UK) by electrophoretic transfer. The membrane was incubated with mouse antiGLUT4 monoclonal antibody (Abcam, Cambridge, MA, USA) (1:1,000 dilution) for 1 h at 37 °C. The specific protein-antibody complex was detected using horseradish peroxidase-conjugated rabbit anti-mouse IgG. Detection by the chemiluminescence reaction was carried using the ECL kit (Pierce, Appleton, WI, USA). The blots were stripped and re-probed with mouse anti-b-actin monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) (1:5,000 dilution) to confirm equal loading.
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Participants One hundred ninety-two Han Chinese individuals, aged 46–62 years, receiving routine physical exams were recruited by the Geriatric Endocrinology Department of PLA General Hospital. The study was approved by the Institutional Review Board of PLA General Hospital and enrolled patients provided informed written consent. They enrolled subjects were divided into three study groups (64 subjects/group): normal individuals [fasting glucose (FG), 4.8–5.2 mmol/L], T2DM susceptible individuals (FG, 6.1–6.9 mmol/L), and diagnosed T2DM patients (FG C 7.0 mmol/L). Individuals in the normal and susceptible groups with the following conditions were excluded from the study: common diabetic complications such as retinopathy, nephropathy, and cardiovascular disorders since it is not known whether they might have latent effect on miRNA expression. Individuals who had previously been diagnosed with DM or had any history of medication for 6 months prior to the study were also excluded. T2DM was diagnosed based on the combination of several parameters: FG level higher than 7.0 mmol/L and 2 h plasma glucose (PG) higher than 11.1 mmol/L in oral glucose tolerance test (OGTT), or diagnosed by other hospitals. RNA isolation and RT-qPCR Peripheral blood was collected via venipuncture into tubes containing sodium EDTA, centrifuged at 1,000 g for 5 min, and the plasma was carefully transferred into RNase-free tubes and stored at -80 °C until use. Total RNA containing miRNAs was isolated from plasma using Trizol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The expression level of miR-31, miR-93, miR-199a, and miR-146a was detected by TaqMan miRNA RT-Real Time PCR. Single-stranded cDNA was synthesized using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and then amplified using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) together with miRNA-specific TaqMan MGB probes: miR31, miR-93, miR-146a, and miR-199a (Applied Biosystems, Foster City, CA, USA). The miR-16 was used for normalization. Each sample in each group was measured in triplicate and the experiment was repeated at least three times for the detection of each miRNA. Cell culture HEK293T cells with high GLUT4 expression, was cultured in Dulbecco modified Eagle’s medium, supplemented with 10 % Fetal bovine Serum (FBS), not essential amino acids
Mol Cell Biochem Fig. 1 MiR-31, miR-93, miR146a and miR-199a repress the GLUT4 expression by targeting 30 UTR. a Schematic diagram for constructing the full length of GLUT4 30 UTR into pGL3 control vector. b Screening the miRNAs directly target GLUT4 30 UTR. pGL3-GLUT4 cotransfected with candidate miRNAs separately, for dualluciferase assay. PRL-TK containing renilla luciferase was co-transfected with 30 -UTR of GLUT4 for data normalization. *P \ 0.05; **P \ 0.01. c Western blot was used to detect GLUT4 expression in HEK293T cells transfected with miR-31, miR-93, miR-146a, and miR-199a
and antibiotics (Penicillin 100 IU/ml, Streptomycin 100 lg/ml). Rat L6 muscle cells were grown in a-MEM supplemented with 10 % FBS 100 units/mL penicillin, 100 lg/mL streptomycin.
Results
Glucose uptake measurement
MiRNAs modulate gene expression by binding to complementary segments present in the 30 UTR of the mRNAs of protein coding genes, and it is predicted that more than 60 % human genes are regulated by miRNA. To investigate which miRNA repress GLUT4 expression and play important roles in T2DM, we first predicted the miRNAs that may repress GLUT4 expression using online bioinformatics tools. We subsequently cloned full length GLUT4 30 UTR downstream of the firefly luciferase reporter gene in the pGL3 control vector to validate whether GLUT4 expression is repressed by the predicted candidate miRNAs (Fig. 1a). HEK293T cells were co-transfected with pGL3-GLUT4, indicated miRNA mimics, and pRL-TK. Nonsense scramble short nucleotide was used as control. As shown in Fig. 1b, miR-31, miR-93, miR-146a, and miR-199a suppressed luciferase activity significantly. These results indicate that miR-31, miR-93, miR146a, and miR-199a target the 30 -UTR of GLUT4, leading to the change of firefly luciferase translation. HEK293T cells were transfected with the four candidate miRNAs separately, and the expression of GLUT4 was detected by western blotting. Compared with corresponding control, the expression of
To measure glucose uptake in rat L6 cells, a nonradioactive assay was performed using a fluorescent deoxyglucose analog, 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4yl) amino)-2-deoxyglucose, Invitrogen, Carlsbad, USA). Briefly, the cells plated on a 96-well plate at a density of 5,000 cells/well were serum depleted for 24 h. Next, the cells were incubated with the indicated drugs for 10 min, and 100 lM 2-NBDG was administrated to each well. After 10 min incubation, the cells were washed with PBS two times, and fluorescence was measured using a microplate reader (ARVOsx; PerkinElmer). Statistical analysis Data were analyzed using SPSS Statistical Package version 16. Independent two group’s analyses are used t test. Statistical analysis for plasma miRNAs expression was performed by one way ANOVA. P \ 0.05 was considered statistically significant.
The expression of GLUT4 was repressed by four miRNAs in HEK293T cells
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Mol Cell Biochem Fig. 2 Mutation analysis identify the miRNAs binding sites. Full length of GLUT4 30 UTR with a three nucleotides mutation in predicted miR-93 binding site b four nucleotides mutation in predicted miR-31 binding site c three nucleotides mutation in predicted miR-199a binding site, and d three nucleotides mutation in predicted miR-146a binding site are cloned into pGL3-control vector for dual-luciferase assay. PRL-TK containing renilla luciferase was co-transfected with 30 -UTR of GLUT4 for data normalization. *P \ 0.05
GLUT4 was significantly suppressed by miR-31, miR-93, miR-146a, and miR-199a (Fig. 1c). Seed sequence mutation clones were also used to further confirm the binding site for miRNAs (Fig. 2). Putative miRNAs binding regions in the 30 -UTR of GLUT4 with 3 or 4 mutant nucleotides (designated as pGL3-GLUT4-Mu) and pGL3-GLUT4-Wt were used as controls, respectively. The histogram in Fig. 2 shows that the enzyme activities were significantly reduced in cells transfected with miR31/miR-93/miR-146a/miR-199a with pGL3-GLUT4-Wt vector compared with relevant pGL3-GLUT4-Mu vectors. These data cumulatively indicate that miR-31, miR-93, miR-146a, and miR-199a may suppress GLUT4 expression by binding to seed sequence in the 30 -UTR of GLUT4. Only miR-199a was up-regulated in T2DM patients’ plasma It is generally accepted that type 2 diabetes is associated with an impaired insulin-stimulated glucose disposal rate, which has been attributed to insulin resistance in skeletal muscle. GLUT4 is primarily expressed in adipose tissues and striated muscle (skeletal and cardiac) that is in turn responsible for
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insulin-regulated glucose transport into the cell and the dysregulation of GLUT4 was found related to T2DM. To investigate whether there are some relations between miRNAs which repress GLUT4 expression, we determined the expression of candidate miRNAs in the plasma of T2DM patients. As no consensus on the use of internal normalization control in plasma was defined for miRNA qPCR, the expression of miR-16, which have been proposed as the internal normalization control, was evaluated in the samples of all participants. Our data demonstrated that miR-16 was detected with high abundance and steadily presented in the plasma of these three groups participants (Fig. 3a). No significant difference was observed in terms of Ct values of miR-16 (P = 0.87). Therefore, miR-16 was selected as normalization control in this experiment. As shown in Fig. 3b, among the four candidate miRNAs, only miR-199a was overexpressed in T2DM patients compared with healthy control (P = 0.037). MiR-199a overexpression reduced the glucose uptake in L6 cells To examine the impact of miR-199a overexpression on glucose uptake in muscle cells, we used the fluorescent glucose
Mol Cell Biochem Fig. 3 Plasma miRNA detection. Box and whiskers plot presented the relative expression of four candidate miRNAs in the plasma samples. Expression levels of the miRNAs were normalized to miR-16. The lines inside the boxes denote the medians. The boxes mark the interval between the 10th and 75th percentiles. Statistical significance was determined using an unpaired t test. *P \ 0.05
analog 2-NBDG. We did the sequence alignment first and found that miR-199a binding site in GLUT4 30 UTR is conserved in human, mouse and rat (Fig. 4a). MiR-199a mimic or miRNA control or miR-199a inhibitor or miRNA inhibitor control was transfected into L6 myoblast cells separately. As shown in Fig. 4b, the expression of GLUT4 was repressed by miR-199a mimic and raised by miR-199a inhibitor meanwhile there was a significantly decrease in 2-NBDG uptake when miR-199a overexpressed and a slightly increase when miR-199a knock down indicates that miR-199a regulate the cell glucose uptake by targeting GLUT4 expression. To confirm the results above, we co-transfected L6 cells with miR199a and a GLUT4 expression vector which not contains the 30 UTR sequence. As shown in Fig. 4c, the overexpressed GLUT4 increase the 2-NBDG uptake which rescued the effect of miR-199a overexpression.
Discussion As a complex metabolic disorder, diabetes mellitus is characterized by chronic hyperglycemia due to defects
in insulin secretion or insulin resistance. It is generally accepted that type 2 diabetes is associated with an impaired insulin-stimulated glucose disposal rate, which has been attributed to insulin resistance in skeletal muscle. Glucose transport across the cell membrane of skeletal muscle is mediated by the glucose transporter proteins GLUT1 and GLUT4 [12]. The GLUT1 glucose transporter isoform is believed to support basal glucose transport, whereas the GLUT4 isoform increases glucose transport in response to insulin and contraction [13, 14]. In this study, we first identified that miR-31, miR-93, miR-146a, and miR-199a repress GLUT4 expression by directly targeting GLUT4 30 UTR in HEK293T cells. Subsequently, to investigate whether there are some relations between those four miRNAs and T2DM pathogenesis, we detected the expression of those four miRNA in the plasma samples of T2DM patients. Our results indicated that miR199a expression was up-regulated significantly compared with healthy control suggest that T2DM patients may have a high level miR-199a that reduce GLUT4 expression and attribute to the insulin resistance.
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Mol Cell Biochem Fig. 4 MiR-199a overexpression decrease glucose uptake of L6 cells. a The sequence of three species GLUT4 30 UTR were aligned by Clustal X. The miR-199a binding site was marked by red box. b Western blot was used to detect endogenous GLUT4 expression in L6 cells transfected with miR-199a mimic or inhibitor. Glucose uptake was measured using 2-NBDG. c Western blot was used to detect GLUT4 expression in L6 cells cotransfected with miR-199a mimic and GLUT4 expression vector. Glucose uptake was measured using 2-NBDG. *P \ 0.05. (Color figure online)
During the 1990s, a large number of studies have searched for the underlying causes of insulin resistance in skeletal muscle. The speculation that decreased GLUT4 expression could contribute to insulin resistance has been rejected by most studies [15–17], except for some studies of the translocation of GLUT4 by insulin or contractions, in which impaired translocation in type 2 diabetic muscle was found [18, 19]. Michael Gaster et al. found GLUT4 was down-regulated in slow muscle fibers of T2DM patients using immunohistochemistry give the first evidence of low GLUT4 level are related to T2DM. In this study, we only found miR-199a overexpressed in patients’ plasma which cannot represent the condition in the muscle cells, further study should be carried out to quantify and position the miR-199a in muscle tissues of T2DM patients. In summary, we first identified miR-31, miR-93, miR146a, and miR-199a repressed GLUT4 expression by targeting the 30 UTR of GLUT4 directly. We found miR-199a overexpressed in T2DM patients’ plasma. Subsequently, we confirmed miR-199a overexpression can also repressed GLUT4 expression in rat L6 myoblasts and decreased the glucose uptake which can be rescued by GLUT4 overexpression. Our results suggest a relation between up-regulated miR-199a and T2DM, which may ultimately lead to
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novel biomarkers for risk estimation and classification and could be exploited for miRNA-based therapeutic interventions of vascular complications associated with this disease.
References 1. Stenbit AE, Tsao TS, Li J et al (1997) GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes. Nat Med 3:1096–1101 2. Brozinick JT Jr, McCoid SC, Reynolds TH et al (2001) GLUT4 overexpression in db/db mice dose-dependently ameliorates diabetes but is not a lifelong cure. Diabetes 50:593–600 3. Maes OC, Chertkow HM, Wang E et al (2009) MicroRNA: implications for Alzheimer disease and other human CNS disorders. Curr Genomics 10:154–168 4. Xu J, Li Y, Wang F et al (2013) Suppressed miR-424 expression via upregulation of target gene Chk1 contributes to the progression of cervical cancer. Oncogene 32:976–987 5. Farazi TA, Hoell JI, Morozov P et al (2013) MicroRNAs in human cancer. Adv Exp Med Biol 774:1–20 6. Mitchell PS, Parkin RK, Kroh EM et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105:10513–10518 7. Arroyo JD, Chevillet JR, Kroh EM et al (2011) Argonaute2 complexes carry a population of circulating microRNAs
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independent of vesicles in human plasma. Proc Natl Acad Sci USA 108:5003–5008 Tanaka M, Oikawa K, Takanashi M et al (2009) Down-regulation of miR-92 in human plasma is a novel marker for acute leukemia patients. PLoS One 4:e5532 Laterza OF, Lim L, Garrett-Engele PW et al (2009) Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury. Clin Chem 55:1977–1983 Wang K, Zhang S, Marzolf B et al (2009) Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci USA 106:4402–4407 Kantharidis P, Wang B, Carew RM, Lan HY (2011) Diabetes complications: the microRNA perspective. Diabetes 60:1832–1837 Bell GI, Kayano T, Buse JB et al (1990) Molecular biology of mammalian glucose transporters. Diabetes Care 13:198–208 Marshall BA, Ren JM, Johnson DW et al (1993) Germline manipulation of glucose homeostasis via alteration of glucose transporter levels in skeletal muscle. J Biol Chem 268:18442–18445 Ren JM, Marshall BA, Gulve EA et al (1993) Evidence from transgenic mice that glucose transport is rate-limiting for
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glycogen deposition and glycolysis in skeletal muscle. J Biol Chem 268:16113–16115 Andersen PH, Lund S, Vestergaard H et al (1993) Expression of the major insulin regulatable glucose transporter (GLUT4) in skeletal muscle of noninsulin-dependent diabetic patients and healthy subjects before and after insulin infusion. J Clin Endocrinol Metab 77:27–32 Pedersen O, Bak JF, Andersen PH et al (1990) Evidence against altered expression of GLUT1 or GLUT4 in skeletal muscle of patients with obesity or NIDDM. Diabetes 39:865–870 Garvey WT, Maianu L, Hancock JA et al (1992) Gene expression of GLUT4 in skeletal muscle from insulin-resistant patients with obesity, IGT, GDM, and NIDDM. Diabetes 41:465–475 Lund S, Vestergaard H, Andersen PH et al (1993) GLUT-4 content in plasma membrane of muscle from patients with noninsulin-dependent diabetes mellitus. Am J Physiol 265:E889– E897 Garvey WT, Maianu L, Zhu JH et al (1998) Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance. J Clin Invest 101:2377–2386
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MiR-199a is overexpressed in plasma of type 2 diabetes patients which contributes to type 2 diabetes by targeting GLUT4 Shuang-Tong Yan • Chun-Lin Li • Hui Tian Jian Li • Yu Pei • Yu Liu • Yan-Ping Gong • Fu-Sheng Fang • Ban-Ruo Sun
•
Received: 15 February 2014 / Accepted: 24 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Decreased GLUT4 expression and impaired GLUT4 cell membrane translocation are involved in type 2 diabetes mellitus (T2DM) pathogenesis so the factors impacting GLUT4 expression may be associated with T2DM. In this study, we identified four miRNAs: miR-31, miR-93, miR-146a, and miR-199a which suppress GLUT4 expression in HEK293T cells. Subsequently, we determined expression of these four miRNAs in plasma samples of T2DM patients, T2DM susceptible individuals, and healthy controls and found miR-199a was overexpressed in patients’ plasma compared with healthy control. Because the miR-199a binding site in GLUT4 30 UTR is highly conserved among vertebrates, we detected the glucose uptake in rat L6 myoblast cells through gain- and loss-offunction of miR-199a. We found that miR-199a can repress glucose uptake in L6 cells, which was rescued by GLUT4 overexpression. These results indicate that T2DM patients may have a high level miR-199a that reduce GLUT4 expression and contribute to the insulin resistance. Hence, miR-199a may be a novel biomarker for risk estimation and classification in T2DM patients. Keywords Type 2 diabetes mellitus GLUT4 MiRNA Glucose uptake Plasma
S.-T. Yan C.-L. Li (&) H. Tian J. Li Y. Pei Y. Liu Y.-P. Gong F.-S. Fang B.-R. Sun Department of Geriatric Endocrinology, PLA General Hospital, Beijing 100853, China e-mail: [email protected]
Introduction GLUT4 is an insulin-responsive facilitative glucose transporter, which is found mostly expressed in adipose, skeletal muscle, and cardiac muscle cells. Under conditions of low insulin, GLUT4 is sequestered in intracellular vesicles in muscle and fat cells. Insulin induces a rapid increase in the uptake of glucose by inducing the translocation of GLUT4 from these vesicles to the plasma membrane. As the vesicles fuse with the plasma membrane, GLUT4 transporters are inserted and become available for transporting glucose, and glucose absorption increases. GLUT4 expression levels are correlated with whole-body insulin-mediated glucose homeostasis, and it is has been shown that the dysregulation of GLUT4 expression is related to diabetes and obesity. GLUT4 heterozygous mutant mice (GLUT4±) have increased serum glucose and insulin, reduced muscle glucose uptake, hypertension, and diabetic histopathologies in the heart and liver [1]. Overexpression of GLUT4 can partially improve the in vivo glucose tolerance in diabetes mice indicated the importance of GLUT4 in the process of diabetes [2]. MicroRNAs (miRNAs) are short non-coding RNAs which modulate gene expression by binding to complementary segments present in the 30 UTR of the mRNAs of protein coding genes. MiRNAs play very important roles in maintaining normal human body physiology conditions, and abnormal miRNA expressions have been found related to many human diseases spanning from psychiatric disorders to malignant cancers [3–5]. Following the discovery that miRNAs were involved in many relevant biological processes, including energy and fat metabolism, these molecules have received growing attention in the fields of diabetes and obesity research. Recently, two research groups highlighted the presence of miRNAs in plasma [6, 7]. These plasma miRNAs are not
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cell associated, but packaged in microvesicles or bound by AGO proteins that protect them from endogenous RNase activity. Interestingly, plasma miRNAs can display unique expression profiles: specific tumor miRNAs were identified in cancer patients, whereas tissue derived miRNAs constitute a marker for injury [8–10]. Anomalous miRNAs expression was also was found in the plasma of patients with diabetes which was highly associated with diabetes and diabetic complications [11]. Hence, the objective of the current study was to determine if miRNA regulate GLUT4 expression and whether such regulation contributes to the disease pathogenesis in type 2 diabetes mellitus (T2DM) patients.
Materials and methods Dual-luciferase assay Full length GLUT4 30 UTR (1,419 bp) was cloned downstream of firefly luciferase coding region in pGL3 vector (Promega, Madison, WI, USA) to generate luciferase reporter vector. For luciferase reporter assays, HEK293T cells were seeded in 48-well plates. MiRNA mimics and luciferase reporter vectors were co-transfected using Lipofectamine 2000 (Life Technologies, Carlsbad, CA USA). pRL-TK vector containing Renilla luciferase was co-transfected for data normalization. Two days later, cells were harvested and assayed with the Dual-Luciferase Assay kit (Promega, Madison, WI USA). Each treatment was performed in triplicate in three independent experiments. The results were expressed as relative luciferase activity (Firefly LUC/Renilla LUC). Western blotting Protein extracts were boiled in SDS/b-mercaptoethanol sample buffer, and 20 lg of samples was loaded into each lane of 10 % polyacrylamide gels. The proteins were separated by electrophoresis, and the proteins in the gels were blotted onto PVDF membranes (Amersham Pharmacia Biotech, St. Albans, Herts, UK) by electrophoretic transfer. The membrane was incubated with mouse antiGLUT4 monoclonal antibody (Abcam, Cambridge, MA, USA) (1:1,000 dilution) for 1 h at 37 °C. The specific protein-antibody complex was detected using horseradish peroxidase-conjugated rabbit anti-mouse IgG. Detection by the chemiluminescence reaction was carried using the ECL kit (Pierce, Appleton, WI, USA). The blots were stripped and re-probed with mouse anti-b-actin monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) (1:5,000 dilution) to confirm equal loading.
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Participants One hundred ninety-two Han Chinese individuals, aged 46–62 years, receiving routine physical exams were recruited by the Geriatric Endocrinology Department of PLA General Hospital. The study was approved by the Institutional Review Board of PLA General Hospital and enrolled patients provided informed written consent. They enrolled subjects were divided into three study groups (64 subjects/group): normal individuals [fasting glucose (FG), 4.8–5.2 mmol/L], T2DM susceptible individuals (FG, 6.1–6.9 mmol/L), and diagnosed T2DM patients (FG C 7.0 mmol/L). Individuals in the normal and susceptible groups with the following conditions were excluded from the study: common diabetic complications such as retinopathy, nephropathy, and cardiovascular disorders since it is not known whether they might have latent effect on miRNA expression. Individuals who had previously been diagnosed with DM or had any history of medication for 6 months prior to the study were also excluded. T2DM was diagnosed based on the combination of several parameters: FG level higher than 7.0 mmol/L and 2 h plasma glucose (PG) higher than 11.1 mmol/L in oral glucose tolerance test (OGTT), or diagnosed by other hospitals. RNA isolation and RT-qPCR Peripheral blood was collected via venipuncture into tubes containing sodium EDTA, centrifuged at 1,000 g for 5 min, and the plasma was carefully transferred into RNase-free tubes and stored at -80 °C until use. Total RNA containing miRNAs was isolated from plasma using Trizol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The expression level of miR-31, miR-93, miR-199a, and miR-146a was detected by TaqMan miRNA RT-Real Time PCR. Single-stranded cDNA was synthesized using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and then amplified using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) together with miRNA-specific TaqMan MGB probes: miR31, miR-93, miR-146a, and miR-199a (Applied Biosystems, Foster City, CA, USA). The miR-16 was used for normalization. Each sample in each group was measured in triplicate and the experiment was repeated at least three times for the detection of each miRNA. Cell culture HEK293T cells with high GLUT4 expression, was cultured in Dulbecco modified Eagle’s medium, supplemented with 10 % Fetal bovine Serum (FBS), not essential amino acids
Mol Cell Biochem Fig. 1 MiR-31, miR-93, miR146a and miR-199a repress the GLUT4 expression by targeting 30 UTR. a Schematic diagram for constructing the full length of GLUT4 30 UTR into pGL3 control vector. b Screening the miRNAs directly target GLUT4 30 UTR. pGL3-GLUT4 cotransfected with candidate miRNAs separately, for dualluciferase assay. PRL-TK containing renilla luciferase was co-transfected with 30 -UTR of GLUT4 for data normalization. *P \ 0.05; **P \ 0.01. c Western blot was used to detect GLUT4 expression in HEK293T cells transfected with miR-31, miR-93, miR-146a, and miR-199a
and antibiotics (Penicillin 100 IU/ml, Streptomycin 100 lg/ml). Rat L6 muscle cells were grown in a-MEM supplemented with 10 % FBS 100 units/mL penicillin, 100 lg/mL streptomycin.
Results
Glucose uptake measurement
MiRNAs modulate gene expression by binding to complementary segments present in the 30 UTR of the mRNAs of protein coding genes, and it is predicted that more than 60 % human genes are regulated by miRNA. To investigate which miRNA repress GLUT4 expression and play important roles in T2DM, we first predicted the miRNAs that may repress GLUT4 expression using online bioinformatics tools. We subsequently cloned full length GLUT4 30 UTR downstream of the firefly luciferase reporter gene in the pGL3 control vector to validate whether GLUT4 expression is repressed by the predicted candidate miRNAs (Fig. 1a). HEK293T cells were co-transfected with pGL3-GLUT4, indicated miRNA mimics, and pRL-TK. Nonsense scramble short nucleotide was used as control. As shown in Fig. 1b, miR-31, miR-93, miR-146a, and miR-199a suppressed luciferase activity significantly. These results indicate that miR-31, miR-93, miR146a, and miR-199a target the 30 -UTR of GLUT4, leading to the change of firefly luciferase translation. HEK293T cells were transfected with the four candidate miRNAs separately, and the expression of GLUT4 was detected by western blotting. Compared with corresponding control, the expression of
To measure glucose uptake in rat L6 cells, a nonradioactive assay was performed using a fluorescent deoxyglucose analog, 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4yl) amino)-2-deoxyglucose, Invitrogen, Carlsbad, USA). Briefly, the cells plated on a 96-well plate at a density of 5,000 cells/well were serum depleted for 24 h. Next, the cells were incubated with the indicated drugs for 10 min, and 100 lM 2-NBDG was administrated to each well. After 10 min incubation, the cells were washed with PBS two times, and fluorescence was measured using a microplate reader (ARVOsx; PerkinElmer). Statistical analysis Data were analyzed using SPSS Statistical Package version 16. Independent two group’s analyses are used t test. Statistical analysis for plasma miRNAs expression was performed by one way ANOVA. P \ 0.05 was considered statistically significant.
The expression of GLUT4 was repressed by four miRNAs in HEK293T cells
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Mol Cell Biochem Fig. 2 Mutation analysis identify the miRNAs binding sites. Full length of GLUT4 30 UTR with a three nucleotides mutation in predicted miR-93 binding site b four nucleotides mutation in predicted miR-31 binding site c three nucleotides mutation in predicted miR-199a binding site, and d three nucleotides mutation in predicted miR-146a binding site are cloned into pGL3-control vector for dual-luciferase assay. PRL-TK containing renilla luciferase was co-transfected with 30 -UTR of GLUT4 for data normalization. *P \ 0.05
GLUT4 was significantly suppressed by miR-31, miR-93, miR-146a, and miR-199a (Fig. 1c). Seed sequence mutation clones were also used to further confirm the binding site for miRNAs (Fig. 2). Putative miRNAs binding regions in the 30 -UTR of GLUT4 with 3 or 4 mutant nucleotides (designated as pGL3-GLUT4-Mu) and pGL3-GLUT4-Wt were used as controls, respectively. The histogram in Fig. 2 shows that the enzyme activities were significantly reduced in cells transfected with miR31/miR-93/miR-146a/miR-199a with pGL3-GLUT4-Wt vector compared with relevant pGL3-GLUT4-Mu vectors. These data cumulatively indicate that miR-31, miR-93, miR-146a, and miR-199a may suppress GLUT4 expression by binding to seed sequence in the 30 -UTR of GLUT4. Only miR-199a was up-regulated in T2DM patients’ plasma It is generally accepted that type 2 diabetes is associated with an impaired insulin-stimulated glucose disposal rate, which has been attributed to insulin resistance in skeletal muscle. GLUT4 is primarily expressed in adipose tissues and striated muscle (skeletal and cardiac) that is in turn responsible for
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insulin-regulated glucose transport into the cell and the dysregulation of GLUT4 was found related to T2DM. To investigate whether there are some relations between miRNAs which repress GLUT4 expression, we determined the expression of candidate miRNAs in the plasma of T2DM patients. As no consensus on the use of internal normalization control in plasma was defined for miRNA qPCR, the expression of miR-16, which have been proposed as the internal normalization control, was evaluated in the samples of all participants. Our data demonstrated that miR-16 was detected with high abundance and steadily presented in the plasma of these three groups participants (Fig. 3a). No significant difference was observed in terms of Ct values of miR-16 (P = 0.87). Therefore, miR-16 was selected as normalization control in this experiment. As shown in Fig. 3b, among the four candidate miRNAs, only miR-199a was overexpressed in T2DM patients compared with healthy control (P = 0.037). MiR-199a overexpression reduced the glucose uptake in L6 cells To examine the impact of miR-199a overexpression on glucose uptake in muscle cells, we used the fluorescent glucose
Mol Cell Biochem Fig. 3 Plasma miRNA detection. Box and whiskers plot presented the relative expression of four candidate miRNAs in the plasma samples. Expression levels of the miRNAs were normalized to miR-16. The lines inside the boxes denote the medians. The boxes mark the interval between the 10th and 75th percentiles. Statistical significance was determined using an unpaired t test. *P \ 0.05
analog 2-NBDG. We did the sequence alignment first and found that miR-199a binding site in GLUT4 30 UTR is conserved in human, mouse and rat (Fig. 4a). MiR-199a mimic or miRNA control or miR-199a inhibitor or miRNA inhibitor control was transfected into L6 myoblast cells separately. As shown in Fig. 4b, the expression of GLUT4 was repressed by miR-199a mimic and raised by miR-199a inhibitor meanwhile there was a significantly decrease in 2-NBDG uptake when miR-199a overexpressed and a slightly increase when miR-199a knock down indicates that miR-199a regulate the cell glucose uptake by targeting GLUT4 expression. To confirm the results above, we co-transfected L6 cells with miR199a and a GLUT4 expression vector which not contains the 30 UTR sequence. As shown in Fig. 4c, the overexpressed GLUT4 increase the 2-NBDG uptake which rescued the effect of miR-199a overexpression.
Discussion As a complex metabolic disorder, diabetes mellitus is characterized by chronic hyperglycemia due to defects
in insulin secretion or insulin resistance. It is generally accepted that type 2 diabetes is associated with an impaired insulin-stimulated glucose disposal rate, which has been attributed to insulin resistance in skeletal muscle. Glucose transport across the cell membrane of skeletal muscle is mediated by the glucose transporter proteins GLUT1 and GLUT4 [12]. The GLUT1 glucose transporter isoform is believed to support basal glucose transport, whereas the GLUT4 isoform increases glucose transport in response to insulin and contraction [13, 14]. In this study, we first identified that miR-31, miR-93, miR-146a, and miR-199a repress GLUT4 expression by directly targeting GLUT4 30 UTR in HEK293T cells. Subsequently, to investigate whether there are some relations between those four miRNAs and T2DM pathogenesis, we detected the expression of those four miRNA in the plasma samples of T2DM patients. Our results indicated that miR199a expression was up-regulated significantly compared with healthy control suggest that T2DM patients may have a high level miR-199a that reduce GLUT4 expression and attribute to the insulin resistance.
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Mol Cell Biochem Fig. 4 MiR-199a overexpression decrease glucose uptake of L6 cells. a The sequence of three species GLUT4 30 UTR were aligned by Clustal X. The miR-199a binding site was marked by red box. b Western blot was used to detect endogenous GLUT4 expression in L6 cells transfected with miR-199a mimic or inhibitor. Glucose uptake was measured using 2-NBDG. c Western blot was used to detect GLUT4 expression in L6 cells cotransfected with miR-199a mimic and GLUT4 expression vector. Glucose uptake was measured using 2-NBDG. *P \ 0.05. (Color figure online)
During the 1990s, a large number of studies have searched for the underlying causes of insulin resistance in skeletal muscle. The speculation that decreased GLUT4 expression could contribute to insulin resistance has been rejected by most studies [15–17], except for some studies of the translocation of GLUT4 by insulin or contractions, in which impaired translocation in type 2 diabetic muscle was found [18, 19]. Michael Gaster et al. found GLUT4 was down-regulated in slow muscle fibers of T2DM patients using immunohistochemistry give the first evidence of low GLUT4 level are related to T2DM. In this study, we only found miR-199a overexpressed in patients’ plasma which cannot represent the condition in the muscle cells, further study should be carried out to quantify and position the miR-199a in muscle tissues of T2DM patients. In summary, we first identified miR-31, miR-93, miR146a, and miR-199a repressed GLUT4 expression by targeting the 30 UTR of GLUT4 directly. We found miR-199a overexpressed in T2DM patients’ plasma. Subsequently, we confirmed miR-199a overexpression can also repressed GLUT4 expression in rat L6 myoblasts and decreased the glucose uptake which can be rescued by GLUT4 overexpression. Our results suggest a relation between up-regulated miR-199a and T2DM, which may ultimately lead to
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novel biomarkers for risk estimation and classification and could be exploited for miRNA-based therapeutic interventions of vascular complications associated with this disease.
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