Physiological Effects of Type 2 Diabetes on mRNA Processing and Gene Expression

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Physiological effects of Type 2 diabetes on mRNA processing and gene expressionLorna W Harries , Karen A Johnstone and Faer S MorrisonExpert Review of Endocrinology & Metabolism. 6.2 (Mar. 2011): p255.Articlehttp://dx.doi.org/10.1586/eem.10.76

Full Text: COPYRIGHT 2011 Expert Reviews Ltd.http://www.expert-reviews.com/loi/eemFull Text:

Author(s): Faer S Morrison 1 , Karen A Johnstone 1 , Lorna W Harries [[dagger] ]2

Keywords

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alternative splicing; diabetes; epigenetic; gene expression; glucolipotoxicity; hyperglycemia; hyperlipidemia;miRNA; oxidative stress

Type 2 diabetes & glucolipotoxicity

Type 2 diabetes mellitus (T2D) is an inflammatory state characterized by hyperglycemia and hyperlipidemia, and byinsulin resistance in insulin-dependent tissues, which include the liver, adipose tissue and skeletal muscle (Figure 1)[1,2] . In the early stages of T2D, insulin resistance is compensated for by increased insulin secretion and an increase

in [beta]-cell mass [3] . However, as glucose and free fatty acid levels rise, insulin sensitivity declines leading to

glucolipotoxicity of the [beta]-cell, resulting in [beta]-cell failure and overt diabetes [3-6] . The deleterious

effects of elevated glucose and fatty acids on [beta]-cell function are thought to be mostly synergistic [7-10] .Evidence indicates that this synergistic effect may be due to a glucose-dependent decrease in oxidation of free fattyacids in the [beta]-cell, alongside increased esterification of fatty acids to neutral lipids in the presence of elevated

glucose and fatty acids [11] . These alterations in lipid metabolism could then result in neutral lipid accumulation oraccumulation of lipid signaling molecules in the cytoplasm, resulting in insulin resistance, [beta]-cell dysfunction

and eventual [beta]-cell apoptosis[11,12]

.Gene-expression changes in T2D as novel drug targets for T2D therapy

Impaired insulin secretion and insulin resistance cause a chronic state of hyperglycemia in diabetes, which, if leftuntreated, results in serious microvascular (retinopathy, nephropathy, peripheral nerve damage) and macrovascular

(coronary artery disease, limb amputation, stroke, heart attack) pathologies [13] . Currently, a combination ofglucose-lowering or incretin-based drug treatments, and changes in lifestyle such as strict diet and exercise, are the

gold standard in diabetes therapy [14,15] . However, with an ever-increasing body of evidence implicating diabetic

physiologies, including high glucose and elevated lipids in aberrant expression of dozens of genes [16-19] , drugstargeted at altering gene expression are attractive candidates for future therapy in T2D, for example those targeted at

epigenetic gene regulation[20]

. In this article, we will consider some of the evidence for aberrant gene expression inT2D, particularly where it relates to hyperglycemia, hyperlipidemia or oxidative stress. We will then consider theevidence for some of the possible mechanisms resulting in aberrant gene expression, with a focus on alternativeRNA processing, epigenetic regulation and regulation by miRNAs.

Widespread gene-expression changes in T2D

A large number of genes have been shown to be differentially expressed in islets isolated from T2D individuals

compared with those isolated from controls [21] . This includes genes known to be important in [beta]-cell function,including hepatocyte nuclear factor 4[alpha] (HNF4A ) and the insulin receptor (INSR ) and a number of genesinvolved in glucose metabolism. Marselli et al. obtained [beta]-cell-enriched islet samples from T2D donors using

laser capture microdissection and found deregulation of multiple cellular pathways [22] . Of particular interest werederegulation of islet transcription factor genes, and genes involved in glucose and lipid metabolism, the oxidativestress response, insulin signaling and [beta]-cell regeneration [22] . There is substantial evidence for aberrant geneexpression in all other tissues affected by T2D. Two groups used gene set enrichment analysis to study the

gene-expression profile in diabetic skeletal muscle compared with nondiabetic skeletal muscle [18,19] . They found a

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coordinate downregulation of genes regulated by transcriptional coactivator peroxisome proliferator-activatedreceptor- (PPAR-) coactivator 1[alpha], which is involved in oxidative metabolism in theskeletal muscle of diabetic patients. This is interesting because mitochondrial dysfunction and defective oxidative

phosphorylation have been suggested to be involved in the pathogenesis of T2D [23-25] . Since then, other groupshave compared gene-expression patterns in diabetic subjects with healthy controls and have found deregulation of

genes involved in metabolism, cell signaling and stress response in diabetic muscle and adipose tissue [22,26] .Meugnieret al. performed global gene-expression analysis on skeletal muscle and adipose tissue of healthy subjects

exposed to 3 h of hyperglycemia while maintaining fasting insulin levels [27] . They found 316 genes deregulated inskeletal muscle and 336 genes deregulated in adipose tissue, with more than 80% of those genes beingdownregulated. Almost all biological processes were affected, with a significant increase in expression of genes inthe antioxidant metallothionine family and forkhead transcription factor (FOXO) family, which has a role in

[beta]-cell function and protection against oxidative stress [28-31] .

Changes in gene expression induced by hyperglycemia or hyperlipidemia

The individual effects of hyperglycemia and hyperlipidemia are more readily studied in animal models of T2D orinvitro . Ghanaat-Pouret al. studied hyperglycemia and hyperlipidemia-induced gene-expression differences in

nondiabetic Wistar rats compared with diabetic and hyperglycemic Goto-Kakizaki (GK) rats [16,17] . Genome-widemicroarray analysis revealed glucose-induced deregulation of gene expression in diabetic islets. The genes found to

be deregulated have diverse functions, including roles in ion transport, metabolism, signal transduction, the cellcycle and apoptosis [16] . Nondiabetic islets exposed to hyperlipidemia in vitroby treatment with the fatty acid

palmitate, also show altered expression of genes with diverse functions, including those involved in insulin gene

expression, inflammation and metabolism [17] . Most significantly, the expression ofPpardwhich is involved in lipidoxidation and cell proliferation, is decreased following palmitate exposure, potentially leading to decreased lipidoxidation and subsequent intracellular lipid accumulation. Kelleret al. compared gene expression in obesity-

induced diabetes-resistant (C57BL/6Lepob/ob ) and diabetes-susceptible (BTBRT+ tf Lepob/ob ) strains of mice[32] . They identified obesity-dependent gene expression differences in islets, liver and adipose of both strains, anddifferential gene expression in islets and adipose that strongly correlated with plasma glucose, including a glucose-dependent increase in Ppara expression. Most significantly, they identified an obesity-dependent compensatoryincrease in expression of cell cycle regulatory genes in the islets of diabetes-resistant C57BL/6, but not diabetic,

BTBR mice, along with increased [beta]-cell proliferation in C57BL/6 mice only [32] . This example illustrates howgenetic background can influence diabetes susceptibility through differential regulation of gene expression inresponse to hyperlipidemia.

Oxidative stress as a potential mechanism of glucotoxicity-induced gene-expression changes

There are several theories explaining how hyperglycemia may cause the pathologies associated with T2D. Onepotentially unifying mechanism behind these theories points to overproduction of superoxide as the main cause

contributing to diabetic complications [13,33,34] . Moreover, production of reactive oxygen species (ROS) is thought

to mediate glucose-induced [beta]-cell dysfunction [3,35] . Mitochondria are the main source of ROS in the cell

and, under normal conditions, ROS act as cell signaling molecules with vital roles in many cellular processes [36] .

They have important roles during development, particularly in programmed cell death [37] , and have recently been

shown to be involved in regulation of glycolysis in skeletal muscle [38] . Oxidative stress occurs when more ROS isproduced than can be scavenged by cellular antioxidant defenses.

[beta]-cells contain lower levels of antioxidant enzymes (e.g., glutathione peroxidase, catalase and superoxide

dismutase) than other tissues, and thus are susceptible to ROS [39-41] . The first direct evidence that glucose

increased intracellular ROS in islets came from Tanaka et al.[42] , and this link between hyperglycemia and

oxidative stress has been strengthened by subsequent experiments [43,44] . Yu et al. suggested that this could be dueto changes in mitochondrial morphology, as they observed mitochondrial fragmentation and hyperpolarization of the

mitochondrial membrane with high glucose exposure [25] . Markers of oxidative stress were found to be higher in

T2D islets compared with normal islets, and this correlated with impaired insulin secretion [45] .

Oxidative stress is a potential mechanism whereby hyperglycemia and hyperlipidemia can cause changes in geneexpression. Yang et al. performed a microarray analysis and found an increase in oxidative stress markers in theliver of high-fat diet-fed mice compared with controls, with decreased expression of fatty acid oxidation genes and


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deregulation of other lipid metabolism and cholesterol synthesis pathways [46] . However, these effects werenormalized by the antioxidant lipoic acid. Similar expression changes were found in the small intestine of mice fed a

high-fat diet [47] , and together, these results indicate that oxidative stress may mediate high-fat diet-induceddyslipidemias.

Cellular components are damaged by higher than normal levels of ROS, with oxidative damage of proteins bycarbonylation, oxidative stress-induced lipid peroxidation and ROS-mediated changes in the DNA base sequence[48,49] . The RNA message is more vulnerable to ROS (especially the very destructive hydroxyl radical) than

proteins, lipids or even DNA, and is subject to various types of RNA base damage [50] . Since control of mRNAsplicing is highly sequence dependent, it is unsurprising that splicing specificity has been shown to be sensitive to

factors such as increased ROS, which may cause base damage [51,52] . There have also been reports of oxidative

stress altering the expression of miRNAs [53,54] . Microarray analysis showed deregulation of 23 miRNAs in human

fibroblasts exposed to hydrogen peroxide (H2 O2 )[54] , including miR-143 , which is elevated in the adipose tissue

of high-fat diet-fed mice [55] , and miR-125a , which is found to be elevated in hyperglycemia [56] . These alterationsin miRNA expression are prevented by the free radical scavenger cysteine, showing that the effects of H2 O2 are

mediated by ROS [54] . Oxidative stress has also been shown to alter epigenetic gene modifications [57] and has beenshown to alter the activity of histone deacetylase 2, which is associated with extracellular matrix protein

accumulation in kidneys leading to diabetic nephropathy [58] . There are numerous alternative mechanisms ofgene-expression regulation apart from alternative mRNA processing, epigenetic and miRNA-mediated regulation ofgene expression. Since the role of oxidative stress in diabetes remains unclear, and the aforementioned mechanismshave been suggested as being responsive to ROS, the remainder of this article is focused on how splicing, miRNAsand epigenetics can be altered in T2D and by hyperglycemia or hyperlipidemia.

Potential changes in alternative splicing of mRNA in T2D

Alternative splicing of mRNA contributes greatly to protein diversity and provides a mechanism for regulation ofgene-expression levels. It is estimated that as many as 95% of multi-exon gene transcripts undergo alternative

splicing [59] . Aberrant splicing caused by mutations in sequences involved in regulating splicing (Figure 2) is a

relatively common disease mechanism [60,61] . Such mutations can inactivate the normal splice site, activate a

cryptic splice site or create an ectopic splice site, and result in exon skipping and/or intron inclusion [62] , leading tothe production of defective mRNAs, which may be targeted for degradation by the nonsense-mediated decay

pathway or be translated into deleterious proteins.

It has been shown that hyperglycemia and hyperlipidemia can have effects on alternative RNA splicing. A smallnumber of genes have been shown to undergo changes in the expression of specific isoforms in T2D and T2Danimal models, and several of these have been shown to specifically respond to either hyperglycemia orhyperlipidemia in vitro . The human insulin (INS) gene contains a cryptic 5 splice site in intron 1, which gives

rise to an alternatively splicedINSisoform with increased translation efficiency [63] . The expression of this

alternativeINSsplice variant was shown to increase with high glucose in vivo and in vitro[63] . A comparableisoform of the mouse orthologIns2 has also been identified and was also shown to have an increased translation

efficiency, thought to be due to an altered secondary structure of the mRNA [63] . The expression ratio of thisisoform relative to nativeIns2 was also shown to increase in diabetic and nondiabetic obese mice and in insulin-resistant nonobese mice. Moreover, incubation of primary mouse islets from lean wild-type mice in high glucose for

70 h increased the ratio of this isoform relative to native insulin by 15-fold [63] . The expression of polypyrimidine

tract-binding protein (PTB) was shown to decrease in response to hyperglycemia in human islets [64] . This change

was postulated to be due to inhibition ofPTB translation by miR-133 , which is induced by high glucose [64] . PTB

is required for stabilization of insulin mRNA by binding to its 3-untranslated region (3-UTR) [65] and is a

negative regulator of alternative mRNA splicing [66] . Therefore, decreased PTB expression in response tohyperglycemia could provide a potential mechanism whereby hyperglycemia can induce alternative splicing ofINS.The insulin receptor also has two alternatively spliced isoforms, differing in affinity for insulin and IGFs, and the

splicing of these isoforms can be regulated by hyperglycemia[67]

.

A number of genes with roles in metabolism and transport have also been shown to have altered isoform expressionprofiles in T2D. The facilitated glucose transporter solute carrier family 2 (SLC2A or GLUT family of glucosetransporters) have a central role in glucose homeostasis. A splice variant of the Slc2a9 gene (Slc2a9b ) was found to


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be upregulated in the kidney and liver of streptozotocin (STZ)-induced diabetic mice, which is postulated to be in

response to hyperglycemia [68] . Harada et al. identified a novel truncated gastric inhibitory polypeptide receptor (Gipr) splice variant, which is upregulated relative to wild-type Giprin isolated islets from high-fat diet-fed obese

mice [69] . This alternative transcript is hypersensitive to gastric inhibitory polypeptide, which stimulates insulin

secretion and is therefore postulated to contribute to hyperinsulinemia and increased fat accumulation [69] . TheATP-binding cassette transporter A1 (ABCA1) has a role in cholesterol homeostasis by transporting phospholipids

and cholesterol across plasma membranes [70] . Singaraja et al. generated humanABCA1 bacterial artificial

chromosome transgenic mice in order to determine the effect of diet on the expression of alternative ABCA1transcripts and found that transgenic mice fed with a high-fat diet expressed higher levels of isoforms containing an

alternative exon 1 (exon 1b, exon 1c or exon 1d) in a variety of tissues [71] . These isoforms do not alter the proteincoding potential ofABCA1 but are likely to indicate alternative promoters that could be differentially regulated.Interestingly, significantly higher levels of the ABCA1 protein were detected in the liver and macrophages of mice

fed a high-fat diet [72,73] . The authors postulate that the PPARs, which are coordinators of lipid metabolism and are

thought to be important in the pathogenesis of T2D [74] , may differentially regulate the alternativeABCA1transcripts [71] . Another gene product, glutamic pyruvate transaminase (GPT), has an important role in linkingcarbohydrate and amino acid metabolism and it is proposed that GPT may have a role in the pathogenesis of T2D[75,76] . The expression of an alternatively spliced isoform ofgptwith altered catalytic activity was found to

increase in the liver of STZ-induced diabetic gilt-head bream, Sparus aurata [77] .

Several genes implicated in the pathogenesis of diabetic complications are aberrantly spliced in T2D. The Slo geneis crucial for erectile function, which is commonly disrupted in men with T2D. The predominant isoform in control

rats is the splice variant SVcyt, which has a cytoplasmic location [78] . Davies et al. identified a novel channel-forming splice variant (SV0 ) that was upregulated in STZ-induced diabetic Fischer rats, with a 40-fold increase in

SV0 compared with SVcytafter 2 weeks and an 80-fold increase after 8 weeks [78] . Diabetes is also associated withincreased expression ofVEGF, especially in the retina and glomeruli, which may contribute to retinopathy andnephropathy, both common diabetic complications. A study by Zygalaki et al. showed that diabetic patients withcoronary artery disease had higher levels of the alternatively spliced VEGFtranscripts VEGF121and VEGF165 inthe left internal mammary artery compared with nondiabetic patients who also had coronary artery disease, which

meant total VEGFexpression was higher in these patients [79] .

Epigenetic modifications of genes in T2D

Epigenetics has been described as 'the structural adaption of chromosomal regions so as to register, signal or

perpetuate altered activity states' [80] . Epigenetic modifications including DNA methylation and histonemodifications influence the selective expression of genes, giving rise to the hundreds of cellular phenotypes inmammals, although each cell making up an organism contains the same genetic material. Epigenetic regulation ofgene expression (Figure 3) emerged as the mechanism underlying 'fetal programming', that is, how environmentalcues in utero , including nutrition, can induce different metabolic phenotypes in the offspring during critical time

windows of development [81] . The role for epigenetic abnormalities in the development of cancer has beenrecognized for more than two decades. It is however becoming apparent that such changes to the genome in response

to various stimuli can occur throughout life, with epigenetic modifications occurring in response to diet and lifestylecontributing to obesity, T2D and cardiovascular disease [82] .

Several studies have shown that hyperglycemia can cause altered histone acetylation and result in increased

expression of inflammatory genes. Diabetes is associated with a 'metabolic memory' [83] , meaning that even aftera subsequent prolonged period of normoglycemia following hyperglycemia, the inflammatory phenotype is still

present. A human acute monocytic leukemia cell line (THP-1) cultured in high glucose showed increased expression

of inflammatory genes [84] . Chromatin immunoprecipitation analysis of NFB-regulated inflammatorygenes showed increased binding of histone acetyltransferases and the NF-B coactivators, E1A binding

protein 300 (EP300) and K(lysine) acteyltransferase 2B (KAT2B), to TNFand cyclooxygenase 2 (COX2 )promoters and increased acetylation of activating histone lysine residues. Validation in vivo was obtained when it

was shown that monocytes from T2D patients showed increased acetylation of histone H3 at the NF-B-dependent inflammatory genes TNFand COX2 compared with monocytes from nondiabetic patients [84] . THP-1monocytes cultured in high glucose also showed altered histone lysine methylation and expression of several genes

involved in pathways relevant to diabetes [85] . Moreover, the histone methylation status of the histone H3 lysine 9


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demethylase - lysine (K)-specific demethylase 4A (KDM4A ) - was altered by high glucose, which could providea mechanism by which high glucose can 'derepress' silenced genes, as histone H3 lysine 9 dimethylation is

generally associated with gene repression [85,86] .

El-Osta et al. have also demonstrated changes to histone modifications in human aortic endothelial cells cultured in

high glucose for 16 h followed by 6 days at physiological glucose concentrations [87] . They observed an increase inNF-B activity associated with increased expression of the NF-Bp65 subunit, along withincreased NF-B-dependent inflammatory gene expression with high glucose, which continued through

normal glucose levels. Following glucose treatment,p65 chromatin adopted a more open formation, with a 2.5-foldincrease in histone H3 lysine 4 methylation of thep65 promoter, mediated by the histone methyltransferase SETdomain containing (lysine methyltransferase) 7 (SETD7) and histone H3 lysine 9 demethylation, mediated by lysine-

specific demethylase 1 (LSD1) [88] . Interestingly, increasedp65 and inflammatory gene expression were preventedby inhibiting mitochondrial superoxide production, and these results were validated in nondiabetic uncoupling

protein 2 transgenic mice, which produce increased intracellular ROS at normal glucose levels [87] .

Vascular inflammation and cardiovascular disease are pathologies commonly associated with diabetes. In a leptin

receptor-deficientLeprdb/db(db/db ) mouse model of diabetes, the activating chromatin modification, histone H3lysine 4 dimethylation, was found to be increased at TNF-[alpha]-dependent inflammatory gene promoters in

vascular smooth muscle cells of diabetic mice compared with controls [89] . There was also a concomitant decrease

in protein levels of LSD1, which is known to demethylate histone H3 lysine 4 at its target promoters [90] . Humanvascular smooth muscle cells treated with high glucose showed the same effect, with increased expression of

inflammatory genes and decreased LSD1 [89] . In a separate experiment, vascular smooth muscle cells from db/dbmice showed decreased histone H3 lysine 9 trimethylation at inflammatory gene promoters, along with aconcomitant decrease in protein levels of the histone methyltransferase histone H3 lysine 9 trimethylase suppressorof variegation 3-9 homolog 1 (SUV39H1). These changes persisted after culture of the db/db mouse vascularsmooth muscle cells for 2 months in physiological glucose concentrations, illustrating the 'memory' displayed by

diabetic cells [91] .

Diabetes is also associated with impaired oxidative metabolism and mitochondrial function. Several studies haveshown changes to oxidative phosphorylation genes, mediated by epigenetic mechanisms. The regulator of oxidative

phosphorylation genes and fatty acid metabolism, PPAR- coactivator 1[alpha] (PPARGC1A ) wasshown to have reduced expression in T2D islets and its promoter was hypermethylated [92] . Barrs et al.

performed a whole-genome promoter methylation analysis on DNA extracted from skeletal muscle of T2D

individuals, and found hypermethylation ofPPARGC1A compared with controls [93] . PPARGC1A hypermethylation

was also seen in human myotubes exposed to the saturated fatty acid palmitate [93] .

Several studies have shown deregulation of various chromatin remodeling complexes and enzymes responsible forepigenetic modifications of DNA and histones. High glucose is associated with activation of the transcriptionalcoactivator and histone acetyltransferase EP300, and is thought to mediate extracellular matrix protein build-up inthe heart in diabetes, through increased activity of NF-B and JUN, leading to cardiomyopathy and heart

failure [94,95] . Deregulation of such enzymes could subsequently cause deregulation of a whole network of genesand contribute to various pathologies associated with T2D.

miRNA-mediated regulation of genes in T2D

MicroRNAs are small noncoding RNAs 20-23 nucleotides in length that act to either fine-tune gene expression orturn gene expression on or off (Figure 4). miRNAs regulate the activity of approximately 50% of protein codinggenes in mammals and coordinate practically every cellular process, including cell viability, lipid and glucose

metabolism, insulin secretion and pancreas development [96-99] , and modify chromatin structure by regulating the

expression of chromatin-modifying enzymes [100,101] . The mechanism by which miRNAs can alter the expression ofgenes depends on the degree of sequence similarity between the miRNA and the target mRNA. If base-pairing isnear perfect between the miRNA and mRNA, this may cause mRNA degradation or, alternatively, with less

sequence similarity, mRNA destabilization and/or inhibition of translation [102,103] . Each miRNA could potentiallyregulate the expression of hundreds of genes, and these gene targets can be predicted bioinformatically using

computer algorithms [104] . miRNAs are involved in the pathogenesis of several diseases, including metabolic

disorders such as T2D, and represent novel therapeutic targets for these diseases [105,106] .


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The expression of miRNAs and their regulation of gene expression in insulin target tissues has recently become thesubject of much research. Inhibition of the liver-specific miRNA, miR-122 , in high-fat diet-fed mice and normalmice using antisense oligonucleotide inhibition caused various alterations in cholesterol and lipid metabolism,including decreased adipose degeneration in the liver, decreased hepatic fatty acid synthesis and increased fatty

acid oxidation, indicating that miR-122 may be a potential therapeutic target in metabolic disease [107,108] .Microarray analysis of gene expression in the liver revealed concordant deregulation of several genes involved in

carbohydrate, cholesterol and fatty acid metabolism [107] . In addition to miR-122 , miR-33 is involved in cholesterol

metabolism [109] . This miRNA, which is more widely expressed than the liver-specific miR-122 , is encoded withinan intron of the sterol regulatory element binding transcription factor (SREBF) gene, which regulates cholesterol andlipid homeostasis [110] . Studies in several human and murine cell lines and mouse models have shown that SREBFand miR-33 expression is inversely correlated with cellular cholesterol levels and that miR-33 targets the ABCA1

protein, which has a role in reverse cholesterol transport and high-density lipoprotein (HDL) generation [110-114] . Inhumans, SREBF1 encodes miR-33a , which is equivalent to mouse miR-33 , and SREBF2 encodes miR-33b , which

is not conserved in mice [109,110] . Since SREBF2 expression is induced by insulin [115,116] , this could mean that inthe insulin-resistant state seen in T2D, increased miR-33b expression could result in decreased ABCA1 and lowHDL levels. Therefore, miR-33b antagonism could be a promising way to increase HDL levels in T2D and improve

the blood lipid profile [109,113] .

In pancreatic islets, Poy et al. demonstrated that the most abundant islet miRNA, miR-375 , regulates insulinsecretion by inhibiting glucose-stimulated insulin secretion, and they later found that diabetic and insulin-resistant

ob/ob mice have increased miR-375 expression in their islets [98,117] . Li et al. subsequently showed that miR-375also has a role in lipoapoptosis of [beta]-cells, demonstrating how miRNAs can target multiple cellular pathways

by regulating multiple genes [118] . Insulin-secreting NIT-1 cells transfected with miR-375 showed enhancedpalmitate-mediated apoptosis, along with significantly decreased levels of the anti-apoptotic V1 protein, which isalso a predicted target ofmiR-375. Antisense oligonucleotide inhibition ofmiR-375 prevented the decrease in V1

protein induced by palmitate [118] . Other miRNAs have been implicated in [beta]-cell dysfunction, includingmiR-34a and miR-146, whose expression in the mouse [beta]-cell line MIN6B1 (a subclone of MIN6 cells)

increased in response to palmitate treatment [119] . In isolated rat islets, palmitate treatment also increasedexpression ofmiR-34a and miR-146, whereas glucose treatment only increased miR-34a expression. Apoptosis was

increased in MIN6B1 cells transfected with miR-34a and miR-146, and transfection with anti-miR-34a andanti-miR-146antisense oligonucleotides was able to partially protect against palmitate-mediated apoptosis.Increased miR-34a expression has been associated with activation of the stress response p53 pathway. Moreover,levels of the miR-34a target vesicle-associated membrane protein 2 (VAMP2) snare protein decreased with miR-34aoverexpression. Deregulation of these miRNAs and others [120] may contribute to [beta]-cell dysfunction,

including secretory defects, defective [beta]-cell exocytosis and [beta]-cell apoptosis [119] .

Secretion of adipokines from white adipose tissue regulates many metabolic processes and has a role in thepathogenesis of metabolic disease and insulin resistance. Insulin-resistant adipocytes have an altered miRNA profile

compared with normal adipocytes [121] . Ling et al. treated adipocytes with high glucose and high insulin to renderthese cells insulin resistant, which increased the expression of 50 miRNAs and decreased the expression of 29

miRNAs [121] . Antisense oligonucleotide inhibition ofmiR-320 improved glucose uptake in response to insulin,showing how miRNAs can contribute to the development of insulin resistance [121] . The miRNA miR-143 has beenshown to regulate adipocyte differentiation, and is also very highly expressed in the white adipose tissue of leptin-

deficient (Lepob/ob ) ob/ob mice [122] . Takanabe et al. found that feeding mice a high-fat diet upregulated miR-143, which correlated with bodyweight and mesenteric fat weight, along with increased signs of impaired glucose

tolerance and insulin resistance [55] . Gallagheret al. showed that the miRNA profile is altered in diabetic skeletal

muscle and implicated altered miRNAs in skeletal muscle insulin resistance [123] .

MicroRNA expression in insulin targets tissues in nonobese, spontaneously diabetic GK rats was compared withmiRNA expression in normal Wistar rats. miRNAs miR-29a , miR-29b and miR-29c were found to be highly

expressed in GK rat skeletal muscle, liver and adipose compared with controls [124] . High glucose and insulintreatment of 3T3-L1 adipocytes significantly increased miR-29a and miR-29b expression and rendered the cellsinsulin resistant. The state of insulin resistance was mimicked by transfection of the cells with miR-29a , miR-29band miR-29c , which decreased insulin stimulated glucose uptake by 50%, due to decreased insulin receptor

substrate 1 phosphorylation and repression of phosphoinositide-3-kinase regulatory subunit 1[alpha] [124] , which


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is upstream of the glucose transporter protein GLUT4. Herrera et al. compared GK rats with normoglycemic BrownNorway rats and found that miR-125a was overexpressed in the liver and adipose tissue of GK rats, along withsignificant alteration of genes involved in fatty acid metabolism, glycolysis and gluconeogenesis, including several

miR-125a target genes [56] . In a subsequent study, they compared three strains of rats: hyperglycemic GK rats,

intermediate glycemic Wistar Kyoto rats and normoglycemic Brown Norway rats [125] . miR-103 and miR-195expression increased in the liver with increasing glycemia. Expression ofmiR-222 and miR-27a increased in adiposetissue with increasing glycemia and miR-10b decreased in muscle with increasing glycemia. One pathway predictedto be affected by deregulation of these miRNAs is the MAPK pathway, which is involved in insulin signaling[125,126] .

Deregulation of miRNAs has also been shown to be directly involved in the pathogenesis of various diabeticcomplications. Overexpression ofmiR-377in human mesangial cells caused increased fibronectin production andincreased oxidative stress, thought to be mediated by the miR-377target genes p21 protein-activated kinase 1 (PAK1) and superoxide dismutase (SOD1/2 ), respectively. Human mesangial cells cultured in high glucose not onlyshowed increased miR-377expression, but also decreased PAK1 , SOD1 and SOD2 gene expression, contributing to

the development of diabetic nephropathy [127] . Du et al. found that miR-29a , mentioned previously in relation to

insulin resistance [124,125] , may also contribute to diabetic nephropathy [128] . miR-29a expression is downregulatedin HK-2 cells cultured in high glucose, along with increased collagen IV production. Transfection ofmiR-29a intoHK-2 cells cultured in normal glucose also caused increased collagen IV production, contributing to

tubulointerstitial fibrosis and diabetic nephropathy [128] .

Aberrant expression of miRNAs may also contribute to diabetic cardiomyopathy and vascular dysfunction. Cultureof the rat cardiomyocyte cell line H9C2 in high glucose conditions increased miR-1 expression, leading to

mitochondrial dysfunction and apoptosis of cardiomyocytes [129] . Transfection ofmiR-1 blocked the protectiveeffect of putative miR-1 target IGF1, showing that miR-1 and IGF1 may mediate the effect of high glucose to cause

apoptosis of cardiomyocytes [129] . miR-1 was also found to be overexpressed in diabetic rabbit heart [130] . Humanumbilical vein endothelial cells cultured in high glucose showed increased expression ofmiR-221 , along withdecreased expression of KIT, which was prevented by antisense oligonucleotide inhibition. Changes in miR-221 andKIT inhibited human umbilical vein endothelial cell migration, which shows that miR-221 may have a role in

vascular dysfunction in diabetes [131] . Moreover, miRNAs are deregulated in the plasma of T2D individuals, which

could potentially contribute to the impaired peripheral angiogenic signaling seen in T2D [132] .

Summary

It is apparent that diabetes-associated factors such as hyperglycemia and hyperlipidemia have many pathologicaleffects on cells. Alterations in gene-expression levels by disruption to the normal splicing patterns of genes,epigenetic changes or deregulation of miRNA expression have the potential to influence progression from impairedglucose tolerance to overt diabetes, and may also influence the development of diabetic complications.

Expert commentary

Much research is being carried out to investigate the changes in gene expression associated with T2D, althoughcause and effect are not always well defined. One potentially unifying mechanism for the gene-expression changes,disruption to normal mRNA processing and abnormal gene regulation may be increased ROS production by themitochondria. This theory seems feasible because hyperglycemia and hyperlipidemia increase intracellular oxidativestress, which can alter splicing, epigenetic regulation and miRNA expression. Together, this research paints a pictureof dramatically altered gene expression in diabetes across almost all biological pathways and in all diabetes-relatedtissues. Especially important is the disruption of metabolic pathways, including glucose and lipid metabolism andtransport, insulin signaling pathways, inflammatory pathways, oxidative phosphorylation and the oxidative stressresponse. Disruption of these pathways may exacerbate the diabetic phenotype, enable metabolic memory andcontribute to diabetic complications such as coronary heart disease, nephropathy and vascular dysfunction.

Five-year view

In the next 5 years, research will identify further genes and pathways that are altered in diabetes due tohyperglycemia and hyperlipidemia. New and effective diabetes therapies will hopefully emerge to preventgene-expression abnormalities. These may include drugs targeting inflammation and inflammatory pathways andchromatin-modifying and epigenetic drugs, such as histone deacetylase inhibitors, which have already been shown to


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be effective in the treatment of schizophrenia. Antioxidant drugs that have cytoprotective properties may beparticularly useful in protecting the [beta]-cell from glucolipotoxicity-induced apoptosis. Another promisingtherapy is the use of antisense oligonucleotide inhibition. With the development of efficient carriers to improvecellular uptake, antisense oligonucleotide inhibition could be used to target aberrant miRNAs and splicing defects indiabetes. In our opinion, the correction of aberrant mRNA processing is most amenable to alteration, and potentialnew therapies utilizing small-molecule moderation of splicing are already in clinical trials for Duchenne muscular

dystrophy [133] . One problem of targeted therapies such as these will be the loss of [beta]-cell mass at the time ofdiagnosis and stem-cell therapies could therefore provide the most promising hope of [beta]-cell renewal.

However, despite the emergence of these promising therapies, what must change over the next half-decade are socialand political attitudes that should do more to encourage a healthy diet and lifestyle, otherwise T2D will continue torise in developing countries.

Key issues

* Type 2 diabetes (T2D) is a complex disorder characterized by chronic hyperglycemia and hyperlipidemia, alongwith insulin resistance, altered insulin secretion and other metabolic disturbances.

* T2D is associated with changes in gene expression across many biological pathways, some of which may beexplained by hyperglycemia and/or hyperlipidemia.

* Alternative splicing of many genes involved in various aspects of metabolism and in the pathogenesis ofdiabetic complications is altered in hyperglycemic or hyperlipidemic states.

* Epigenetic regulation of gene expression is altered in diabetes, including deregulation of chromatin-remodelingmachinery. Epigenetic deregulation is associated with inflammation, metabolic memory, mitochondrial dysfunctionand various diabetic pathologies.

* miRNA expression is also deregulated in diabetes, resulting in deregulation of target gene expression. Aberrantexpression of miRNAs is implicated in insulin resistance, [beta]-cell dysfunction and diabetic complications.

* A potential mechanism mediating these changes may be mitochondrial dysfunction and increased intracellularoxidative stress, since hyperglycemia and hyperlipidemia increase the production of reactive oxygen species.

* Targeting the mechanisms that underlie the observed changes in gene expression may provide potential newtherapeutic targets for the treatment of T2D.

CAPTION(S):

Figure 1. The pathophysiology of Type 2 diabetes.

Clinical hyperglycemia in diabetes results from a vicious cycle of events involving several organs. The endocrinefunction of the pancreas is to maintain glucose homeostasis by secretion of insulin and glucagon from the islets ofLangerhans. Insulin is secreted after glucose ingestion and stimulates glucose uptake by insulin-responsive tissuesand glucose utilization and storage, whereas glucagon is secreted in response to low blood glucose and stimulates

hepatic gluconeogenesis. Decreased pancreatic [beta]-cell mass and impaired islet function in diabetes result inimpaired insulin secretion from the pancreatic [beta]-cells. This occurs alongside impaired suppression of

glucagon secretion from the islet [alpha]-cells [60] , which exacerbates the already high basal hepatic glucose

production associated with Type 2 diabetes [134] . The driving force of hyperglycemia is insulin resistance of allinsulin-dependent tissues, which is manifested by reduced metabolic clearance of glucose by peripheral tissues,

defective glucose transport into tissues and subsequent defective metabolism, storage and utilization of glucose [60]

. In addition, altered fatty acid metabolism and accumulation of lipids in muscle, liver and pancreas, specifically

intracellular lipotoxic metabolites [135] , results in an increase in peripheral insulin resistance and subsequentglucolipotoxicity of the [beta]-cell, further impairing insulin secretion.

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Author Affiliation(s):

[1] Institute of Biomedical and Clinical Science, Peninsula College of Medicine and Dentistry, University of Exeter,Exeter, EX2 5DW, UK

2 [email protected]

Author Note(s):

[dagger] Author for correspondence

Acknowledgement

The authors would like to thank Jonathan Locke for his critical appraisal of the manuscript.

Financial & competing interests disclosure

The authors thank the Peninsula College of Medicine and Dentistry for providing funding. Lorna W Harries is

supported by Wellcome Trust grants number 081278/Z/06/Z and 099845/Z/09/Z. The authors have no other

relevant affiliations or financial involvement with any organization or entity with a financial interest in or

financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Faer S Morrison, Karen A Johnstone, Lorna W Harries

Source Citation (MLA 7th Edition)Harries, Lorna W, Karen A Johnstone, and Faer S Morrison. "Physiological effects of Type 2 diabetes on mRNA

processing and gene expression."Expert Review of Endocrinology & Metabolism 6.2 (2011): 255+.AcademicOneFile. Web. 11 June 2013.

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Physiological Effects of Type 2 Diabetes on mRNA Processing and Gene Expression