Characterization of main UTRs data bases.
Insulin resistance (IR) is defined as a condition, in which regular amount of insulin is insufficient to develop physiological respond of the cell. For this reason there is constantly great need for increased level of this hormone within insulin resistant body. Very important factors leading to insulin resistance development are environmental components such as inappropriate diet and sedentary life style. A great important role in insulin resistance pathogenesis plays genetic background, as IR develops more frequently in families with positive history of metabolic disorders. There are severe anomalies in expression level of genes playing role in regulation of insulin action detected in patients with impaired insulin sensitivity. Regulation of gene expression can be exerted at either transcriptional level or post-transcriptional level. The former is related to the primary gene sequence located in the promoter region and it is responsible for controlling whether a gene is transcribed or not. The later utilizes regions of transcript not being translated, located at 5’ and 3’ end of the mRNA named the Untranslated Regions (UTRs). The main roles of UTRs are transcript stability control, initiation or inhibition of translation and sub-cellular localization in the cytoplasm. Regulation by UTRs is mediated in several ways, mainly by interaction of regulatory motifs in UTRs with numerous proteins as well as regulation by microRNA.
1.1. IR: Insulin resistance
The main tissues affected by IR are adipose tissue, skeletal muscle and liver (Hernandez-Morante et al., 2008; Karlsson & Zierath, 2007). Insulin resistance also affects lymphocytes and other peripheral blood leucocytes (Maratou et al, 2007; Piątkiewicz et al. 2007). The first diagnosed symptom of insulin resistance development is the decrease in glucose utilization by skeletal muscles (Patti, 2004), what is mediated by decrease in glycogen synthase (GYS) activity. Furthermore the expression rate and the phosphorylation state of numerous kinases (mainly PI-3K) of insulin pathway is decreased. The phosphorylation of serine residues of IRS-1 and IRS-2 is increased (Boura-Halfon & Zick, 2002). Impaired activity of GYS leads to insulin resistance in liver. Next, the number and metabolism of mitochondria decline (Morino, 2006). At this stage, the glucose utilization becomes impaired in adipose tissue. In parallel, the lipids metabolism deregulation, with increased FFA and TG levels, further impair insulin sensitivity in adipose tissue. Insulin resistance is characterized by dysfunction in GLUT4 translocation and glucose uptake in all cells, where insulin is essential. Despite long and intense studies, the origin and pathomechanism of insulin resistance remain unknown. It is believed that both environmental and genetic factors play role in its pathogenesis. IR causes increase in insulin production and secretion as a compensatory mechanism. The prolong demand for insulin results in decreased in pancreatic β-cells efficiency and insulin secretion. This is a theoretical pathomechanism of type 2 diabetes (T2DM) development.
1.1.1. Insulin resistance, mutations and genes expression
The body of literature reports that at insulin resistant state, severe anomalies in gene expressions encoded proteins involved in insulin pathway are diagnosed. In subjects with insulin resistance the decreased levels of
The downstream kinases such as IRS proteins, PI-3K, Akt have been also shown to be decreased in insulin resistant patients. (Hansen & Shafrir, 2002). Numerous studies provide data for impairments in
The last part of insulin pathway is GLUT4 activation and translocation into cell membrane what is essential for insulin-dependent glucose uptake. There are few polymorphisms in
1.1.2. Insulin resistance and active kinases dephosphorylation – Protein Tyrosine Phosphatases (PTPs)
The initial animal models experiments performed during last decade allowed for accurate assessment of the PTPs role in insulin action and glucose metabolism. Further evaluation of PTPs action in humans correlated their activity with obesity, metabolism dysfunction and impairment in insulin action (LeRoith et al, 1996). Several PTPs have been implicated in insulin signal regulation. The most important in insulin signaling is Protein Tyrosine Phosphatase 1B (PTP1B) encoded by
Another phosphatase that has been correlated with insulin resistance is Leukocyte Antigen-Related Phosphatase (LAR). Main data suggesting role of this phosphatase in insulin resistance pathogenesis came from knockout animal model studies that demonstrated severe insulin resistance in animals with decreased glucose uptake rate by skeletal muscles and decreased PI-3K activity (Zabolotny et al., 1999). Studies in humans showed similar pattern, that is significantly higher abundant of mRNA and protein in skeletal muscles and adipose tissue in obese subject, that positively correlated with obesity and insulin resistant state (Worm et al., 1995).
1.1.3. Insulin resistance and obesity
The average human body contains from 10 to 15 kg of adipose tissue that performs diverse functions ranging from energy storage to endocrine secretion. The excess accumulation of adipose tissue impairs insulin sensitivity by (1) excessive secretion of FFA into blood stream and its oxidation, (2) secretion of numerous cytokines that modulate insulin sensitivity, (3) chronic inflammatory state induction (George, 1996).
Abundant FFA level and the metabolite of its oxidation (Acylo-CoA) impair insulin action mainly by IRS-1 and IRS-2 serine/threoine residues phosphorylation, what is mediated through NF-кB pathway (Ragheb et al., 2009). Furthermore, NF-кB pathway activation is associated with increased rate of proinflammatory cytokines production.
Adipose tissue is known as an active endocrine organ producing and secreting into blood stream various cytokines like lepin, adiponectin, RBP4, resistin. Thanks to adipocytokines, adipose tissue connects with the central nervous system (CNS) and regulates energy balance. Some cytokines are implicated in insulin sensitivity regulation (George, 1996). Dysregulation in adipocytokines production, what has place in obesity, causes impairment in phosphorylation rate of numerous important kinases involved in insulin signal transduction (Cohen et al., 2002; Greenspan & Baxter, 1994). Adipose tissue in obese subjects is highly infiltrated by macrophages that change their phenotype into pro-inflammatory cells, secreting pro-inflammatory cytokines like IL -1, 6, 10 (Interleukin 1, 6, 10), TNF-α (Tumor Necrosis Factor-α), MCP-1 (Monocyte Chemotactic Protein-1). Mentioned cytokines are well known factors leading to JNK kinase activation, serine residues phophorylation and insulin sensitivity impairment (Müssig et al., 2005).
Macrophages are not the only cells causing inflammatory state. Some reports provide information that adipocytes hypertrophy is associated with preadipocytes differentiation deregulation and changing their phenotypes into pro-inflammatory cells secreting, similar to macrophages, various cytokines. These cells become typical pro-inflammatory cells with markers expression characteristic for immune cells like CD68 (Gustafson et al., 2009).
1.2. UTRs: Localization, structure and function in post-transcriptional regulation of gene expression
The UTRs are localized at both ends of transcript (mRNA), but are not transcribed to proteins (do not encode proteins). The 5’UTR is localized upstream the start codon AUG (Met), on the other hand 3’UTR is placed downstream the stop codons UAA, UAG, UGA. The average size of 5’UTR in humans is 210,2 nt (nucleotides) with maximal size 2803 nt and minimal size equal 18 nt. The average size for 3’UTR is 1027 nt with maximal size 8555 nt and minimal size equal 21 nt (Mignone et al., 2002). The characteristic feature of those regions is GC content with higher amount of GC in 5’UTR about 60%, on the other hand in 3’UTR GC contents is about 45% (Pesole et al., 1999). Very interesting feature has been seen by Pesole (Pesole et al., 1999), who observed that the higher amount of GC the shorter 3’UTR.
UTRs make numerous conformational structures and tridimensional loops that interact with proteins and other functional and regulatory compounds like ribosomes or microRNA. Within the UTRs a specific functional motifs can be observed, that play important role in function and transcription control (Carmody & Wente, 2009). The most important 5’UTR motif is 5’7-methylguanin (5’7mG), which is added to the transcript just after the transcription initiation, before the whole transcript synthesis is completed (Mignone et al., 2002). Within 5‘UTR other motifs can be distinguished like region IRES (Internal Ribosomal Entry Site), which stands for the 5’UTR region that interact with ribosome during translation initiation, numerous hairpins that interact with various proteins controlling transcripts stabilization and ORF (Open Reading Frames). Furthermore others motifs like IRE (Iron Respons Element) that is composed of about 30 nucleotides and has a stem-loop structure (Pickering & Willis, 2005) can be found. The most important motifs localized in 3’UTR are CPE (Cytoplasmic Polyadenylation Element), MRE (microRNA Regulatory Element) and poli(A) tail (Mignone et al., 2002; Carmody & Wente, 2009; Conne et al., 2000).
UTRs play their role thanks to many motifs, conformational loops and hairpins that interact with numerous proteins and others factors like microRNA or ribosomes. The main role of 5’UTR is controlling of translation initialization as well as transcript stabilization, on the other hand 3’UTR is mostly implicated in regulation of transcript stabilization and its localization in cytoplasm (Carmody & Wente, 2009; Pickering & Willis, 2005). Furthermore 3’UTR is the place of microRNA action via MRE. Depending on the cell type various mechanisms and regulatory motifs might regulate transcript stability. For example, IRE element regulates iron homeostasis. On the other hand regulatory proteins, growth factors as well as proto-oncogenes posses long 5’UTRs, what inhibits translation initiation and as a consequence, protein synthesis (Pickering & Willis, 2005).
1.2.1. Regulation of translation initiation
The main regulatory element of translation regulation is the regulation of translation initiation. This process requires interaction of ribosomal 40 S subunit with 5’7-methylguanin (5’7mG). The resulting complex (so called 43 S) further interacts with translation initiation factors like eIF2, eIF4F, eIF4G, eIF3. Next, eIF1A and eIF3 facilitate binding the 43 S subunit with eIF2-GTP-Met-tRNA, what begins the mRNA scanning process and searching for the initiation (start) codon (AUG) in 5’→3’ direction (Carmody & Wente, 2009; Meijer & Thomas, 2002). Once the start codon has been achieved, the eIF5 facilitates the 40 S and 60 S subunits joining resulting in 80 S ribosomal subunit. The 80 S subunit initiates the protein synthesis and elongation. In the regulation of efficiency of translation initiation and 5’UTR scanning the secondary structures of 5’UTR play a great role. However, the effect on translation initiation has been shown for structures posses the bounding energy higher ΔR < -50 kcal/mol (Svitkin et al., 2001). In the initiation translation process the most important is 5’7mG and its interaction with 40 S ribosomal subunit as well as with translation initiation factors. The mRNA binding with 40 S subunit is facilitated also by the IRES motif and this process dominates in situation when the translation initiation by 5’7mG is impaired by stress, apoptosis or suppressed by cell-cycle stage (Pickering & Willis, 2005;
Meijer & Thomas, 2002). This mechanism is common for mRNA encoding growth factors or transcription factors. IRES is localized close to AUG codon and the IRES-mediated translation regulation depends on the secondary and tertiary structure (Peng et al., 1996) as well as on the complementarity to 18 S rRNA (Chappell et al., 2000). Many genes posses the IRES in 5’UTR e.g. genes involved in apoptosis like
Very important role in translation initiation plays the nucleotide sequence flanking start codon with following sequence: GCCRCCAUGG. The R stands for purine, usually adenine. The purine in -3 position and G in +4 position is the rule of start codon determination and is strong consensus sequence present in animals and plants (Svitkin et al., 2001). The presence of AUG in 5’UTR and false determination of the main ORF decrease the translation initiation process via assignment of upstream open reading frames (uORF). The uORF results in translation initiation and synthesis of false proteins (Mignone et al., 2002; Svitkin et al., 2001). The fate of 40 S subunit that recognized the wrong ORF depends on the size of uORF. The 40 S subunit might dissociate and restart scanning, however, if the uORF is greater than 30 codons, the rescanning is not possible (Peng et al., 1996).
The mechanisms mediated translation initiation vary depending mostly on environmental condition and, in situation, when one mechanism is inhibited, the second is active. For example when the cap-dependent mechanism of translation initiation is inhibited by hypoxia or apoptosis, the translation is initiated by IRES-dependent mechanism. The same changeable mechanisms might be seen for translation regulation via uORF. It is believed that this switch hypothesis is an adaptive mechanism of gene expression regulation in various cellular conditions (Meijer & Thomas, 2002).
1.2.2. Transcript stability
The mRNA stability is mostly regulated via 3’UTR, especially by elements rich in AU repeats – ARE (AU – Rich Elements) (Griffin et al., 2004). AREs are classified into 3 groups depending on AUUUA repetitive units, regulation mechanisms and degradation efficiency. However, the result of its action is fast deadenylation and mRNA degradation (Peng et al., 1996). mRNA degradation is also regulated by numerous endonuclease enzymes, that hydrolyze the poli(A) tail of transcript with following fast degradation of the whole mRNA (Mignone et al., 2002). In the transcript stability regulation important role play hnRNPs (ribonucleoproteins) that stabilize the mRNA and is responsible for its localization in the cells. The recognition site for hnRNPs is located in the 3’UTR (Mignone et al., 2002).
The 5’UTR also plays role in the regulation of transcript stability by process named Nonsense-Mediated mRNA Decay (NMD) (Nicholson et al., 2010). The mechanism of regulation is connected with the proper identification of uORF as the false reading frame and its degradation. It is also responsible for accurate identification of stop codon and the translation termination in a proper position. In physiological conditions the premature translation termination codons (PTCs) are produced in variety of organisms. To prevent from the production of protein lacking C-terminal domains, those transcripts are recognized and subsequently degraded by NMD (Nicholson et al., 2010). The NMD mechanisms are also responsible for controlling the abundance of physiological full length transcripts (Mendell et al., 2004; Rehwinkel et al., 2005).
1.2.3. Regulation of transcript localization in cytoplasm
Subcelluar transcripts localization depends mainly on the type of protein encoded by gene and its demand in the cell. The mRNAs in cytoplasm are connected with ribonucleoproteins (Mignone et al., 2002). Three main mechanisms regulating subcellular localization of mRNA in the cell are known. The prevalent mechanism relies on the active transcript transport into the particular compartments of the cell by cytoskeleton elements and specific proteins interacting with mRNA (in particular with the 3’UTR). The second mechanism is connected with interaction of various proteins with motifs located in 3’UTR thus influencing the transcript localization. The third mechanism relies on the diffusion of mRNA (Mignone et al., 2002).
1.2.4. Post-transcriptional regulation via microRNA
MicroRNAs are short, single stranded classes of RNAs of 19-25 nucleotides (nt) in length. MiRNAs are produced from longer precursors containing hairpin structure (pre-miRNAs) that are generated from pri-miRNAs by nuclear RNase III Drosha. Pre-miRNAs are then transported into the cytoplasm and processed by Dicer RNase III complex to produce about 22 nt mature miRNAs (Kim, 2005). Mature miRNAs appear in the cell as complexes with proteins known as miRNP (miRNA containing ribonucleoprotins complex), or mirgonaute or miRISC (miRNA containing RNA induced silencing complex) (Kim, 2005). Generally one strand of miRNA is cleaved whilst one strand stands for active strand of mature miRNA. In animals including humans miRNAs act by imperfect pairing to the MRE (MiRNA Regulatory Element) in 3’UTR of target transcripts. Because of the mismatch between miRNA and target site in mRNA, one miRNA might target numerous different mRNAs, on the other hand one transcript might be regulated by various miRNAs (Jackson & Standart, 2007). There are several mechanisms of miRNA-dependent gene expression regulation, mainly through translation repression or mRNA decay (Jackson & Standart, 2007; Shuang & Fang, 2009; Zhao & Liu, 2009).
Translation repression might occur at initiation stage or after initiation stage. Three distinct mechanisms mediate translation repression by miRNA. First mechanism relies on blocking translation initiation by repression the assembly of ribosome that is the 60 S subunit to form complete 80 S translation active form (Thermann & Hentze, 2007). Second mechanism targets the translation initiation by repression the translation complex formation, mainly by blocking the eIF4E assembly to 5’7mG (Shuang & Fang, 2009). Third mechanism by which miRNA modulated translation initiation is connected with PolyA Binding Proteins (PABPs) action, or rather with the PolyA tail deadenylation (Wakiyama et al., 2007). There are various post-initiation mechanisms that influence translation initiation mediated by miRNA. It has been shown that microRNA represses the IRES depended translation initiation, inhibits the LIN4 protein synthesis or causes timely ribosome drop-off and early translation termination (Shuang & Fang, 2009).
Numerous studies provide data reported mRNA degradation as a main aspect of gene expression repression mediated by miRNAs. MiRNA acts, in contrary to siRNA not by endonucleolytic cleavage, but rather by deadenylation and decapping of target mRNA and its subsequent degradation (Wu et al., 2005). The process of mRNA decay by miRNA has place in a cytoplasms foci named P-bodies (Processing bodies) that contain miRNAs, target mRNAs and enzymes required for mRNA decay (Jackson & Standart, 2007).
1.2.5. UTRs data bases
In order to classify the knowledge and to comprehensively understand the mechanisms of post-transcriptional regulation of gene expression mediated by UTRs, various data bases were created. Data bases provide information about functions and regulation mechanisms on the basis of primary and secondary structure of regulatory motifs. All data assembled in UTRs data bases have been determined by experimental studies and published (Mignone et al., 2005; Huang et al., 2006). Data bases contain information about sequence and structure of regulatory motifs like IRES, IRE, MRE, ARE, indicating the region of transcript that the particular motif appears. Furthermore, data bases provide information about regulatory factors interacting with particular motif (transcription factor, regulatory protein, miRNA). All data bases stand for useful tool for therapeutic possibility anticipating and searching. Examples of the most common UTRs data bases with short characterization are presented in table 1.
|ERPIN (||http://tagc.univ.mrs.fr/erpin/||Identification of a wide range of secondary structure, orientation and regulatory motifs in mRNA sequence.|
|MicroInspector||http://bioinfo.uni-plovdiv.bg/microinspector/||Tools for miRNA target site prediction as well as for searching miRNAs of analyzed mRNA.|
|RegRNA||http://regrna/mbc.nctu.edu.tw/||Identification of regulatory motifs and elements in mRNA sequence with functional effects.|
|UTRdb, UTRsite||http://utrdb.ba.itb.cnr.it/tool/utrscan||Identification regulatory motifs in 3’ and 5’UTRs important in post-transcriptional regulation.|
1.3. Single nucleotide polymorphisms in UTRs and insulin resistance
5’ and 3’ UTRs are highly rich in polymorphisms like Alu elements or long polymorphisms LINE. In UTRs are present also mini and microSTRs as well as SNPs in a high abundance. The heterogeneity regions of UTRs for human are approximately 36% for 3’UTR and 12% for 5’UTR (Mignone et al., 2002). Single Nucleotide Polymorphism (SNP) is the replacement, deletion or insertion of a single base in genome sequence and is the most common change in human genome. The effects of nucleotide replacement vary, depend on the place, where the nucleotides have been changed. In most cases, SNPs do not have the phenotypic effects, and stand for the genomic heterogeneity within or between distinct populations. On the other hand SNPs in coding region of the genome (cSNPs – coding SNPs) might result in amino acids replacement and finally changes in protein structure and function. SNPs located in introns might influence the splicing process with effect on ensuing transcript. The great influence on mRNA and proteins synthesis possess SNPs located in regulatory regions like promoter region of the gene or in UTRs. Changes in those regions are associated with deregulation in gene expression at transcriptional and post-transcriptional levels (Doss et al., 2008).
The body of literature associate SNPs located in UTRs with gene expression regulation (Mendell et al., 2005; Chen et al., 2006). The proper nucleotide sequence in motifs described above ensures accurate function of these regions and gene expression regulation (Mendell & Dietz, 2001). Nucleotide changes in the functional transcripts regions influence the mRNA synthesis, splicing, transcripts stabilization and decay. Other mechanisms that SNPs might influence post-transcriptional gene expression regulation are the translation initiation or uORF generation. Nucleotide changes cause conformation changes in UTRs, especially in 5’UTR what notably influence the efficiency of translation initiation, the 5’UTR scanning and start codon searching. Furthermore changes in motifs that interact with 40 S ribosomal subunit, proteins, transcription factors or miRNA abolish the binding sites for these factors thus impair regulatory mechanisms.
So far, majority of investigators have focused mainly on genetic variants located in coding region. In recent decade polymorphisms in functional region have been emphasized. The growing interest dues to the fact that most diseases have been associated with abnormalities in gene expression rate as the main cause, thus the regulatory mechanisms have been widely investigated. Initially, investigators focused on genetic variants in regulatory regions as the cause of abnormities in gene expression during carcinogenesis. Next, more diseases have been correlated with changes in those regions (Conne et al., 2000; Pickering & Willis, 2005; Chen et al., 2006; Sethupathy & Collins, 2008; Halvorsen et al., 2010).
1.3.1. SNP in 5’ and 3’UTRs and insulin resistance
The correlations between insulin resistance and genetic variants in UTRs have been reported previously by many investigators (Xia et al., 1999; Chen et al., 2006; Nelsøe et al, 2006). The associations linking genetic variations in UTRs of such genes as
1.3.2. SNPs in microRNA genes/ microRNA target sites and insulin resistance
Gene expression regulation via microRNAs is crucial for maintaining body homeostasis. Dysregulation of this process might be a reason for various metabolic diseases (Sethupathy & Collins, 2008). Numerous factors affect miRNA translation regulation such as mutations in the proteins involved in miRNA processing and maturation (
In our previous study we have shown difference in genotype distribution of two SNPs located in 3’UTR of
INSR (Insulin Receptor) is a transmembrane glycoprotein formed by four chains: 2 α and 2 β subunits. The α subunit is responsible for ligand (insulin) binding, whilst the β subunit possesses the activity of tyrosine kinase that autophosphorylate tyrosine residues of β subunit and further kinases (IRS proteins) (Hubbard et al., 1994).
The present work is devoted to evaluation of the influence of two investigated SNPs (rs3756668 of
2. Material and methods
The experimental protocols were approved by ethical review boards at Wroclaw Medical University, No. KB – 556/2008, November 30, 2008.
2.1. Peripheral lymphocytes and adipose tissue collection
Visceral adipose tissue biopsies were taken during abdominal surgery after receiving written agreement. Samples were immediately preserved in RNA
Adipose tissue biopsies were collected from 15 patients with T2DM (6 men and 9 women) and from 24 controls (11 men and 13 women) in similar age (56±8 years for patients and 49±10 years for healthy subjects). Adipose tissue donors were inpatients of First Department and Clinic of General, Gastroenterological and Endocrinological Surgery, Wroclaw Medical University and of Provincial Specialist Hospital, Kamieńskiego in Wroclaw. The aims of abdominal surgeries were mainly cholecystectomy, surgery of abdominal hernia or gastric surgery. Lymphocytes were collected from 34 type 2 diabetic patients (21 men and 13 women) and from equal number of control subjects (17 men and 17 women) in similar age (mean age of diabetic patients was 58±7 years, controls 52±8 years). T2DM patients were inpatients of Department of Angiology, Hypertension and Diabetology, Wroclaw Medical University. Control subjects were selected based on fast glucose level below 106 mg/dl, lack of diabetes in family history, additionally for women no gestational diabetes in the past. Diabetic patients were divided into two subgroups depending on the insulin sensitivity:
2.2. BMI and insulin resistance ratios
BMI was assessed dividing weight in kilograms by square of height in meters [kg/m2]. Overweight was assigned with BMI > 25 kg/m2, obesity with BMI > 30 kg/m2.
Insulin resistance rate was estimated by insulin resistance ratios, calculated using following formulas:
HOMA-IR – [(glucose [mmol/l] * insulin [µU/ml])/22.5],
QUICKI – [(log glucose [mg/dl] + log insulin [µU/ml])].
Insulin resistance state was diagnosed with HOMA-IR > 2.5 and QUICKI < 0.321 (Ruano et al., 2006).
2.3. Bioinformatics analysis of investigated SNPs
The bioinformatical analyses of investigated SNPs were done with the use of bioinformatics tools available on-line: http://utrdb.ba.itb.cnr.it/tool/utrscan. The analyses were done to assess the localization of investigated SNPs in functional motifs of
2.4. RNA isolation and gene expression level
RNA from peripheral lymphocytes was isolated with the use of mirVana™ miRNA Isolation Kit (Ambion) according to manufactures protocol dedicated for total RNA isolation. RNA from visceral adipose tissues was isolated using TriPure Isolation Reagent (Roche) according to manufactured protocol. The tissues were homogenized using 2,0 mm Zirconia Beads (BioSpec Products, Inc). After homogenization tissues were centrifuged at max speed for 5 min in 4°C in order to collect the fat depot at the top of tube. The fat was discarded and the homogenate was extracted with 200 l of chloroform, briefly vortexed and centrifuged for 15 min at max speed in 4°C. The aqueous phase was collected and RNA was precipitated with 500 l of isopropanol, centrifuged for 10 min at max speed and washed with 1 ml of 70% ethanol in DEPC-treated water. RNA pellet was suspended in RNase-Free water and stored in -75°C.
Reverse transcription was performed with the use of High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to manufactured protocol. The
2.5. Statistical analysis
Statistical analysis was done using STATISTICA8 software. Statistical significance was considered with p < 0.05. The association of investigated SNPs with clinical parameters and
3.1. Anthropometrical and biochemical characterization of analyzed groups
67.5% of all diabetic patients were insulin resistant (
In previous study the correlation between genotype and insulin resistant phenotype has been presented (Malodobra et al., 2011). Furthermore we have noticed higher frequency of G/G genotypes of both SNPs in
3.2. Bioinformatics analysis of investigated SNPs
The bioinformatics analysis was done in order to evaluate the localization of investigated SNPs in regulatory motifs of
|Gene name||rs ID||Type of change||Region||Position in mRNA||Regulatory Element||The wild type reading frame||The mutation type reading frame|
INSR and PIK3R1 genes expression rate measurements
3.4. Genotype association with the
INSR and PIK3R1 genes expression levels
In order to analyze whether the genotype of investigated SNPs located in 3’UTRs of
On the other hand there was statistically significant relationship between rs3745551 located in 3’UTR of
3.5. Correlation between
INSR and PIK3R1 genes expression levels, biochemical parameters and insulin resistant phenotype
The genetic predispositions are large components that trigger the IR and T2DM risk and pathogenesis. SNPs in functional region are in great interests of numerous investigators and are associated with variety of diseases pathogenesis. The relationships between IR and SNPs in UTRs have been reported by many investigators (Xia et al., 1999;
Chen et al., 2006; Nelsøe et al et al., 2006). Especially 3’UTR is considered as a “hot spot” of pathology and polymorphic sites located within 3’UTR are associated with increased risk of numerous diseases (Conne et al., 2000). Taking into consideration the fact that IR is characterized by deregulations in numerous genes expression rates encoding important for insulin signaling kinases, the SNPs located in UTRs of these genes were particularly under investigation. Numerous SNPs located in
In present work, the
The present work contains as well the influence the G/G haplotype of rs3745551 (
Previously described results (Malodobra et al., 2011) demonstrated that two out of seven genotyped SNPs showed the association with insulin resistant phenotype. Those two SNPs displayed as well increased risk of IR development. Thus in present work thanks to increasing the number of analyzed subjects we were able to evaluate the relationship between two SNPs haplotype (rs3756668 G/G and rs3745551 G/G) and insulin resistant phenotype. Very interesting correlation has been observed with progressively increased insulin resistance state (assessed by clinical parameters: fasting glucose and insulin concentrations, HOMA-IR and QUICKI ratios) depending on number of G/G genotypes. The higher insulin resistant state has been seen for carriers of both G/G genotype, moderate values of measured parameters have been seen for carriers one of at risk genotypes. The lowest values of measured parameters possessed subject not affected by at risk G/G genotype.
In contradictory to results described by others investigators (Maratou et al., 2007; Piatkiewicz et al., 2007), we did not detect impairments in insulin signaling in patients with T2DM both in
The pathomechanism of impairment in insulin sensitivity in adipose tissue is quite different than in other tissues (skeletal muscles, liver) (George, 1996). In adipose tissue the main dysfunction leading to insulin resistance is the adipogenesis deregulation favoring differentiation towards pro-inflammatory cells (Gustafson et al., 2009). In addition hypertrophy and hyperplasia of adypocytes further lead to insulin sensitivity impairment. The visceral adipose tissue is especially implicated in metabolic syndrome pathogenesis including IR (Preis et al., 2010), for that reason this type of adipose tissue has been collected for analysis. Adipose tissue is characterized by deregulation in expression as well as phosphorylation rate of numerous genes and kinases (Ahmad et al., 1995; Andreelli et al., 2000; Patti, 2004; Rasouli & Kern, 2008).
In presented study in type 2 diabetics adipose tissues,
It has been proved by many investigators that SNPs in UTRs might affect mRNA stability and translation initiation processes (Mendell et al., 2005; Chen et al., 2006). In order to assess whether investigated SNPs in
Despite the fact we performed evaluation how particular genotype of investigated SNPs affects the gene expression rate. We did not notice significant changes in
Similar analysis has been done for
The rs3756668 located in 3’UTR of
Described in present study results provide the first evidence for association of SNPs in UTRs of
Concluding genes expression measurements, presenting results negate the dysfunction in insulin signaling in peripheral lymphocytes, at least at mRNA level. On the other hand the expression rate of genes implicated in insulin action is decreased in adipose tissue of patients with T2DM. The rs3745551, that in previous study showed correlation with insulin resistance, in present work, displayed relationship with
The present study also showed the effect of two SNPs haplotype influence on insulin resistant phenotype.
This project was supported by Ministry of Science and Higher Education of Poland, Grant No: N N401 009436.
Ahmad F. Azevedo J. L. Cortright R. J. Dohm G. L. Goldstein B. J. 1997Alteration in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistance human obesity and diabetes. , 100 2 449 458 0021-9738
Ahmad F. Li P. M. Meyerovitch J. Goldstein B. J. 1995Osmotic loading of neutralizing antibodies defines a role for protein-tyrosine phosphatase 1B in negative regulation of the insulin action pathway. , 270 35 20503 20508 0021-9258
Almind K. Delahaye L. Hansen T. van Obberghen E. Pedersen O. Kahn C.R. 2002Characterization of the Met326Ile variant of phosphatidylinositol 3-kinase p85α. PNAS, 0027-8424 4 99 2124 2128
Andreelli F. Laville M. Vega N. Riou J. P. Vidal H. 2000Regulation of gene expression during severe caloric restriction: lack of induction of p85alpha phosphatidylinositol 3-kinase mRNA in skeletal muscle of patients with type 2 (non-insulin-dependent) diabetes mellitus. , 43 3 356 363 0001-2186X
Barroso I. Luan J. Middelberg R. P. S. Harding A. H. Franks P. W. Jakes R. W. Clayton D. Schafer A. J. O’Rahilly S. Wareham N. 2003Candidate gene associated study in type 2 diabetes indicates a role for genes involved in β-cell function as well as insulin action. , 1 1 41 55 1545-7885
Baynes K. Beeton C. A. Panayotou G. Stein R. Soos M. Hansen T. Simpson H. O’Rahilly S. Shepherd R. P. Whitehead J. P. 2000Natural variants of human p85 alpha phosphoinositide 3-kinase in severe insulin resistance: a novel variant with impaired insulin stimulated lipid kinase activity. , 43 3 690 693 0001-2186X
Bennet A. M. Näslung T. I. Morgenstern R. De Faire U. 2001Bioinformatic and experimental tool for identification of single nucleotide polymorphisms in genes with a potential for the development of the insulin resistance syndrome. , 249 2 127 136 0954-6820
Boura-Halfon S. Zick Y. 2009Phosphorylation of IRS protein, insulin action and insulin resistance. Am. J. Physiol. Endocrinol. Metab., 0193-1849 296 581 591
Carmody S.R. Wente S.R. 2009mRNA nuclear export at a glance. J. Cell. Sci., 0021-9533 12 122 1933 1937
Chappell S. A. Edelman G. M. Mauro V. P. 2000A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. ., 97 4 1536 1541 0027-8424
Chen J. M. Férec C. Cooper D. N. 2006A systematic analysis of disease-associated variants in the 3’ regulatory regions of human protein-coding genes II: the importance of mRNA secondary structure in assessing the functionality of 3’ UTR variants. ., 120 3 301 333 0340-6717
Cheung A. Kusari J. Jansen D. Bandyopadhyay D. Kusari A. Bryer-Ash M. 1999Marked impairment of protein tyrosine phosphatase 1B activity in adipose tissue of obese subjects with and without type 2 diabetes mellitus. J. Lab. Clin. Med 0022-2143 2 134 115 123
Cohen B. Novick D. Rubinstein M. 1996Modulation of insulin activities by leptin. , 274 5290 1185 1188 0036-8075
Coldwell M. J. Mitchell S. A. Stoneley M. Mac M. Farlane A. E. Willis 2000Initiation of Apaf-1 translation by ribosome entry. , 19 7 899 905 0950-9232
Conne B. Stutz A. Vassalli J. D. 2000The 3’ untranslated region of messenger RNA: A molecular “hotspot” for pathology? ., 6 6 637 641 1078-8956
Di Paola R. Frittitta L. Miscio G. Bozzali M. Baratta R. Centra M. Spampinato D. Santagati M. G. Ercolino T. Cisternino C. Soccio T. Mastroianno S. Tassi V. Almgren P. Pizzuti A. Vigneri R. Trischitta V. 2002A variation in 3’ UTR of increases specific gene expression and associated with insulin resistance. Am. J. Hum. Genet., 70 3 806 812 0002-9297
Doss C. G. P. Sudandiradoss C. Rajasekaran R. Choudhury P. Sinha P. Hota P. Batra U. P. Rao S. 2008Application of computational algorithm tools to identify functional SNPs. , 8 4 309 316 0143-8793X
Duman B. S. Cagatay P. Hatemi H. Öztürk M. 2007Association of resistin gene 3’-Untranslated region EX4-44 G→A polymorphism with obesity- and insulin-related phonotypes in Turkish type 2 diabetes patients. , 4 1 49 55 1614-0575
Dupuis J. Langenberg C. Prokopenko I. et al. 2010New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk. , 42 2 105 120 1546-1718
Ferland Mc D. Collough S. E. Ozanne K. Siddle A. E. Willis M. Bushell 2010The involvement of microRNAs in Type 2 Diabetes. ., Trans. 38 6 1565 1570 1470-8752
Garvey W.T. Maianu L. Zhu J.H. Brechtel-Hook G. Wallace P. Baron A.D. 1998Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance. J. Clin. Invest., 0021-9738 11 101 2377 2386
George A. B. 1996Endocrinology and metabolism clinics of North America. Obesity. In: , 25 (4).
Glinsky G. V. 2008An SNP-guided microRNA map of fifteen common human disorders identifies a consensus disease phenocode aiming at principal components of the nuclear import pathway. , 7 16 2570 2583 1551-4005
Greenspan F. S. Baxter J. D. 1994Basic & Clinical Endocrinology. In: , Fourth edition, Norwalk, Connecticut, USA.
Griffin M. E. Hamilton B. J. Roy K. M. Du M. Willson A. M. Deenan B. J. Wang X. W. Nichols R. C. 2004Post-transcriptional regulation of glucose transporter-1 by an AU-rich element in the 3’UTR and by hnRNP A2. , 318 4 977 982 0000-6291X
Gustafson B. Gogg S. Hedjazifar S. Jenndahl L. Hammarstedt A. Smith U. 2009Inflammation and impaired adipogenesis in hypertrophic obesity in man. Am. J. Physiol. Endocrinol. Metab., 1522-1555 297
Halvorsen M. Martin J. S. Broadaway S. Laederach A. 2010Disease-associated mutations that alter the RNA structural ensemble. , 6 8 e1001074 1553-7404
Hansen B. Shafrir E. 2002Insulin resistance and insulin resistance syndrome. In: , New York, NY, USA
Hernandez-Morante J.J. Milagrot F.I. Lujan J.A. Martinez J.A. Zamora S. Garaulet M. 2008Insulin effect on adipose tissue (AT) adiponectin expression is regulated by the insulin resistance status of the patients. Clin. Endocrinol., 1365-2265 3 69 412 417
Højlund K. Wojtaszewski J.F.P. Birk J. Hansen B.F. Vestergaard H. Beck-Nielsen H. 2006Partial rescue of in vivo insulin signalling in skeletal muscle by impaired insulin clearance in heterozygous carriers of a mutation in the insulin receptor gene. Diabetologia, 0001-2186 8 49 1827 1837
Holcik M. Korneluk R. G. 2000Functional characterization of the X-linked inhibitor of apoptosis (XIAP) internal ribosome entry site element: role of La autoantigen in XIAP translation. , 20 13 4648 4657 0270-7306
Huang H. Y. Chien C. H. Jen K. H. Huang H. D. 2006RegRNA: an integrated web server for identifying regulatory RNA motifs and elements. ., 34 429 434 1362-4962
Hubbard S. R. Wei L. Ellis L. Hendrickson W. A. 1994Crystal structure of the tyrosine kinase domain of the human insulin receptor. , 372 6508 746 754 0028-0836
Jackson R. J. Standart N. 2007How do microRNA regulated gene expression? , 367 1 13 1525-8882
Jamshidi Y. Snieder H. Wang X. Pavitt M. J. Spector T. D. Carter N. D. O’Dell S. D. 2006Phosphatidylinositol 3-kinase p85α subunit gene haplotype is associated with body fat and serum leptin in a female twin population. Diabetologia, 49 11 2659 2667 0001-2186X
Karlsson H. K. R. Zierath J. R. 2007Insulin signaling and glucose transport in insulin resistant human skeletal muscle. , 48 2 103 113 1085-9195
Kim V. N. 2005MicroRNA biogenesis: coordinated cropping and dicing. , 6 5 376 385 1471-0072
Kusari J. Verma F. B. Henry R. R. Olefsky J. M. 1991Analysis of the gene sequences of the insulin receptor and the insulin sensitive glucose transporter (GLUT4) in patients with common type non-dependent diabetes mellitus. ., 88 4 1323 1330 0021-9738
Le Roith D. Taylor S. I. Olefsky J. M. 1996Diabetes Mellitus. A fundamental and clinical text. In: , Philadelphia, PA
Malodobra M. Pileckam A. Gworys B. Adamiec R. 2011Single nucleotide polymorphisms within functional regions of genes implicated in insulin action and association with the insulin resistance phenotype. , 349 1-2 187 193 1573-4919
Maratou E. Dimitriadis G. Kollias A. Boutati E. Lambadiri V. Mitrou P. Raptis S. A. 2007Glucose transporter expression on the plasma membrane of resting and activated white blood cells. ., 37 4 282 290 0014-2972
Meijer H. A. Thomas A. A. M. 2002Control of eukaryotic protein synthesis by upstream open reading frames in the 5’-untranslated region of an mRNA. 367 1 11 0264-6021
Meiron M. Anunu R. Scheinman E. J. Hashmueli S. Levi B. Z. 2001New isoforms of VEGF are translated from alternative initiation CUG codons located in its 5’UTR. ., 282 4 1053 1060 0000-6291X
Mendell J. T. Dietz H. C. 2001When the message goes awry: Disease-producing mutations that influence mRNA content and performance. , 107 4 411 414 0092-8674
Mendell J. T. Sharifi N. A. Meyers J. L. Martinez-Murillo F. Dietz H. C. 2004Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. , 36 10 1073 1078 1061-4036
Mignone F. Gissi C. Liuni S. Pesole G. 2002Untranslated regions of mRNAs. ., 3 3 1 10 1465-6906
Mignone F. Grillo G. Licciulli F. Iacono M. Liuni S. Kersey P. J. Duarte J. Saccone C. Pesole G. 2005UTRdb and UTRsite: a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs. , 33 141 146 1362-4962
Morino K. Petersen K. F. Shulman G. I. 2006Molecular mechanisms of insulin resistance in humans and their potential links with mitochondrial dysfunction. 55 2 9 15 0012-1797
Müssig K. Fiedler H. Staiger H. Weigert C. Lahmann R. Schleicher E. D. Haring H. U. 2005Insulin-induced stimulation of JNK and the PI 3-kinase/mTOR pathway leads to phosphorylation of serine 318 of IRS-1 in C2C12 myotubes. , 335 3 819 825 0000-6291X
Nelsøe R. L. Hamid Y. H. Pociot F. Paulsen S. Andersen K. M. Borch-Johnsen K. Drivsholm T. Hansen T. Pedersen O. Mandrup-Poulsen T. 2006Association of a microsatellite in FASL to type 2 diabetes and of the FAS-670G>A genotype in insulin resistance. ., 7 4 316 321 1466-4879
Nicholson P. Yepiskoposyan H. Metze S. Orozco R. Z. Kleinschmidt N. Mühlemann O. 2010Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors. ., 67 677 700 1420-9071
Patti M.E. 2004Gene expression in human with diabetes and prediabetes: what have we learned about diabetes pathophysiology. Curr Opin Clin Nutr Metab Care, 1363-1950 4 7 383 390
Peng S. S. Chen C. Y. Shyu A. B. 1996Functional characterization of a non-AUUUA AU-rich element from the c-jun proto-oncogene mRNA: evidence for a novel class of AU-rich elements. , 16 4 1490 1499 0270-7306
Pesole G. Bernardi G. Saccone C. 1999Isochore specificity of AUG initiator context of human genes. , 464 1-2 60 62
Pfaffl M.W. 2001A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res, 1362-4962 9 29 2002 2007
Piątkiewicz P. Czech A. Tatoń J. 2007Glucose transport in human peripheral blood lymphocytes influenced by type 2 diabetes. ., 55 2 119 126 0000-4069X
Pickering B. M. Willis A. E. 2005The implications of structured 5’ untranslated regions on translation and disease. ., 16 39 47 1084-9521
Poy M. N. Spranger M. Stoffel M. 2007microRNAs and the regulation of glucose and lipids metabolism. , 9 2 67 73 1462-8902
Preis S.R. Massaro J.M. Robins S.J. Hoffmann U. Vasan R.S. Irlbeck T. Meigs J.B. Sutherland P. D’Agostino R.B. O’Donnell C.J. Fox C.S. 2010Abdominal subcutaneous and visceral adipose tissue and insulin resistance in the Framingham heart study. Nature, 1930-7381 11 18 2192 2198
Ragheb R. Shanab G.M.L. Medhat A.M. Seoudi D.M. Adeli K. Fantus I.G. 2009Free fatty acid-induced muscle insulin resistance and glucose uptake dysfunction: Evidence for PKC activation and oxidative stress-activated signaling pathways. Biochem. Biophys. Res. Commun., 1090-2104 2 3589 211 216
Rasche A. Al-Hasami H. Herwig R. 2008Meta-analysis approach identifies candidate genes and associated molecular networks for type 2 diabetes mellitus. BMC Genomics 1471-2164 9 310 327
Rasouli N. Kern P. A. 2008Adipocytokines and the metabolic complications of obesity. , 93 11 64 73 0002-1972X
Rehwinkel J. Lutenic I. Raes J. Bork P. Izaurralde E. 2005Nonsense-mediated mRNA decay factors act in concert to regulate common mRNA targets. , 11 10 1530 1544 1355-8382
Ruano M. Silvestre V. Castro R. Garcia-Lescun M. C. G. Aguirregoicoa E. Marco A. Rodriguez A. Garcia-Blanch G. 2006HOMA, QUICKI and MFfm to measure insulin resistance in morbid obesity. , 16 5 549 553 0960-8923
Salonen J. T. Uimari P. Aalto J. M. et al. 2007Type 2 diabetes whole genome association study in four population. The DiaGen Consortium, , 81 2 338 345 0002-9297
Sethupathy P. Collins F. S. 2008MicroRNA target site polymorphisms and human disease. Trends. Genet., 24 10 489 497 0168-9525
Shepherd P. R. Khan B. B. 1999Glucose transporters and insulin action. Implications for insulin resistance and diabetes mellitus. ., 341 4 248 257 0028-4793
Shuang Z. Fang L. 2009Mechanisms of microRNA-mediated gene regulation. Sci. China, C, Life Sci 1862-2798 12 52 1111 1116
Stentz F. B. Kitabchi A. E. 2007Transcriptome and proteome expressions involved in insulin resistance in muscle and activated T-lymphocytes of patients with type 2 diabetes. , 5 3-4 216 235 1672-0229
Stoneley M. Chappell S. A. Jopling C. K. Dickens M. Mac M. Farlene A. E. Willis 2000c-Myc protein synthesis in initiated from the internal ribosome entry segment during apoptosis. , 20 4 1162 1169 0270-7306
Svitkin Y. V. Pause A. Haghighat A. Pyronnet S. Witherall S. Belsham G. J. Sonenberg N. 2001The requirement for eukaryotic initiation factor 4A (eIF4A) in translation is in direct proportion to the degree of mRNA 5’ secondary structure. , 7 3 382 394 1355-8382
Tan M. S. Chang S. Y. Chang D. M. Tsai J. C. R. Lee J. Y. 2003Association of resistin gene 3’Untranslated Region +64G→A polymorphisms with type 2 diabetes and hypertension in a Chinese population. J Clin Endocrinol Metab., 88 3 1258 1263 0002-1972X
Thermann R. Hentze M. W. 2007Drosophila miR-2 induces pseudo-polysomes and inhibits translation initiation. , 447 7146 875 878 1476-4687
Venable C. L. Frevert E. U. Kim Y. B. Fischer B. M. Kamatkar S. Neel B. G. Kahn B. B. 2000Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/protein kinase B activation. , 275 24 18318 18326 0021-9258
Villuendas G. Botella-Carretero J. I. López-Bermejo A. Gubern C. Ricart W. Fernández-Real J. M. San J. L. Millán H. F. Escobar-Morreale 2006The ACAA-insertion/deletion polymorphism at the 3’ UTR of the IGF-II receptor gene is associated with type 2 diabetes and surrogate markers of insulin resistance. 155 2 331 336 0804-4643
Wakiyama M. Takimoto K. Ohara O. et al. 2007Let-7 microRNA-mediated mRNA deadenylation and translation repression in a mammalian cell-free system. Genes Dev., 0890-9369 21 1857 1862
Wolford J. K. Hanson R. L. Kobes S. Bogardus C. Prochazka M. 2001Analysis of linkage disequilibrium between polymorphisms in the gene with type 2 diabetes mellitus in Pima Indians. Mol Genet Metab, 73 1 97 103 1096-7192
Worm D. Vinten J. Staehr P. Henriksen J. E. L. Handberh A. Beck-Nielsen H. 1995Altered basal and insulin-stimulated phophotyrosine phosphatase (PTPase) activity in skeletal muscle form NIDDM patients compared with control subjects. , 39 10 1208 1214 0001-2186X
Wu L. Fan J. Belasco J. G. 2005MicroRNAs direct rapid deadenylation of mRNA. , 103 11 4034 4039 0027-8424
Xia J. Bogardus C. Prochazka M. 1999A type 2 diabetes associated polymorphic ARE motif affecting expression of is involved in RNA-protein interaction. Mol. Genet. Metab., 68 1 48 55 1096-7192
Zabolotny J. M. Kim Y. B. Peroni O. D. Kim J. K. Pani M. A. Boss O. Klaman L. D. Kamatkar S. Shulman G. I. Kahn B. B. Neel B. G. 2001Overexpression of the LAR (Leukocyte Antigen Related) protein-tyrosine phosphatase in muscle causes insulin resistance. , 98 9 5187 5192 0027-8424
Zeggini E. Scott L. J. Saxena R. Voight B. F. 2008Meta-analysis of genome wide association data and large scale replication identifies additional susceptible loci for type 2 diabetes. Nature Genetics, 40 5 634 645 1546-1718
Zhang H. Jia Y. Cooper J. J. Hale T. Zhang Z. Elbein S. C. 2004Common variants in Glutamine Fructose-6-Phosphate Amidotransferase 2 (GFPT2) gene are associated with type 2 diabetes, diabetic nephropathy and increased GFPT2 mRNA levels. , 89 2 748 755 0002-1972X
Zhao S. Liu M.F. 2009Mechanisms of microRNA mediated gene regulation. Sci China Ser C-Life Sci, 1862-2798 12 52 1111 1116