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86 MicroRNA, 2014, 3, 86-97

Micro RNA: An Epigenetic Regulator of Type 2 Diabetes

Vinitha Kadamkode and Gautam Banerjee*

Unilever R & D India, 64, Whitefield Main Road, Bangalore, India

Abstract: Type 2 Diabetes is a complex disease with multifactorial pathogenesis. The interplay between genes and life-style changes complicates the etiology of the disease. In spite of growing number of attempts to unravel the mechanism of this metabolic disease, the molecular nature of type 2 Diabetes is not fully understood. The discovery of a new class of non-coding RNAs, micro RNAs and their role in regulation of gene expression have paved the way for better understand-ing of disease pathogenesis. Increasing number of evidences suggest crucial role of miRNAs in insulin resistance and �cell dysfunction, which are the two main features of type 2 Diabetes. This review summarizes the role of miRNA in elici-tation and progression of type 2 Diabetes.

Keywords: � cell, biomarker, ER stress, insulin resistivity.

INTRODUCTION

Diabetes mellitus is a global epidemic of recent time. Currently more than 300 million people are affected globally with impaired glucose tolerance and are at greater risk for developing the disease. The global projection of Diabetes in 2035 is about 471 million [1]. Dysfunction of pancreatic �cell and Insulin resistance is the main hallmark of type 2 Diabetes [2-4]. Type 2 diabetes is characterized by impaired insulin response and insulin secretion. The genetic and life-style changes contribute to the development of insulin resis-tance in insulin responsive tissues like adipose tissue, skele-tal muscle and liver. The positive calorie balance as a result of lifestyle changes results in expansion of adipose tissue and subsequent release of free fatty acids into circulation. The pro-inflammatory milieu along with vicious fatty acid further worsens the systemic insulin resistance. The major metabolic pathways that get affected in insulin responsive tissues in-clude insulin signaling, mitochondrial function, adipocytes differentiation, fatty acid oxidation etc. Emerging scientific evidences suggest a pivotal role of miRNAs in all these metabolic pathways which are directly related to diabetes pathogenesis.

Persistent hyperglycemia can lead to micro vascular complications like diabetic nephropathy, neuropathy, reti-nopathy and macro vascular complications like coronary artery disease, peripheral arterial disease and stroke [5]. There are growing evidences suggesting the role of epi-genetic regulation in type 2 Diabetes development. The in-terplay between genes and environment and its effect on disease development is well explained by epigenetic factors. The major epigenetic regulations include DNA methylations, histone modifications and microRNAs. The detailed analysis

*Address correspondence to this author at the Unilever R & D, 64 India, Whitefield Main Road, Bangalore, India; Tel: +91 80 39831068; Fax: +91 80 28453086; E-mail: [email protected]

of specific epigenetic changes associated with diabetes is beyond the scope of this review and we are focusing only on the role of miRNA in regulating the epigenetic changes like DNA methylation, histone modification etc.

The growing interest and research findings on endoge-nous small RNA, micro RNA (miRNA) as large classes of gene regulators and also epigenetic regulators have accentu-ated the role of these in etiology and pathogenesis of Type 2 Diabetes [6-12]. Currently limited information is available on the role of miRNAs on regulation of other epigenetic modifications like DNA methylation, chromatin remodeling etc. Hence further studies are required to elucidate the im-pact of miRNA on other epigenetic regulators.

WHAT IS MICRO RNA?

Micro RNA (miRNA) are endogenous small non coding RNA of approximately 18-23 nucleotides. Gene expression is modulated by miRNAs by binding to 3’untranslated region (UTR) or coding sequences or 5’ UTR of target messenger RNAs (mRNAs) and subsequently inhibition of translation of mRNAs or degradation of mRNA [13-15]. A single miRNA may target several genes and regulate more than 30% of human protein coding genes [16, 17]. Biological processes like cell growth, proliferation, differentiation, apoptosis, metabolism etc are tightly regulated by miRNAs [18-21]. Investigations on miRNA attained significant pro-gress in recent time with the advent of new tools and tech-nologies like high throughput sequencing. The miRNAs are now considered as a major gene regulators [22]. As miRNAs are part of various biological processes, their deregulation has been linked to many diseases like cancer, cardiovascular diseases, autoimmune diseases, neurodegenerative diseases etc [23-29]. This review summarizes the multiple role of miRNA in epigenetic regulation on pathogenesis of Type 2 Diabetes mellitus and also its emerging role as biomarker for early prediction of the disease.

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Micro RNA: An Epigenetic Regulator of Type 2 Diabetes MicroRNA, 2014, Vol. 3, No. 2 87

Micro RNAs and Epigenetic Regulation of Type 2 Diabetes

The developmental origin or the fetal origin of type 2 Diabetes is based on the metabolic programming that occurs during intrauterine growth and also depends on the nutri-tional exposures in post natal life. The changes in prenatal and post natal nutrition and predisposition to metabolic dis-eases have been well substantiated in many epidemiological studies. Epigenetic regulations like DNA methylation, chro-matin remodeling and micro RNA contributes to the molecu-lar changes associated with metabolic programming which predisposes to type 2 Diabetes [30-35]. The data mining analysis done by Wren et al. highlights the importance of epigenetic regulation in the etiology of type 2 Diabetes [36]. Poor in utero conditions and subsequent changes in DNA methylation pattern contribute to impaired � cell develop-ment, function and reduced insulin sensitivity, which eventu-ally manifests as type 2 Diabetes. One of the strongest evi-dences suggesting the role of epigenetics in developmental origin of type 2 Diabetes is Intrauterine growth retardation and down regulation of pancreatic and duodenal homeobox 1 (pdx1), a transcription factor involved in � cell development by promoter DNA methylation, histone modifications or chromatin protein binding [37]. Adverse prenatal environ-ment leading to changes in DNA methylation and early life insulin resistance was observed in Dutch Hunger winter families study [38-40]. The reversal of histone deacetylation in prenatal undernourished newborn resulted in partial resto-ration of � cell transcription factor, PDX-1. These findings are indicative of the existence of regulatory proteins like SIRT4 or miR-9 which might be involved in chromatin modifications [37]. Altered expression of miRNAs in insulin responsive tissues have been observed in undernourished fetus and in low birth weight individuals. The miR-483-3p was shown to be up regulated in subcutaneous adipose tissue of low birth weight individuals [41]. The expression of Let-7c was reduced in liver of maternal high fat fed off springs. The miR-709, an abundant miRNA which targets methyl-CpG binding protein 2 was also down regulated [42]. In a recent study conducted by Vasumathi et al, a specific cluster of miRNA, DLK1-MEG3 was found to be down regulated in pancreatic islets from diabetic donors and hypermethylation of the MEG3 promoter was also observed. These differen-tially regulated miRNA cluster targets major proteins in-volved in � cell apoptosis [43]. Currently limited information exists on the role of miRNAs in regulating other epigenetic modifications like DNA methylation, chromatin remodeling etc. Hence further studies are required to elucidate the im-pact of miRNAs on other epigenetic regulators.

miRNAs in Pathogenesis of Type 2 Diabetes

Type 2 diabetes is characterized by impaired insulin re-sponse and insulin secretion. The genetic and lifestyle changes contribute to the development of insulin resistance in insulin responsive tissues like adipose tissue, skeletal muscle and liver. The positive calorie balance as a result of lifestyle changes results in expansion of adipose tissue and subsequent release of free fatty acids into circulation. The pro-inflammatory milieu along with vicious fatty acid further worsens the systemic insulin resistance. The major metabolic

pathways that are affected in insulin responsive tissues in-clude insulin signaling, mitochondrial function, adipocytes differentiation, fatty acid oxidation etc [44-48].

The other important feature of the disease is � cell dys-function, clinically manifested as reduced insulin secretion in response to glucose. The increasing insulin demand due to the increased insulin resistance leads to compensatory ex-pansion of � cell mass. Recent studies have shown that miRNAs are involved in the regulation of transcription fac-tors involved in � cell expansion. The proinflammatory mi-lieu prevailing in circulation due to insulin resistance also exerts pro-apoptotic effect on � cells. The inflammatory cy-tokines and free fatty acids can also hamper the insulin syn-thesis and secretory machinery [49-51]. The miRNAs are known to regulate the expansion, apoptosis, insulin synthesis and secretion in � cells.

Micro RNA and � Cell Dysfunction

The involvement of miRNA in Type 2 Diabetes was first described with the discovery of miR-375 in regulation of insulin secretion [52]. Recent studies demonstrated that miRNAs play important roles in insulin production and se-cretion, pancreatic islet development, �-cell differentiation and insulin resistance in various tissues like skeletal muscle, adipocytes etc [53-58]. Some miRNAs as mentioned below are well investigated and characterized in the context of �cell dysfunction.

miR-375

The miRNA- 375 is one of the most widely investigated islet specific miRNA. The miR-375 was identified and cloned from pancreatic � cell line MIN6 [30]. The miR-375 plays a significant role in genesis of pancreas. The role of miR-375 in development of pancreas was first studied in Zebrafish [59]. The miR-375 regulates both � and � cell mass and the expression of miR-375 increases during pan-creatic organogenesis [60-61].

The role of miR-375 is known in both insulin secretion and glucose homeostasis. Myotrophin (Mtpn), a protein im-plicated in actin depolymerization and vesicular fusion is considered to be the target of miR-375 . The miR-375 down regulates the expression of Mtpn thereby suppresses glucose stimulated insulin release (GSIS) and inhibition of miR-375 enhances glucose stimulated insulin release. The other pre-dicted target of miR-375 is 3’-phosphoinositide–dependent protein kinase-1 (PDK1), and thus decreases glucose stimu-lated insulin gene expression and DNA synthesis [62, 63]. Glucose treatment reduces the level of precursors of miR-375 [63]. Though down regulation of miR-375 enhances insulin secretion, it is observed that mice lacking miR-375 gene exhibits severe hyperglycemia and increased total pan-creatic �-cell numbers, plasma glucagon levels and hepatic glucose output. Mice lacking miR-375 is characterized by reduced � cell mass and impaired cell proliferation. Pancre-atic islets of obese mice (ob/ob) (with increased � cell mass) exhibit increased expression of miR-375. Genetic deletion of miR-375 in obese mice diminishes the proliferative capacity of pancreas that leads to diabetes [60]. The miR-375 is also known to regulate � cell apoptosis. It is shown that pancre-atic � cells line (NIT-1 cells) with ectopic miR-375 expres-

88 MicroRNA, 2014, Vol. 3, No. 2 Kadamkode and Banerjee

sion are more susceptible to palmitate-induced apoptosis. The miR-375 enhances palmitate-induced apoptosis in insu-lin-secreting NIT-1 cells by repressing myotrophin (V1) pro-tein expression [64]. The miR-375 level in circulation is high in streptozotocin (STZ) treated C57BL/6 mice and non obese diabetic (NOD) mice. The extracellular miR-375 level is high in islets treated with cytokines or STZ and the level reduced with cell death inhibitors [65]. The level of miR-375 expression in pancreas is higher in Type 2 Diabetes popula-tion and is also associated with pancreatic islet amyloid for-mation and � cell deficit [66]. The level of miR-375 in serum is higher in subjects with pre-Diabetes and Type 2 Diabetes [67]. Taken together it may be concluded that miR-375 is a negative regulator of glucose stimulated insulin release, but it is essential for � cell proliferation in response to the in-creasing insulin demand during insulin resistance.

miR-124

Three isomers of the miR-124a is well documented e.g. miR-124a1, a2 and a3 that are abundant in the pancreatic �-cells. The miR-124a2 is significantly up regulated in late pancreas development. Over expression of miR-124a2 in MIN6 cells down regulates the expression of Foxa2 and also pancreatic duodenum homeobox-1 (Pdx-1) which is a down-stream target. It also down regulates the ATP-sensitive K+ (KATP) channel subunits, Kir6.2 and Sur-1 [68]. Inhibition of FoxA2 by miR-124a2 results in lowering of glucose stimulated insulin release and inhibition of � cell develop-ment and insulin production. Over expression of miR-124a, increases the basal levels of insulin but decreases glucose stimulated insulin release by down regulating exocytosis regulators such as Rab-GTPase family 27a (Rab 27a), and Noc2 and up regulating Snap 25, Rab3a and Synapsin 1a [69, 70]

miR-9

The miR-9 expressed in pancreatic � cells is known to regulate insulin release in � cells through interactions with transcription factor oc2 (onecut-2). Over expression of miR-9 in INS1E cell line results in reduction of glucose stimu-lated insulin release by suppression of Oc2 which in turn, negatively regulates granuphilin/Slp4, Rab GTPase effectors associated with insulin secretory granules [71]. The miR-9 also decreases Sirt1 levels in � cells and reduces GSIS. Sirt1 activation is known to down regulate Ucp2 which is a nega-tive regulator of GSIS. The inhibitory effect of miR-9 on GSIS could be due to down regulation of Sirt1 and the sub-sequent up regulation of Ucp-2 [72].

Other islet specific mi RNAs in T2D

The miR-96 negatively regulates insulin exocytosis by increasing the levels of granuphilin, a negative modulator of insulin exocytosis and decreasing the expression of Noc2 [69].

The miRNA-195, miR-15a, miR-15b, and miR-16 are highly expressed in regenerating pancreas than the develop-ing mouse pancreas. Neurogenin3 (NGN3) is found to be the predicted target of these differentially expressed miRNAs. Over expression of these miRNAs in developing pancreas results in lesser number of hormone producing cells. These

miRNAs enable the regeneration of pancreas in a neuroge-nein independent pathway [73].

Acute exposure of MIN6 cells to high glucose enhances level of miR-15a, whereas the prolonged exposure reduces level of miR-15a. Insulin biosynthesis is also regulated in similar way as that of miR-15a. The uncoupling protein-2 (Ucp2) is the predicted target of miR-15a. The Ucp2 reduces ATP level and thus decreases ATP/ADP ratio and this leads to decreased glucose stimulated insulin secretion (GSIS). The miR-15a down regulates Ucp2 and thereby increases GSIS. Over expression of miR-15a increases insulin biosyn-thesis in MIN6 cells. These studies clearly indicate that miR-15a is an important regulator of insulin secretion [74]. The miR-15a level is significantly reduced in plasma of type 2 Diabetes subjects. These human studies indicate that miR-15a could be associated with � cell dysfunction or reduced insulin synthesis in type 2 Diabetes [75].

Metabolic disorders are primarily inflammatory in nature. Several studies have shown release of inflammatory cytoki-nes in type 2 Diabetes and obese population. Pancreatic �cells exposed to pro-inflammatory cytokines like interleukin-1� and TNF� induces marked up regulation of miR-34a, miR-146a and miR-21. The insulin content, insulin promoter activity, or pro insulin mRNA levels are not reduced by over expression of miR-21 or miR-146a. Minor reduction of insu-lin content and insulin promoter activity is observed when miR-34a is over expressed. The insulin content reduced by IL-1� treatment is not restored by blocking of miRNAs using anti-miRNAs. Over expression of miR-21 decreases glucose stimulated insulin secretion and also the expression of VAMP2 (vesicle associated membrane protein 2), essential for insulin exocytosis [76]. Prolonged exposure of MIN6 cells to saturated fatty acids increases the expression of miR-34a and 146 [77].

The compensatory expansion of � cell mass during obe-sity is regulated by several miRNAs. Prediabetic db/db mice and high fat diet fed mice (with compensatory � cell expan-sion) exhibits reduction in the expression of miR-383-3p. Blocking of miR-383-3p with anti miR-383-3p in INS832/13 cells results in increased � cell protection and improved sur-vival. The miR-383-3p targets G-protein coupled receptor 30 (GPCR 30) and GLP-1 receptor (GLP-1) which are known to promote � cell protection and proliferation. Inhibition of miR-383-3p also protected � cells against pro inflammatory cytokines (IL-1�, TNF-�, IFN-�) mediated apoptosis. These studies clearly indicate that miR-383-3p can be used as a target for protecting pancreatic � cells against apoptosis [78].

The miR-30d stimulates insulin production through down regulation of mitogen activated protein 4 kinase 4 (MAP4K4) and in turn activates transcription factor MafA. Islets of diabetic db/db mice have reduced levels of miR-30d [79].

The miR-21 level is elevated in islets of NOD and db/db mice [76, 77, 80]. Blocking of miR-21 with anti-miR 21 pre-vents cytokine induced � cell dysfunction and apoptosis [76]. On contrary, increased level of miR-21 down regulates the expression of programmed cell death protein-4 (PDCD4) which induces cell death through BAX family of apoptotic proteins [80, 81]. These varying results need to be confirmed with further experiments.


Role of Micro RNAs in Endoplasmic Reticulum (ER) Stress Mediated Death of � Cells

The endoplasmic reticulum is the central organelle for protein folding and maturation. Increased folding demand if overwhelms the folding capacity of ER, this compartment’s homeostasis is disrupted and its functions is compromised. This phenomenon is generally accompanied by the accumu-lation of improperly folded proteins in the lumen of ER lead-ing to ER stress. Chronic and prolonged ER stress is known to induce � cell death and also impairs glucose stimulated insulin secretion��

In response to the nature, intensity and duration of ER stress, cells initiate the unfolded protein response (UPR), which helps in balancing the ER protein load and ER folding capacity [82-85]��

The miRNAs, one of the largest classes of regulators of gene expression are known to play important role in manag-ing cellular response to ER stress. Recent evidences indicate that the ER stress response and the translation of UPR effec-tors can be modulated by miRNA�[86, 87]��

The mammalian UPR is composed of three ER trans-membrane sensors: protein kinase RNA activated like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol requiring enzyme 1 (IRE1)[88]��

Activation of IRE-1 results in unconventional cytosolic splicing of XBP-1 mRNA and subsequent generation of XBP-1(s) (Spliced XBP-1). The spliced XBP-1 induces UPR target genes like ER chaperones which help in adapting to the ER stress by increasing the folding capacity of ER. Sev-eral studies have identified the relationship between miRNAs and XBP-1 [87, 89, 90]��

Among the UPR pathway, IRE-1 pathway is the most important pro-adpative ER stress response pathway. Pan-creas and placenta express higher level of IRE1�. The down-stream targets of IRE-1� include ER-associated protein deg-radation genes such as ER degradation-enhancing a-mannosidase-like protein (EDEM) and genes that are impor-tant for protein folding such as protein disulfide isomerase-P5 [91-93]�� Prolonged activation of IRE1 causes activation of JNK kinases, which can lead to apoptotic cell death [94, 95]��

A study conducted by Lipson et al. in 2006 elucidated the role of IRE-1� in regulation of insulin biosynthesis. Tran-sient activation of IRE-1� has beneficial effect on pancreatic � cells. In contrast, chronic exposure of � cells to high glu-cose induces ER stress and hyper activation of IRE1, which leads to suppression of insulin gene expression and apoptosis [94]�� Thus IRE1 signaling may be considered as a potential target for modulating ER stress response in pancreatic �cells.

IRE-1�, the transmembrane ER stress sensor also exhib-its RNAase activity upon activation. Upton and colleagues [96]� recently reported cleavage of miRNAs by IRE-1. Sus-tained IRE1� RNAase activation causes rapid decay of miRNAs -17, -34a, -96, -125b which are translational repres-sors of Caspase-2 mRNA. The cleavage of pro adaptive miRNAs increases caspase-2 levels and subsequent mito-chondrial apoptotic pathway�

In pancreatic � cells during chronic ER stress, IRE1� re-duces miRNA-17 which increases the TXNIP (pro-oxidant thioredoxin interacting protein) mRNA stability. TXNIP activates the NLRP3 inflammasome, which cleaves pro-caspase-1 to its active form, in turn causing maturation and secretion of interleukin-1�, thus promoting inflammation and programmed cell death [97]� This finding suggests that miRNA-17 could be used as a potential target for prevention of � cell death.

Unlike miRNA-17 there are no studies on the regulation of other IRE-1� target miRNAs (miR-34a, miR-96, miR-125b) in pancreatic � cells during chronic ER stress. In the above mentioned study by Lerner et al. ER stress is induced by thapsigargin. In case of of type 2 Diabetes, the major ER stress inducers are high glucose, high saturated free fatty acids and inflammatory cytokines. Hence further studies need to be done on understanding the regulation of these specific miRNAs during physiological ER stress. These find-ing would help in identifying the specific miRNAs which are the key regulators of � cell apoptosis.

Role of miRNAs in Adipose Tissue Function in Insulin Resistance

The importance of miRNAs in adipocyte biology origi-nated from the pioneering works of Esau et al. in 2004. The miR-143 is shown to be up regulated during differentiation of adipocytes [98]. Inhibition of miR-143 with anti miRs reduces the expression of adipocytes specific genes e.g. GLUT-4, aP2, HSL and PPAR�2. Later in 2009, it is ob-served that induction of miRNAs during adipogenesis is down regulated in adipocytes from leptin deficient ob/ob and diet-induced obese mice [99]. An inverse correlation is ob-served between miRNAs induced during adipogenesis. Ec-topic expression of miR-103 and miR-143 up regulates adi-pogenesis markers. The inverse correlation among miRNAs induced during adipogenesis and obesity indicates the asso-ciation between obesity and loss of miRNAs that character-ize fully differentiated and metabolically active adipocytes. TNF� treatment down regulates many miRNAs in 3T3L1 that are normally up regulated during adipogenesis [99].

Abundance of miR-27b decreases during adipogenesis of human multi-potent adipose-derived stem (hMADS) cells [100]. Over expression of miR-27b down regulates the ex-pression of PPAR� and CEBP/� and thus inhibits adipocytes differentiation.

The miR-27a and 27b levels are also down regulated fol-lowing adipogenic induction of human adipose-derived stem cells (ASC), while the mRNA level of PHB (prohibitin) is up regulated. Prohibitin is known to play crucial role in adipo-cyte differentiation and mitochondrial function. Over expres-sion of miR-27a and miR-27b inhibit PHB expression and adipocytes differentiation. Using PHB 3'-untranslated region (3'-UTR) luciferase reporter assay, it is shown that miR-27a and miR-27b directly target PHB in human ASC. Ectopic expression of miR-27a and mir-27b impairs mitochondrial biogenesis and structure integrity and complex I (Cox I) ac-tivity accompanied by excessive reactive oxygen species (ROS) production [101]. PPAR� has also been found to be targeted and repressed by miR-130 [102].


A recent study by Mete Civelek et al, demonstrates that natural variation of miRNA expression in adipose tissue in a population of 200 men is characterized for metabolic syn-drome phenotypes. Total 24 miRNAs are found to be signifi-cantly associated with metabolic syndrome. The outcome of this study suggests a regulatory role for miR-204-5p which is predicted to be the inhibitor the acetyl-coenzyme A carboxy-lase beta (ACACB), a key fatty acid oxidation enzyme that has been shown to play a role in regulating body fat and in-sulin resistance in adipose tissue [103].

The miR-519d level in subcutaneous adipose tissue is significantly higher in obese subjects. PPAR-�, is identified as the target of miR-519d and regulates the accumulation of lipid during adipogenic differentiation [104].

Up-regulation of the expression of miR-103 and miR-107 are observed in ob/ob mice and also in diet induced obese mouse model. Silencing of miR-103 and 107 improves glu-cose homeostasis and increases insulin sensitivity. Caveolin-1 is identified as the direct target of miR-103 and 107. In obese mice increased levels of miR-103 and 107 down regu-late caveolin-1 there by leading to destabilization of insulin receptor and thus causing impaired insulin signaling. Silenc-ing of miR-103 and 107 enhance insulin signaling and smaller insulin sensitive adipocytes [105].

The miR-378/378* is an intronic miRNA located within the PPAR gamma coactivator- 1 (PGC-1�) that regulates lipid metabolism. The miR-378/378* contained within the intron of PGC-1 � is induced during adipogenesis and when over-expressed in mesenchymal precursor cell line (ST2), size of the lipid droplets increases significantly and a set of lipogenic genes are upregulated due to increased expression of PPAR�2. Knock-down of miRNA378 and/or miRNA378* decreases accumulation of triacylglycerol [106].

Mice genetically lacking miR-378 and miR-378* are re-sistant to high-fat diet-induced obesity and exhibit enhanced mitochondrial fatty acid metabolism and elevated oxidative capacity of insulin-target tissues. One of the targets of these miRNAs are carnitine O-acetyltransferase, a mitochondrial enzyme involved in fatty acid metabolism, and MED13, a component of the Mediator complex that controls nuclear hormone receptor activity, and these mRNAs are repressed by miR-378 and miR-378*, respectively [107].

The miR-221 down regulates Adiponectin receptor 1 (ADIPOR1). In addition, it is also observed that leptin and TNF-� treatments down regulate miR-221 in cultured human pre-adipocytes [108]. The adipose tissue of patients with poly cystic ovary syndrome (PCOS) has high levels of miR-93. GLUT4 expression is down regulated by miR-93, which could induce insulin resistance in these patients [109].

Micro RNAs contribute significantly to the development of insulin resistance in adipose tissue via interacting with insulin resistance responsible genes. The miR-29 family of miRNAs (miR-29a, miR-29b, and miR-29c) is up regulated in insulin sensitive tissues of Goto-kakizaki (GK) rats [110-112]. Over expression of miR-29a, miR-29b and miR-29c suppresses insulin stimulated glucose uptake in 3T3L1 adi-pocytes via indirectly inhibiting the AKT pathway [110]. The miR-320 induces insulin resistance in 3T3L1 adipocytes by targeting p85 subunit of PI3K [113].

3T3L1 adipocytes secretes milk fat globule epidermal growth factor 8 (MFG-8) associated micro vesicles. Bio-chemical and proteomic analysis reveals that these vesicles have exosomal properties and hence referred as adiposomes. The release of adiposomes is high in cells cultured in high glucose medium. Insulin and TNF-� also up regulates MFG-E8 in the micro vesicles [114]�

Adiposomes play important role in inducing systemic in-sulin resistance. The exosome like vesicles from obese mice when injected into wild type mice induces insulin resistance in wild type mice. The wild type mice after receiving the adiposomes from obese mice shows high levels of inflamma-tory cytokines, TNF� and IL-6 [115]�� This study indicates the role of adiposomes in systemic insulin resistance��

Micro vesicles released from large adipocytes of rat har-boring glycosylphosphatidylinositol anchored proteins con-tains specific transcripts and miRNAs and are transferred into acceptor adipocytes thus stimulating lipid synthesis in small acceptor adipocytes. The transcripts enclosed within the adiposomes are specific for fatty acid esterification, lipid droplets biosynthesis and adipokines. The findings from this study demonstrates the role of exosomal transcripts and miRNA in cell –cell communication [116]�

It has been shown recently that adiposomes contain ap-proximately 7000 mRNAs and 140 miRNAs [117]�� Among the miRNAs, the abundance of let-7b, miR-103, miR-146b and miR-148a is up regulated in exosomes post differentia-tion as in donor adipocytes [116]��

Studies have reported that specific miRNAs like miR-16; miR-27a, miR-146b and miR-222 are differentially regulated during adipocyte differentiation [118]. Interestingly, these miRNAs are abundantly expressed in adiposomes derived from large adipocytes compared to small adipocytes [116].

The study conducted by Parra et al., also observed that miR-222 levels increases in white adipose tissue of mice fed with high fat diet [119]. These findings clearly indicate that adiposomal miRNAs differ among small and large adipo-cytes, and hence these can be considered as potential marker to identify adipocytes hypertrophy though the exact role of these miRNAs in causing insulin resistance is not known. Future studies are recommended to understand the role of exosomal miRNA in Type 2 Diabetes pathogenesis.

Micro RNAs and Skeletal Muscle Insulin Sensitivity

Several human and animal studies have shown the impor-tance of miRNAs in skeletal muscle insulin sensitivity. The comparative expression profile of microRNAs in human skeletal muscle biopsies before and after a 3-hr euglycemic hyperinsulinemic clamp indicates that insulin down regulates the expression of 39 distinct skeletal muscle miRNAs. Mus-cle specific miRNAs e.g. miR-1, miR-133a, and miR-206 are down regulated by insulin. Insulin down regulates the ex-pression of miR-1 and miR-133a in healthy subjects also. The levels of both miR-1 and 133a are higher in STZ in-duced diabetic mice compared to normal mice. In this study by Granjon et al, it was proposed that activation of SREBP1c by insulin and subsequent down regulation of MEF2C which then decreases the levels of miR-1 and 133a [120] . Lower level of miR-133a is observed in skeletal muscle of diabetic


patients [121]. In a study conducted by Gallagher et al. in2010, it is observed that type 2 Diabetes subjects have lower levels of miR-133a, miR-206 and miR-1 when compared to healthy subjects. The Level of miR-133a decreases with in-creased glucose level but in mice model of spontaneous Dia-betes an increase in the levels of miR-133a is observed. These results indicate that hyper-insulinemia is the major contributor for reduction in miR-133a levels and this miRNA might indicates the degree of insulin resistance.

The miR-24 is significantly down regulated in in skeletal muscle of GK and Wistar rats [122]. The level of miR-24 is lower in the plasma of diabetic patients [75]. Down regula-tion of lin-28 and over expression of let-7 in skeletal muscle induces insulin resistance and impaired glucose tolerance. Let-7 down regulates multiple components of the insulin-PI3K-mTOR pathway, including IGF1R (insulin like growth factor 1 receptor (Igf1r), INSR insulin receptor (Insr), and Insulin receptor substrate 2 (Irs2) [123]. Over expression of let-7 in mice results in impaired glucose tolerance and re-duces GSIS. Anti-miR treatment of high fat fed mice in-creases lean mass and muscle mass [124].

TNF� up regulates miR-494 in C2C12 myoblasts. Over expression of miR-494 in C2C12 myoblasts suppresses insu-lin action by down-regulating phosphorylations of GSK-3�/�, AS160 and p70S6K, downstream of Akt, and also down regulates Slc2A4, a coding gene for Glut4 [125].

Insulin resistant obese Zucker (OZ) rats exhibit reduced expression of miR-1 and miR-133 in skeletal muscle [126].

The miR-23a/b, miR-24, miR-126, miR-130a,miR-424 and miR-450 are down regulated and miR-307 and let-7f up regulated in skeletal muscle of Goto–Kakizaki (GK) com-pared to Wistar rats [111, 122]. The miR-10b is down regu-lated in muscle tissue of hyperglycemic Goto–Kakizaki (GK) compared to normal Wistar rats [111].

Micro RNAs and Hepatic Insulin Sensitivity

Up regulation of miR-143 is observed in the liver of dia-betic rat [127] and obese mouse [128]. The miR-143 down regulates oxysterol-binding-protein-related protein (ORP) 8 and impairs the ability of insulin to induce AKT activation.

The miR-802 level is increased in the liver of obese mouse models. Over expression of miR-802 induces im-paired glucose tolerance and decreases insulin sensitivity. The miR-802 down regulates the expression of Hnf1b in liver which further leads to glucose intolerance, impaired insulin signaling and increased hepatic gluconeogenesis [129].

The miR-181a level is higher in insulin resistant cultured hepatocytes and in the serum of Diabetic patients. Over ex-pression of miR-181a decreases Sirt1 protein level and activ-ity and causes insulin resistance in hepatocytes [130].

Mitochondrial dysfunction in hepatocytes induces miR-96 expression and impairs insulin signaling. The miR-96 targets Insulin receptor substrate (IRS-1) and decreases gly-cogen synthesis in hepatocytes [131]. The miR-122 is the most abundant microRNA in liver. It accounts for about 70% of total liver miRNAs. The miR-122 is down-regulated in the liver of STZ-induced diabetic mice [132].Micro RNA-122

inhibition in normal mice reduces plasma cholesterol levels, increases hepatic fatty-acid oxidation and decreases rates of hepatic fatty-acid / cholesterol synthesis rates and . activates central metabolic sensor AMPK. The miR-122 inhibition in a diet-induced obesity mouse model decreases plasma choles-terol levels and shows significant improvement in liver stea-tosis, accompanied by reductions in several lipogenic genes [133].

Trajkovski et al. (2011) observed that expression of miR- 103 and miR-107 are up-regulated in the liver of ob/ob and diet-induced obese (DIO) mice [105]. Over expression of miR-103 and 107 impair glucose homeostasis. The miR-125a is up regulated in liver of spontaneously diabetic Goto-Kakizaki (GK) rats [112]� The miR-126 is up regulated in hepatocytes with mitochondrial dysfunction. The predicted target of miR-126 is IRS-1 and miR-126 induces insulin re-sistance by degradation of IRS-1 [134]��

The altered expression of miRNAs in various insulin re-sponsive tissues is depicted in Fig. (1).

ROLE OF miRNAs IN CROSS TALK AMONG INSU-LIN RESPONSIVE TISSUES

A large number of studies highlighted the importance of resident miRNAs and their roles in gene regulation within the cell. Recent studies have demonstrated that miRNAs are not only intracellular, but could be also extracellular in na-ture, and often detected in various body fluids like serum, plasma, saliva, urine and cerebrospinal fluid [135-137]�These extracellular miRNAs are either contained within small membranous vesicles like exosomes, exosome like vesicles, microparticles and apoptotic bodies or packaged with in high density lipoprotein and RNA binding proteins and nucleophosmin�[138-143]��

The miRNAs which are exported in exosomes, micro particles or associated with high density lipoproteins play important role in cell-cell communication [144-147]��Recent studies have shown that miRNAs in circulation reflect the subtle changes within the tissue of its origin like the meta-bolic status, extent of tissue injury etc [148]��

Non exosomal miRNA are also known to take part in metabolic cross talk. A recent study by Wand et al. identified the role of miR-130b in mediating the adipose skeletal mus-cle cross talk. The miR-130b is secreted into culture medium during 3T3L1 differentiation and a slight increased level is observed within the cells. It is also observed that miR-130b released from adipose tissue is taken up by skeletal muscle. The predicted target of miR-130b is PGC-1� (peroxisome proliferator activated receptor � coactivator-1�) and these findings indicate that repression of PGC-1� by miR-130b in skeletal muscle of obese individuals might contribute to the reduction of oxidative capacity and mitochondrial dysfunc-tion and lead to metabolic disorder [149].

CIRCULATING miRNAs AS BIOMARKERS OF TYPE 2 DIABETES Zampetaki and colleagues are the pioneers to identify the miRNA profile in serum of type 2 Diabetes patients. In their prospective study blood miRNA profile of 800 individuals were analyzed and miR-15a, miR-28-3p, miR-29b, miR-126


Fig. (1). Differential expression of miRNAs type 2 diabetes.

Table 1. Important miRNAs in pancreas and its target genes involved in glucose homeostasis.

miRNA Target Genes Function References

miR-375 Mtpn (myotrophin) GSIS*� [42]

miR-124a2 FoxA2, CREBP GSIS�Inhibition of � cell development

[46]

miR-9 Oc2 (onecut-2) GSIS� [49]

miR-96 Noc2 Lower the response to secretagogues [47]

miRNA-195, miR-15a, miR-15b,

miR-16

NGN3 (Neurogenin 3) Inhibition of � cell development [51]

miR-15a UCP-2 (uncoupling protein-2) Increased insulin biosynthesis [52]

miR-21 VAMP2 PDCD4 (programmed cell death protein-4)

GSIS�apoptosis�

[54, 58, 59]

miR-383-3p GPCR 30 (G-protein coupled receptor 30) GLP-1 (GLP-1 receptor) apoptosis�

[56]

miR-30d MAP4K4 (mitogen activated protein 4 kinase 4) insulin biosynthesis� [57]

*GSIS- Glucose stimulated insulin secretion

and miR-223 exhibited differential regulation in 80 partici-pants with either Pre diabetes or Type 2 Diabetes. Among these differentially regulated miRNA, miR-28-3p is up regu-lated in Type 2 Diabetes subjects. The changes in the expres-sion pattern of blood miRNA are observed 5-10 years before the onset of the disease. One of the important finding of the study is that the miR-126 which regulates vascular integrity is lower in circulation of pre diabetes and a further reduction are observed in Diabetics [75].This study provided the initial

evidence for using miRNA as biomarkers for early identifi-cation of the disease.

In another study by Kong et al. in 2011 the level of 7 Diabetes related miRNAs (miR-9, miR-29a, miR-30d, miR34a, miR-124a, miR146a and miR375) are studied. The levels of these miRNAs known for their role in insulin secre-tion are studied among normal glucose tolerant (NGT), im-paired glucose tolerant (IGT) and type 2 Diabetes subjects. In Diabetes subjects, all 7 miRNAs are significantly

miR�375miR�124a2miR�96miR�15amiR�30d

miR�124a2miRNA�195,miR�15a,�miR�15b,miR�16,�miR�21,�miR�383�3p

� cell�development�and�apoptosisPancreas

Adipose�tissue

miR�27a/bmiR�130miR�519d

Differentiation

miR�204�5pmiR�103/107miR�378miR�320

Insulin�resistance

miR�133aLin�28Let�7

Insulin�resistance

Skeletal�Muscle

Liver

miR�143miR�802miR�181amiR�96miR�126

Insulin�resistanceInsulin�secretion�and�synthesis

Fig�1.�Vinitha�and�Banerjee,�2014


Table 2. Important miRNAs in adipose tissue and its target genes involved in glucose homeostasis.

miRNA Target Genes Function References

miR-27b PPAR �CEBP/�

prohibitin

differentiation� [62, 63]

miR-27a prohibitin differentiation� [63]

miR-130 PPAR � Differentiation� [64]

miR-204-5p ACACB (acetyl-coenzyme A carboxylase beta)

insulin resistance� [65]

miRNA-519d PPAR � Adipogenesis� [66]

miR-103/107 Caveolin-1 insulin resistance� [67]

miR-378 Carnitine-O-acetyl transferase fatty acid metabolism� [69]

miR-320 P85 subunit of PI3-kinase insulin resistance� [75]

up-regulated compared with NGT and five are significantly up-regulated compared with pre-diabetes, while miRNA expression is not significantly different between NGT and pre-diabetes [67].

In a study published in 2012, Karolina et al. measured the miRNAs present in the blood and exosomes of 265 pa-tients with different health conditions associated with the metabolic syndrome. Significant up regulation of miR-27a, miR-150, miR-192, miR-320a and miR-375 is observed in patients with diabetes and a strong correlation between higher glucose levels and increased levels of miR-27a and miR-320a is identified [150].

In the recent study conducted by Pescador et al, serum miRNAs, (miR-138, miR-376a and miR-15b) are identified as potential predictive biomarkers in obesity. The miR-138 or miR-376a levels could be used to distinguish obese pa-tients from normal healthy controls, diabetic patients and obese diabetic patients. The combination of miR-503 and miR-138 could be used to distinguish diabetic from obese diabetic patients [151].

These pioneering studies demonstrated the potential of miRNAs as biomarkers for diabetes but the heterogeneity of the results obtained reinforces the need for large prospective studies to identify reliable miRNA signatures for diagnosing diabetes.

FUTURE PROSPECTS

Emerging scientific evidences accentuates the role of miRNA in regulation of glucose and lipid metabolism. Sev-eral human and animal studies have shown differential regu-lation of specific miRNAs in type 2 Diabetes. The miRNAs directly target genes involved in �-cell survival, insulin exo-cytosis, insulin signaling etc. Silencing using antagomirs and locked nucleic acids have shown that miRNAs could be used as targets for glucose control [63, 77, 105]. However these approaches have been proven only by in vitro studies and are far from clinical use. Hence modulation of miRNAs still remains as a challenge.

Another potential area of research interest is the applica-tion of circulating miRNAs as biomarkers for type 2 Diabe-tes diagnoses and prognosis. There are limited studies on serum mi- RNA profile in human. The miRNAs have all the characteristic features of a stable blood based biomarker like specificity, stability and reflective of minor and subtle changes within the cell, and hence they can be a powerful predictive marker for early identification of disease. Some studies have identified the strong predictive nature of miRNA thus preempting the disease onset but the heteroge-neity in the results obtained from multiple research groups indicate that further studies on profiling of serum miRNA and its validation has to be done across different populations to arrive at a signature profile of miRNAs during different stages of type 2 Diabetes onset and progression.

CONFLICT OF INTEREST The authors confirm that this article content has no con-

flict of interest.

ACKNOWLEDGEMENTS Declared none.

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Received: October 17, 2014 Revised: November 13, 2014 Accepted: November 18, 2014

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