Transcriptional and translational regulation of cytokine signaling in inflammatory β-cell...

Archives of Biochemistry and Biophysics 528 (2012) 171–184

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Archives of Biochemistry and Biophysics

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Review

Transcriptional and translational regulation of cytokine signaling in inflammatoryb-cell dysfunction and apoptosis

Guy W. Novotny a, Morten Lundh a,1, Marie B. Backe a,1, Dan P. Christensen a,1, Jakob B. Hansen a,1,Mattias S. Dahllöf a,1, Emil M.H. Pallesen a,1, Thomas Mandrup-Poulsen a,b,⇑a Section of Endocrinological Research, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmarkb Department of Medicine and Surgery, Karolinska Institute, Stockholm, Sweden

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 August 2012and in revised form 20 September 2012Available online 9 October 2012

Keywords:IL-1IFNgDiabetesKDACmicroRNAROSHDAC

0003-9861/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.abb.2012.09.014

⇑ Corresponding author. Address: Section of Endocment of Biomedical Sciences, Panum Institute 12Copenhagen N, Denmark. Fax: +45 35327537.

E-mail address: [email protected] (T. Mandrup-Po1 Equal contribution, random order.2 Abbreviations used: IL-1,interleukin-1; KDACs, lysine

deacetylases; KATs, lysine acetyltransferases; HDAC, hiactivated kinase; MAPK, mitogen-activated protein koxide synthase; NO, nitric oxide; TSA, trichostatin Apolypyrimidine tract binding protein; Pdx1, pancreatiMafA, musculoaponeurotic fibrosarcoma oncogene hoprotein; PGC1a, peroxisome proliferator-activated realpha; FoxO1, forkhead box O1A; ROS, reactive oxygeONOO�, generate peroxynitrite; H2O2, hydrogen peroGpx, gluthatione peroxidase; Cat, catalase; SOD, suoxidase, nicotine amide adenine dinucleotide phosDiabetic Fatty;TRX, thioredoxin; TXNIP, thioredoxin-acetyl-l-cysteine; UPR, unfolded protein response;RNAInduced Silencing Complex; DNMT1, DNA methgrammed cell death 4.

Disease is conventionally viewed as the chaotic inappropriate outcome of deranged tissue functionresulting from aberrancies in cellular processes. Yet the patho-biology of cellular dysfunction and deathencompasses a coordinated network no less sophisticated and regulated than maintenance of homeo-static balance. Cellular demise is far from passive subordination to stress but requires controlled coordi-nation of energy-requiring activities including gene transcription and protein translation that determinethe graded transition between defensive mechanisms, cell cycle regulation, dedifferentiation and ulti-mately to the activation of death programmes. In fact, most stressors stimulate both homeostasis andregeneration on one hand and impairment and destruction on the other, depending on the ambient cir-cumstances. Here we illustrate this bimodal ambiguity in cell response by reviewing recent progress inour understanding of how the pancreatic b cell copes with inflammatory stress by changing gene tran-scription and protein translation by the differential and interconnected action of reactive oxygen andnitric oxide species, microRNAs and posttranslational protein modifications.

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Introduction

The pancreatic b cell is by virtue of its unique ability to releaseinsulin in immediate response to nutrients at the same time amiraculous blessing to metabolic homeostasis and a potentialcurse to health, since both excess and inadequate function is life-threatening. The b cell is despite its extreme specialization a stun-

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rinological Research, Depart-.2.10, Blegdamsvej 3, 2200

ulsen).

deacetylases; HDACs, histonestone deacetylase; JAK, janus-inase; iNOS, inducible nitric; ER, estrogen receptor; PTB,c and duodenal homeobox 1;mologue A; UCP, uncouplingceptor-gamma coactivator 1-n species; NO⁄, nitric oxide;xide; OH⁄, hydroxyl radical;peroxide dismutase; NADPHphate oxidase; ZDF, Zuckerinteracting protein; NAC, N-HO, haemeoxygenase; RISC,yltransferase 1; Pdcd4, pro-

ning paradox in many regards. The approximate 1 g of insulin pro-ducing tissue is distributed throughout the pancreatic organ inabout 1 million mini-organ back-ups, but although the small b-cellmass is indispensable, b-cell replication is extremely limited [1].Even though the stimulus secretion coupling depends on glucoseoxidation and generation of reactive oxygen species, b-cell oxida-tive defense is lower than in most cells [2,3]. Despite an exquisitesensitivity to inflammatory insults, the b cell has the highestexpression of receptors for the master inflammatory cytokineinterleukin-1 (IL-1)2 [4]. And as if in contempt for death the b cellis the only endocrine islet cell that expresses inducible nitric oxidesynthase [5], the catalyst of the formation of the toxic reactive NOspecies.

These apparent self-contradictions contain a common message:that there is nothing so bad that it is not good for something andthat too much of a good thing is bad. In other words, due to its un-ique importance, the b cell has to integrate a multitude of physio-logical and pathophysiological stimuli to adapt to insulin needs. Asan example, the requirements for substrate mobilization and theensuing insulin resistance as well as the anabolic demand of tissuerepair and remodeling during acute phase responses of inflamma-tion and tissue trauma needs to be sensed by the b cell to restorenormal organ function. It therefore makes perfect sense that theb- cell is equipped with IL-1 receptors enabling the cell to detectthe low-signal strength of femto-molar circulating IL-1b. The price

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172 G.W. Novotny et al. / Archives of Biochemistry and Biophysics 528 (2012) 171–184

paid by the b cell is susceptibility to damage when autoimmunityor over-nutrition causes intra-islet persistent inflammation.

The aim of this article is to review how the interconnected ac-tion of reactive oxygen and nitric oxide species, microRNAs andposttranslational protein modifications regulates cellular genetranscription and protein translation in a differentiated mannerto accommodate to ambient conditions by using the pancreaticb-cell response to inflammatory stress as example (Fig. 1).

Lysine deacetylases in gene transcription and translation

Lysine deacetylases (KDACs), also known as histone deacety-lases (HDACs), the term still used in the nomenclature of the indi-vidual enzymes, play a vital role in b-cell function and viability.KDACs catalyze the removal of acetyl-residues from histone andnon-histone proteins, thus antagonizing the lysine acetyltransfer-ases (KATs). Of note KDACs are regulators of many more non-his-tone than histone proteins [6]. Two distinct families of KDACsexist; the classical KDACs (Histone Deacetylase (HDAC) 1–11)and the Sirtuins (SIRT1–7). KDACs are phylogenetically subdividedinto four different classes: Class I (HDAC1, -2, -3 and -8), Class IIa(HDAC4, -5, -7 and -9), Class IIb (HDAC6 and -10), class III(SIRT1–7) and Class IV (HDAC11) [7]. The classical KDACs containa Zn2+ ion in their active site and are inhibited by various smallmolecules with very different chemical structures such as cyclicpeptides (e.g. HC Toxin), aliphatic acid- (e.g. valproic acid),hydroxamic acid- (givinostat, vorinostat) or benzamide-derived

Fig. 1. The b cell and exposure to proinflammatory cytokines. (Top) Exposing the b ceexposure to small amounts of cytokines (depicted by the small, single yellow lightning bsignaling pathways. Long exposure to higher levels of cytokines (depicted by the two larggene expression and protein levels), ultimately promoting b- cell dysfunction and death. (transcription, translation and protein activity will be described in regards to the actionsgamma), TNFa (Tumor necrosis factor alpha), PLD (Phospholipase D), PKC (Protein kinaseN terminal kinase), p38 (mitogen-activated protein kinase p38), ERK (Extracellular sigsynthase), JAK/STAT1 (Janus tyrosin kinase/Signal transducer and activator of transcriapoptotic members of the B-cell lymphoma protein family), KDACs (Lysine deacetylases

structures (e.g. entinostat) [8]. The Sirtuins also have a Zn2+ bind-ing pocket, however this is located away from the catalytic sitein contrast to the classical KDACs [5,9]. In addition the Sirtuinsare dependent on NAD+ as a cofactor, thereby making them indi-rect sensors of the metabolic state of the cell as reviewed else-where [10]. Whereas all of the classical KDACs have deacetylaseactivity, not all Sirtuins share this feature as SIRT4 does not possessthis property. On the other hand SIRT4, SIRT6 and probably alsoSIRT1–3 have mono-ADP-ribosyltransferase activity [11].

Both KDAC families are implicated in cytokine-induced b-celldysfunction and apoptosis. As described in the following sections,inhibition of the classical KDACs reduces cytokine-induced b-cellapoptosis and restores insulin secretion [12], and overexpressionor activation of SIRT1 prevents cytokine-induced b-cell demise[13]. With respect to the protective effect of inhibiting the classicalKDACs or overexpressing SIRT1 in cytokine-exposed b-cells, thetargets of KDACs are largely unknown. In light of the widespreadinvolvement of KDACs upon cellular processes (Fig. 2), we here re-view possible mechanisms on the transcriptional, translational andpost-translational level based largely on non-b cell studies.

KDACs and cytokine signaling

Sensing of numerous extracellular factors such as nutrients,ions, and hormones, but also cytokines and other stress and dangersignals is essential for the homeostatic functions of the b cell, andintracellular integration and actuation of signals is necessary for

ll to proinflammatory cytokines alters protein activity and gene expression. Shortolt), results in stimulation of b-cell function through activation of the PLD and PKCe yellow lightning bolts), results in a range of changes (transcription factor activity,

Bottom) In this review cytokine signaling in the b cell and the resulting alterations inof KDACs, ROS species and microRNAs. IL-1b (Interleukin 1 beta), IFNc (Interferon-C), NFjB (Nulcear factor kappa light chain enhancer of activated B cells), JNK (c-JUN

nal-regulated kinase), ROS (Reactive oxygen species), iNOS (inducible nitric oxideption) pathway, UPR (Unfolded protein response), Bcl-2 proteins (pro- and anti-).

Fig. 2. Widespread involvement of KDACs in cellular processes. Traditionally, theKDACs are viewed as histone deacetylases, regulating gene transcription throughaltered transcription-factor accessibility to chomatin DNA. However as described inthis review, the part KDACs play in regulating b-cell function may additionally bedue to their role in altering protein activity and modulating mRNA stability andRNA splicing.

G.W. Novotny et al. / Archives of Biochemistry and Biophysics 528 (2012) 171–184 173

the fine-tuned insulin secretory response. Acetylation and deacet-ylation of histones and nonhistone proteins is a means to regulateexpression of genes downstream to signaling events. Inflammatorystress, modeled by exposure of b cells to cytokines invokes agraded sequence of responses: short term or low concentrationexposure to IL-1b as encountered during the acute phase responseinduces pre-pro-insulin mRNA expression which is promoted byhistone acetylation [14], while higher concentrations or longerexposure as seen in islet inflammation results in detrimental ef-fects including production of inflammatory chemokines as wellas activation of intracellular apoptotic pathways [15], (and re-viewed in [16,17]), illustrating the bimodal effects of cytokineson b cells (Fig. 1).

Detrimental cytokine signaling is also regulated by the activitiesof KDACs, [12,18–22]. While several thousands of acetylation-prone cellular protein targets have been identified [6] little isknown about b-cell targets of acetylation.

The most important cytokines for cytokine-induced b-cell toxic-ity include IL-1b, the type II interferon IFNc and TNFa, whichin vivo likely act in synergy to achieve b-cell death during develop-ment of diabetes [16]. TNFa acts through modulating immune cellresponse as well as activating JNK/p38 and NFjB pathways (re-viewed in [23]). In immune-cell free experiments upon b cells,TNFa may be excluded as the JNK/p38 and NFjB pathways are alsoactivated by IL1-b. In the b cell IFNc signals through its receptorsIFNcRI and IFNcRII, leading to Janus-activated kinase (JAK) depen-dent phosphorylation, dimerization and nuclear translocation ofthe transcription factor STAT1 [17], and STAT1 appears necessaryfor promoting cytokine induced b-cell death [24–26]. In colorectalcarcinoma cells, KDAC activity promotes IFNc induced STAT1 phos-phorylation, and KDAC inhibition reduces STAT1 dimerization andnuclear translocation [27], but the role of KDACs in b-cell IFNc sig-naling has not been investigated.

IL-1b signaling via the Type 1 receptor mainly involves activa-tion of the mitogen-activated protein kinase (MAPK) (JNK/p38/

ERK) and NFjB pathways. Of the many genes under NFjB controlis inducible nitric oxide synthase (iNOS) that catalyzes productionof nitric oxide (NO) which is toxic to b cells. NFjB activation isassociated with phosphorylation of inhibitor of NFjB (IjB)a, whichtargets IjBa to degradation and releases NFjB to translocate to thenucleus [28]. Upon cytokine exposure a biphasic phosphorylationpattern of IjBa is observed, the second wave of which is bluntedby KDAC inhibition, hence, expression of iNOS and NO productionis facilitated by KDACs in the b cell [12,22]. The activity of NFjB isregulated by its acetylation status. Thus acetylation of NFjB on onehand inhibits its binding to IjBa, promoting nuclear translocation[29]; on the other hand reduces DNA binding leading to nuclear ex-port of NFjB [30]. SIRT1 deacetylates the NFjB subunit RelA/p65 inb cells, and SIRT1 overexpression protects b cells against cytokine-induced iNOS expression and NO production [13] suggesting thatin b cells, p65 deacetylation inhibits NFjB signaling. SIRT1 alsoregulates the activity of the transcription factor FoxO1 [31]. Whendeacetylated, FOXO1 induces transcription of DNA repair genes,and when acetylated expression of apoptotic genes [31]. SIRT1 alsoactivates its own transcription in a positive feedback loop [32].SIRT1 is therefore an attractive pharmacological target in b cells.

RelA/p65 is also excluded from the nucleus by complex forma-tion with HDAC1 and 2 [33], but whether this depends on theenzymatic activities of these KDACs is uncertain. High concentra-tions of KDAC inhibitors allow acetylated STAT1 to form a complexwith p65 leading to nuclear exclusion of p65 [34], thus coordinat-ing the shut-down of the cellular proinflammatory response. Acet-ylation and deacetylation of p65 is regulated by other post-translational modifications. Thus, acetylation of p65 lysine 310 isdetermined by prior phosphorylation of p65 serines 276 and 536[35], and phosphorylation of p65 inhibits its binding to HDAC1[36]. These findings illustrate the complexity of the post-transla-tional ‘‘protein code’’.

Two recent independent studies suggested differential roles ofindividual KDACs in cytokine-induced b-cell demise. Using smallmolecules and knock-down studies HDAC3 was found to mediatethe cytotoxic effects of cytokines in both studies [18,20]. Lundhet al. additionally found a protective effect of knocking downHDAC1. Interestingly, knock-down of HDAC3 was found to restoreglucose-stimulated insulin secretion [18] and knock-down ofHDAC1 preserved accumulated insulin secretion [20]. Mechanisti-cally, knock-down of HDAC3, but not HDAC1, reduced cytokine-in-duced NFjB-binding to the iNos promoter [20]. Thus, combinedtargeting of HDAC3 and HDAC1 preserves both b-cell viabilityand function.

KDACs and b-cell gene transcription

DNA is tightly condensed in the nucleus of eukaryotic cellsbound to histones, the scaffold for chromatin packaging, therebysecuring both compaction of DNA as well as preventing accessibil-ity of the transcriptional machinery to DNA. Posttranslational his-tone modification is a key regulatory process in transcription. Inthe classical paradigm histone acetylation shields ionic bonds be-tween DNA backbone and acetylation-prone lysine residues, thusloosening chromatin packaging and allowing transcription. Severalstudies, however, question this concept in favor of a model of com-plex interdependencies between histone methylation, phosphory-lations and acetylation, the so-called ‘‘histone code’’, as well asmethylation of DNA, yielding differentiated patterns of packagingallowing for transcriptional control of individual genes [37,38], re-viewed in [39,40]. For instance, acetylation of histone H4 lysinesfavors recruitment of TAFII250, a subunit of the transcription factorcomplex TFIID, to the histone by interaction with the bromodo-main of TAFII250, facilitating transcription initiation of DNA in


the vicinity [41]. In contrast to the classical model, the only gene sofar described to be affected by pharmacological inhibition of KDACactivity in cytokine-stressed b cells is iNOS and KDAC inhibitiondecreases iNOS expression.

Pancreatic histone H3 acetylation is similar between diabeticand non-diabetic mice [42], suggesting that if KDACs partake inthe pathogenesis of diabetes at histone acetylation levels, the lev-els of such changes are not persistent [14]. At diabetes diagnosismost b cells have been destroyed, which could explaine why tracesof acetylation events may be unobservable, however Susick et al.showed no increase in H4 acetylation on INS-1 cells exposed toIL-1b [21], rendering this explanation less likely.

Still, at the insulin promoter changes in acetylation of H3 andH4 result in altered expression [14], and reducing glucose levelsin b-cell growth media recruits HDAC1 and -2 to the insulin pro-moter via complex formation with the transcription factor PDX-1, reducing insulin mRNA transcription through histone H4 deacet-ylation [43]. Thus, further investigations are necessary to clarifythe effects of KDACs on b-cell gene transcription under various cel-lular conditions. At present, it appears that the regulatory roles oflysine deacetylases in cytokine induced b-cell demise and malfunc-tion are only in part mediated through histone modification, indi-cating that non-histone proteins are important targets forinvestigating the KDAC effects upon cytokine exposure.

KDACs and translation

The stability of some but not all mRNAs is also controlled byKDAC activity as KDAC inhibition altered the splicing of 683 differ-ent genes [44], and the non-selective KDAC inhibitor trichostatin A(TSA) was shown to decrease the half-life of DNA methyltransfer-ase (DNMT)-3B mRNA from 4 to 2.5 h [45], claudin-1 mRNA from18 to 7.5 h [46], and the estrogen receptor (ER) mRNA half-lifefrom 4 to 1.5 h [47]. In the b cell the level of preproinsulin mRNAis regulated via mRNA stability [48,49]. The mechanism involvesthe expression of the mRNA-stability protein polypyrimidine tractbinding protein (PTB), which is downregulated in rat islets afterexposure to high glucose, likely through induced expression ofmicroRNA miR-133a targeting Ptb mRNA [50]. Cytokines werenot found to affect miR133a or Ptb-levels; however a role forKDACs in glucotoxicity in b cells has yet to be investigated. Genesinvolved in cytokine-induced b-cell death are subject to changes inmRNA-stability in non-b-cells. The antiapoptotic Bcl-2 protein BCL-2 is downregulated by proinflammatory cytokines [51] and overex-pression of BCL-2 protects the b cell from cytokine-induced deathand impaired insulin secretion [52,53]. The expression of BCL-2protein is regulated by changing the mRNA stability [54]. HoweverTSA has been shown to regulate BCL-2 protein without affectingmRNA half-life [55], questioning the importance of regulation ofBcl-2 mRNA stability in cytokine and KDACi exposed cells.

The enzyme iNOS is central to cytokine-induced b-cell death inrodents. Interestingly, iNos mRNA is likely to be regulated in both adeacetylase-dependent and a deacetylase-independent manner.Broad KDAC inhibition has been shown to decrease the cytokine-induced production of iNOS protein [12,19] underlining the impor-tance of KDAC activity in cytokine induced b-cell dysfunction.

However, HDAC2 was seen to regulate mRNA translation in adeacetylase-independent manner [56]. Importantly knock-downof eIF5A, an important protein initiation translation factor, im-paired iNos translation in a streptozotocin-induced diabetes model[57]. Though a connection between KDACs and eIF5A has not beeninvestigated yet, current literature indicates a likely interplay be-tween these enzymes in the regulation of iNOS as knock-down ofHDAC1, -2 and -3 individually decreases cytokine-induced iNosmRNA levels [20], (despite these effects might be through the loss

of protein and thereby activity per se). If so, targeting both theactivity of selected KDACs and their complex formation might fur-ther protect cytokine-stressed b cells.

KDACs and b-cell function

The b cell releases insulin in response to nutrients (glucose),hormones and other humoral mediators and neuronal signals tomaintain glucose homeostasis and lipid and protein metabolism.Insulin is a peptide hormone synthesized as preproinsulin thatconsists of four peptide chains (signal peptide and, A, B and C). Pre-proinsulin has to undergo three successive processing steps to as-sume the mature form. The first step removes the signal peptideforming proinsulin. The second step cleaves and releases the con-necting C-peptide, leaving A and B linked by disulfide bonds. Thethird step further processes the A–B peptide to produce the matureform. Glucose induced insulin secretion is controlled by the so-called stimulus-secretion coupling. Briefly, glucose is taken up byb cells via glucose transporter (GLUT) 2 and then metabolized byglycolysis and the Krebs cycle to yield an increase in the cytoplas-mic ATP to ADP ratio. This closes ATP-sensitive potassium channelsand leads to cell membrane depolarization and subsequently open-ing of voltage-gated calcium channels. As a result, the free cyto-plasmic calcium concentration increases and triggers fusion ofinsulin-containing vesicles with the cell membrane releasing insu-lin to the blood stream. This closely related chain of events allowstight coupling between glucose levels and insulin secretion. Be-sides inducing insulin release, glucose also stimulates insulin genetranscription [58]. Other substances known to stimulate insulin re-lease include the amino acids arginine and leucine, acetylcholinereleased from parasympathetic nerves, cholecystokinin, and theincretin hormones glucagon-like peptide 1 and glucose-dependentinsulinotropic peptide. Glucose induced preproinsulin transcrip-tion depends on at least three b cell specific transcription factors:pancreatic and duodenal homeobox 1 (Pdx1), NeuroD1, and V-maf musculoaponeurotic fibrosarcoma oncogene homologue A(MafA) [59].

At high ambient glucose levels Pdx1 associates with the HATp300, leading to an increase in histone H4 acetylation in the insulinpromoter and eventually increased proinsulin transcription[43,60–63]. In contrast, when ambient glucose is low insulin pro-duction is no longer active and the histone H4 is deacetylated, pre-sumably via Pdx1-mediated recruitment of HDAC1 and 2 to theinsulin promoter [43,61]. Thus it seems that acetylation by a spe-cific KAT favors insulin expression and it follows that KDAC activitydecreases insulin expression. Indeed, inhibitors of the classicalZn2+-dependent KDACs (KDACi) increase histone H4 acetylationand insulin expression at low glucose levels (3 mM) but have no ef-fect at high glucose levels (30 mM) [61]. In isolated human islets,KDACi stimulate insulin release when incubated at low glucose(2.8 mM) [64]. Sirt4, a family member of the NAD+-dependent sir-tuins represses amino acid- and glucose-induced insulin secretionfrom isolated mice islets [65]. By repressing the activity of gluta-mate dehydrogenase in the mitochondria Sirt4 limits the metabo-lism of glutamate and glutamine to generate ATP. However, thismechanism is not dependent on deacetylation, as Sirt4, as men-tioned previously, has no deacetylase activity. In contrast, Sirt1positively regulates glucose stimulated insulin secretion in vitrovia repression of the gene encoding uncoupling protein (UCP) 2[66] and increased expression of key b-cell functional genes suchas Glut2, glucokinase, and Pdx1 [67].

Cytokine-induced inhibition of insulin secretion involves de-creased expression of insulin [68] and proteins important for insu-lin exocytosis [69] and NO-mediated attenuation of glucoseoxidation in the b cell [70]. Several KDACi (such as trichostatin A,


vorinostat and givinostat) inhibit the cytokine-induced reductionin accumulated insulin release [12,19,21,22], possibly by inhibitingNO production. Notably, givinostat not only inhibits cytokine-med-iated reduction in insulin release but in fact increases insulin re-lease to above the level of control islets [19].

Recently it was shown that, of the three class I KDACs HDAC1–3,HDAC1 in particular is involved in cytokine-induced inhibition ofinsulin secretion [20]. In contrast to the classical KDACs, whichmediate cytokine toxicity, overexpression or increased activationof Sirt1 protects isolated rat islets from cytokine-induced reductionin glucose stimulated insulin secretion [13]. The molecular mech-anism involves deacetylation of p65 but it cannot be ruled out thatthere are mechanisms unrelated to deacetylase activity, such assubstrate sensing via peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC1a) and AMP kinase.

Insulin resistance is used to describe the physiological conditionwhere insulin signaling is impaired. Although controversies stillexist, autocrine insulin feedback is involved in b-cell function[71]. Further, the mechanisms leading to insulin resistance in theclassic insulin sensitive target tissues liver, muscle, and fat alsocause b-cell insulin resistance coupled with b-cell dysfunctionand apoptosis reviewed in [71]. In the liver, HDAC2 has beenshown to deacetylate insulin receptor substrate 1 and dampeninsulin signaling, an effect that could be partially reversed afterHDAC2 knockdown [72], and KDAC activity inhibits GLUT4 func-tion, leading to insulin resistance [73–77]. However, GLUT2 is themajor transporter in b cells and it is, to our knowledge, not knownif KDAC activity inhibits GLUT2.

As previously described the pro-inflammatory cytokine IL-1balone or in combination with IFNc and/or TNFa leads to b-celldeath and has been implicated in the pathogenesis of both T1DMand T2DM. Based on animal studies, IL-1b also plays an importantrole in the destruction of transplanted islet grafts [78–82] andblocking cytokines in clinical islet transplantation has been sug-gested as a future intervention to prevent graft destruction [83].Originally, our lab reported that exposure of isolated rodent isletsor an insulin-producing cell line (INS1) to non-specific KDACi caninhibit cytokine-induced b-cell destruction [12], findings thatnow have been confirmed by our and other labs [19,21,22]. As pre-viously mentioned, all classical KDACs are expressed in the pancre-atic b cell, albeit at different levels, and cytokines and KDACidifferentially regulate the expression of individual KDACs [19].On the basis of differential regulation by cytokines and KDACi,HDAC1, 2, 6, and 11 have been proposed as key promoters of proa-poptotic cytokine signaling, and recently it was shown that HDAC1and 3 in particular promote proapoptotic cytokine signaling[18,20] (Fig. 3). Further, cytokine-induced b-cell death, NO produc-tion and iNos expression are inhibited by overexpression or phar-macological activation of Sirt1 [13]. The protection is speculatedto involve inhibitory deacetylation at lysine 310 of the key inflam-

Fig. 3. Classical KDACs involved in cytokine-induced b-cell toxicity. Proinflammatory cpromoted by activity of the classical KDACs, HDAC 1, 2, 3, 6 and 11. Inhibition ofproinflammatory cytokine action.

matory transcription factor NFjB. Additionally, a recent study sug-gested that the fate of b cells in response to cytokine-induced NOproduction is controlled by Sirt1 activity, such that Sirt1 activityshifts the response of the b cell from pro-apoptotic to protectiveby deacetylating the transcription factor forkhead box O1A (FoxO1)[31]. Presently, it is unknown how the activity of Sirt1 is regulated.

Notably, it appears that no single rule of thumb can be put for-ward in relation to whether deacetylase activity is good or bad ina pro-inflammatory setting as both inhibition (of classic KDACs)and activation (e.g. of Sirt1) can preserve b-cell function. Thus isappears essential to distinguish between the two families of lysinedeacetylases and seemingly also between single enzymes withineach family. In depth investigations on interaction partners andimpact of individual deacetylases under varying settings are war-ranted to reveal the mechanisms behind this paradoxical relation.

In conclusion the classical KDACs and the Sirts play importantroles in both b-cell physiology and pathophysiology, while the im-pact of the specific KDACs is largely an unexplored field in the b-cell. Interestingly, not all KDACs or Sirts have deacetylase activityand it remains to be determined if enzymatic activity is importantfor the functions discussed above.

Reactive oxygen species (ROS)

Redox reactions are pivotal for the cell to maintain respiration,metabolism and energy supply. The mitochondrion is a major sitefor redox processes to generate ATP through the electron transportchain. A consequence of mitochondrial oxidative phosphorylationis the generation of the free radical superoxide (O��2 ) (Fig. 4) inthe inner mitochondrial membrane where it reacts with other rad-icals, such as nitric oxide (NO⁄) to generate peroxynitrite (ONOO�),or iron clusters in some proteins to generate hydrogen peroxide(H2O2) and subsequently under favorable conditions the hydroxylradical (OH⁄). These are termed reactive oxygen species (ROS),which modulate cell signaling including transcriptional activityand protein synthesis, and adversely affect DNA, proteins, phos-pholipids, and carbohydrates, leading to cellular dysfunction anddeath [2,84–86].

During cellular respiration, three steps are important for gener-ation of superoxide: increased glycolytic flux, decreased ADP con-centrations, and increased Ca2+ concentrations. The cellulardefense against ROS is composed of different antioxidant enzymessuch as glutathione reductase, gluthatione peroxidase (Gpx), cata-lase (Cat), superoxide dismutase (SOD), and mitochondrial manga-nese-dependent SOD (MnSOD) located in different cellcompartments [84]. Paradoxically, the SOD-catalyzed neutraliza-tion of superoxide generates hydrogen peroxide, which can reactwith free divalent metal ions such as iron and copper in the Fenton

ytokine exposure leads to b-cell dysfunction and death, consequences which areKDACs (KDACi) with broad inhibitors such as givinostat protects b cells from

Fig. 4. Generation and removal of reactive oxygen species (ROS). During mitochondrial respiration, escaping electrons react with oxygen to generate the ROS superoxide(O��2 ), which is quickly converted by mitochondrial manganese-dependant superoxide dismutase (Mn-SOD) to generate the ROS hydrogen peroxide (H2O2). H2O2 in turn canbe neutralized to water through gluthatione peroxidase (Gpx). In the cytosol, O��2 O��2 can be generated from oxygen through activation of nicotine amide adenine dinucleotidephosphate oxidase (NADPH) and, as in the mitochondria, be converted to H2O2 by the cytosolic superoxide dismutase enzyme Cu/Zn-SOD, and neutralized by Gpx activity.H2O2 can move from different cellular compartments, or through the cell membrane, via aquaporin transport. Reactive oxygen species are marked with RED text.


reaction to generate another highly toxic ROS, the hydroxyl radical[87] (Fig. 4).

As mentioned, mitochondrial activity generates a significantamount of ROS. However, it is important to note that ROS are alsoderived from cytosolic enzymes of which NADPH oxidases are theonly enzymes known to function as ROS generating enzyme per se[88]. NADPH oxidases are membrane-bound proteins present in al-most every cell type, they catalyze generation of superoxide fromoxygen (Fig. 4), and are used by phagocytotic leukocytes to medi-ate host-defense [89,90].

ROS and the b cell

The b cell is a highly metabolically active cell, thus being con-stantly challenged by ROS. Although expression of ROS inactivatingenzymes in the b cell is low, especially Cat and Gpx, [2,3], it hassufficient protection to deal with the load of ROS produced throughthe normal physiological situations of glucose metabolism: ATPgeneration and insulin production.

Interestingly there is experimental evidence indicating that b-cell function is dependent upon ROS availability such that thesmall amounts of ROS generated during normal glucose metabo-lism promotes insulin secretion (Fig. 5). In islets treated with anti-oxidants, thereby lowering ROS levels, insulin secretion is reduced,correlating with decreased Ca2+ influx, suggesting that ROS mayregulate Ca2+ flux [91]. One way the b cell is able to control ROSand thereby insulin secretion is through UCP2. A study with b-cellspecific knockout of UCP2 suggested that ROS may be increasedwith a subsequent increase in glucose stimulated insulin secretion

[92]. Further it was demonstrated that ROS-mediated insulinsecretion is dependent on ATP production, suggesting ROS to bean amplifying signal for insulin secretion [92]. UCP2 mRNA isupregulated by metabolic factors such as high glucose and fattyacids as well as pro-inflammatory cytokines, suggesting that thisprotein is part of the defense system combating ROS-mediated celldamage and dysfunction [93]. Investigation of the NADPH oxidaseadds further complexity to the effect of ROS on insulin secretion.Inhibition of NADPH oxidase in rat pancreatic islets either pharma-cologically, or through antisense targeting of the p47phox subunit ofNADPH oxidase, resulted in decreased ROS levels. Additionally, re-duced insulin secretion as well as impaired glucose metabolismwas observed, indicating that the ROS producing enzyme NADPHoxidase is important for maintaining optimal b-cell function [94].

Cytokine signaling and ROS

Exposing b cells to pro-inflammatory cytokines (IL-1b, IFN-cand TNF-a) induces accumulation of reactive oxygen species [2].Accordingly, rhIL-1b, injected into the pancreatic artery of Wistarrats, resulted in elevated blood sugar, and reduced plasma insulin.These patho-physiological changes correlated with increased oxi-dative stress as measured by increased plasma hydroperoxides,and decreased expression of islet MnSOD, as well as decreased isletaconitase activity, suggesting that increased oxidative stress is partof IL-1b-mediated b-cell destruction in vivo [95].

Pro-inflammatory cytokines induce production of the reactivecompound nitric oxide (NO) in b cells, which has detrimental ef-fects on the b cell [96]. Interestingly, pro-inflammatory cytokine

Fig. 5. ROS influence on cellular processes. ROS increase can be either beneficial or detrimental to the b-cell. Blue box: A non-toxic increase, for example ROS generatedthrough normal glucose metabolism, stimulates insulin release through a rise in H2O2 levels. Red box: In contrast, toxic increases in ROS levels due to exposure to highglucose, fat or cytokine levels reduce b-cell function. High levels of mitochondrial superoxide (O��2 ) leads to increased NFkB activity and increased IL-1b secretion. Increasedmitochondrial H2O2 generated through conversion of O��2 by Mn-SOD can also lead to a higher Bax/Bcl2 ratio, promoting apoptosis. The generation of H2O2 by SOD leads toactivation of JNK and p38. H2O2 can be converted to the highly reactive hydroxyl radical (OH⁄), either through the Fenton reaction if the required Fe++ is present, or through areaction with nitric oxide (NO⁄) if present for example due to cytokine exposure. Besides activating cellular pathways, the reactive nature of H2O2 and OH⁄ causes nucleotide,lipid and protein damage. Cumulatively, the result is impaired b-cell function and viability. Pdx1 (Pancreatic and duodenal homeobox 1), GLUT-2 (Glucose transporter 2), IRS-1 and -2 (Insulin receptor substrate-1 and -2), UPR (Unfolded protein response), Bax (Bcl-2–associated X protein), Bcl-2 (B-cell lymphoma 2).


(IL-1b, IFN-c and TNF-a)-induced NO production has been shownto be responsible for increasing ROS production in the colonic cellline HT29 [97]. This suggests that some of the toxic effect of the NOradical in b cells may stem from increased ROS production; how-ever this is controversial as IL-1b alone has been shown to induceROS independent of NO production (Todaro et al. [95]).

In rodent islets as well as b-cell lines, cytokines (IL-1b, IFN-c,and TNF-a) induce expression of the nicotine amide adenine dinu-cleotide phosphate oxidase (NADPH oxidase) subunit p47phox

[98,99], to generate cytosolic ROS. Also Rac1, another componentof the NADPH oxidase holoenzyme, is transiently upregulated uponpro-inflammatory cytokine exposure in INS832/13 cells [100].Upon pharmacological inhibition of NADPH oxidase or the Rac1component, cytokine induced ROS levels were reduced [99], corre-lating with increased mitochondrial membrane potential [100]. Inaddition to cytokines, other b-cell stress factors such as high glu-cose, palmitate, oleate and even hydrogen peroxide exposure alsoinduce expression of p47phox thereby increasing ROS production[98,99,101]. In vivo, the islets of the diabetic Zucker Diabetic Fatty(ZDF) rat show increased levels of ROS and ROS producing enzymes(phosphorylated p47phox, active Rac1, Nox activity), associatedwith increased downstream JNK phosphorylation and caspase 3activity [102].

Transcriptional and post-translational regulation by ROS

ROS activates the NFjB and MAPK pathways in b-cells. NFjBactivity is regulated by mitochondrial ROS, as stimulation of mito-chondrial ROS induces NFjB translocation to the nucleus in b cells.MnSOD knockdown (leading to increased mitochondrial O��2 ) inRINm5F cells resulted in increased NFjB translocation, while over-expression of MnSOD reduced IL-1b mediated translocation ofNFjB to the nucleus and DNA binding. This mechanism was notobserved after overexpression of the cytoplasmic ROS scavengingenzymes Cu/ZnSOD (O��2 ) or Gpx or Cat (H2O2), suggesting thatmitochondrial ROS is the mediator of a feed-back loop enhancingand sustaining NFjB translocation [103]. Importantly, ROS-medi-

ated NFjB activation in b cells does not appear to be mediatedby hydrogen peroxide (H2O2) itself, as treatment of b-cells withhydrogen peroxide failed to induce NFjB DNA binding [104], sug-gesting superoxide (O��2 ) to be the primary mediator of NFjB acti-vation. Interestingly, b-cell oxidative stress upon IL-1b exposure isin part due to NFjB time-dependent ROS formation, as pharmaco-logical inhibition of NFjB using an inhibitor of IKKb, decreased ROSin IL-1b exposed islets [105]

While hydrogen peroxide does not affect the NFjB pathway, itdoes induce JNK and p38MAPK phosphorylation in islets, furtherindicating that ROS mediates the MAPK pathway [106]. Adenoviraloverexpression of dominant negative JNK protected against hydro-gen peroxide-induced phosphorylation of c-Jun and protectedagainst hydrogen peroxide mediated glucose-stimulated insulinsecretory dysfunction and nuclear Pdx1 extrusion [106,107]. Aftertransplantation of islets transfected with dominant negative JNKunder the kidney capsule of Swiss nude mice made diabetic witha single dose STZ, blood glucose significantly decreased [106]. Bothhigh and low concentrations of glucose can induce ROS and b-cellstress. Induction of ROS in MIN6 cells by long-term incubation inlow glucose was able to induce phosphorylation of JNK as well asp38, and treatment with antioxidants reduced the phosphorylationof JNK [108]. Knock-down of the NOX 2 subunit of the NADPH oxi-dase with siRNA restored palmitate and oleate mediated reductionin Akt phosphorylation and insulin content, and reduced phos-phorylated PTEN, JNK, p38 and p53 in insulin producing cells, con-firming that ROS activates both the NFjB and the JNK pathway[101].

Mitochondrial ROS activates the inflammasome to mediate IL-1b secretion, thereby contributing to a vicious cycle of cytokinemediated b-cell dysfunction. In islets, activation of ROS by stimula-tion with high glucose activates the NLRP3 inflammasome bymediating the dissociation of thioredoxin (TRX) from thiore-doxin-interacting protein (TXNIP) where after TXNIP activatesthe NALP3 inflammasome to process pro-IL-1b to mature IL-1b[109]. High-glucose induced islet IL-1b production was preventedby incubating with either a ROS inhibitor or knocking-out TXNIPor NLRP3 [109]. Accordingly, mice transgenic for human islet


amyloid polypeptide (hIAPP), which induces ROS in islets [110], in-creased IL-1b processing [111].

ROS and b-cell dysfunction

Although ROS production during normal respiration is benefi-cial to b-cell function, b-cell stress (cytokine exposure, high glucoseor fat) increases ROS to levels exceeding that which the b cell cansafely manage (Fig. 5). High levels of ROS contribute to the dys-function of the b cell via repression of insulin synthesis, transloca-tion of the b-cell specific transcription factor Pdx1 from thenucleus to the cytoplasm, reduction of glucose sensitivity by de-creased GLUT-2 expression, reduction of insulin sensitivity byrepression of insulin receptor substrate (IRS) 2 and by increasingthe Bax/Bcl2 ratio [51,112–115] (Fig. 5). Additionally, in a rat mod-el ROS production was stimulated through overexpression of therate-limiting enzyme in generation of angiotensin II renin, whichleads to stimulation of cytosolic ROS through NADPH oxidase, re-sulted in reduced islet insulin sensitivity by decreased IRS-1 andIRS-2 expression [116]. IL-1b-mediated decreased insulin signalingalso appears to be dependent on NO. Thus, in iNOS deficient isletsas well as in wild type islets treated with an iNOS inhibitor, the IL-1b-mediated repression of IRS-2 was normalized or improved,respectively [115].

Exposure of islets to hydrogen peroxide causes Pdx1 extrusionfrom the nucleus, and decreased insulin content, and treatmentwith the antioxidant N-acetyl-L-cysteine (NAC) prevented hydro-gen peroxide mediated Pdx1 extrusion and maintained Pdx1 inthe nucleus [106,107]. Further, treatment of ZDF rats with NAC re-sulted in improved plasma glucose [117]. These data implies ROSand NO as general mediators of b-cell dysfunction. Additionally,ROS alter insulin structurally as measured by decreased antigen–antibody recognition [118]. This process is likely to take place onlyin the periphery, because insulin is packed with zinc in secretorygranules and zinc acts as an antioxidant either by protecting sul-phydryl groups of proteins from free radical attack or antagonizingredox-active metals [87].

Cytokines induce ROS mediated mitochondrial dysfunction, un-folded protein response (UPR) and DNA damage [119–121]. In-creased ROS, due to deletion of the ER-associated redox enzymeNADH-cytochrome b5 oxireductase (Ncb5or), resulted in elevatedb-cell oxidative stress at 5 weeks of age, which correlated with de-creased mitochondrial area and swollen ER measured by electronmicroscopy [122]. Furthermore, b-cell ER-stress markers such asBiP and Xbp1s were significantly upregulated at 5 weeks of age[122]. This correlated with decreased glucose tolerance and dra-matically increased fasting blood glucose levels [122]. Cytokine-mediated ER-stress is also regulated by NO since NO inhibits theER Ca2+ pump SERCA2B. Accordingly, CHOP expression in cytokineexposed islets from iNOS knockout mice was completely blocked[119,123].

A number of experiments indicate that cytokine induced b-celldysfunction can be reduced through inhibition of ROS production,or removal of the produced ROS. In RINm5F cells, overexpression ofmitochondrial catalase (Mito-Cat) and suppression of MnSOD(thereby lowering OH⁄� production) reduced cytokine inducedROS and NO production [120]. Overexpression of Mito-Cat com-pletely blocked cytokine induced mitochondrial hydrogenperoxideformation, whereas MnSOD knock down only partially reducedmitochondrial hydrogenperoxide levels [120]. This correlated withDNA oxidation and hydroxyl radical formation, where Mito-Catoverexpression inhibited both after cytokine exposure, but MnSODknock down cells were only partially protected [120]. In a trans-genic mouse model overexpressing both MnSOD and Mito-Cat, is-lets were protected from ROS and the mice were protected from

multiple low-dose streptozotocin treatment [124]. Mito-Cat over-expressing cells are however not able to reduce cytokine-mediatedCHOP expression or caspase-12 activity as a measure of UPR [85].

Lowering cytosolic ROS have potential benefits in b-cell sur-vival. Accordingly, knock-out of NOX 2, a subunit of NADPH oxi-dase, decreased plasma superoxide levels, and islet superoxideproduction and apoptosis. Upon multiple low-dose streptozocintreatment, which causes islet inflammation, NOX 2 knockoutshowed a rescue of b-cell function and mass [125]. Additionally,reducing ROS levels by upregulating the oxidative stress protectiveenzyme haemeoxygenase (HO) 1 [104], and islet specific overex-pression of HO-1 in the non obese diabetic mouse (NOD) delayedthe onset of diabetes and reduced b-cell ROS and cytokine inducedislet apoptosis [126]. Reducing ROS levels through inhibition ofNFjB improved plasma insulin, blood glucose and Hba1c levelsin a type 2 diabetic rat model [105], while in isolated human isletswith increased nuclear translocation of NFjB due to post isolationstress, treatment with an antioxidant reduced translocation ofNFjB revealed by EMSA stain [127].

In conclusion, evidence indicates that ROS are involved in main-taining b-cell function, such as potentiating insulin secretion. How-ever when ROS levels exceed what the b cell can handle theybecome detrimental. Paradoxically, as the b cell is a metabolicallyactive cell, it has poor antioxidant defense probably due to itsdependency on ROS for normal function. In stress situations ROSregulates the NFjB and MAPK pathways to mediate transcriptionof genes involved in b-cell dysfunction and death (Fig. 5).

microRNA

microRNAs (miRNAs) are a class of small, non-coding RNAs thatover the past decade have received much attention due to theirrole in regulating protein expression at the translational level. miR-NAs are initially contained as hairpin structures in long primarytranscripts (pri-miRNA) which are processed in the nucleus by aprotein complex containing the RNAseIII enzyme Drosha to a hair-pin precursor of appr. 80 nucleotides (pre-miRNA) which are thentransported to the cytoplasm [128–131]. The mature miRNA (�22nucleotides long) is excised from the hairpin structure by the RNA-seIII enzyme Dicer [132] and incorporated into the RNAInducedSilencing Complex (RISC), where the miRNA is used to target com-plementary messengerRNAs (mRNA) in a sequence specific man-ner, thereby interfering with synthesis of the encoded protein[133,134]. Translational inhibition of mRNAs through miRNA/RISCeventually results in transport of the inhibited mRNA to processingbodies (P-bodies) where it is either degraded, or under certain cir-cumstances such as cellular stress, can be released again to thecytoplasm for renewed translation [54].

Recognition of mRNAs by the loaded miRNA/RISC occurs at tar-get sites that are complementary to the seed sequence, a �8-nucle-otide long sequence in the 5’ end of the microRNA [134,135].Target sites have historically most often been searched for in the3’ untranslated region (3’UTR) of the messenger RNA; however thismay neglect a novel mechanism of how miRNAs function as recentresults indicate that functional targets may be found across thelength of the mRNA [136,137]. The short length of the seed se-quence has the consequence that target sites to each microRNAare present in hundreds of different mRNAs, thereby complicatingthe elucidation of miRNA regulation of cellular functions.

Since their discovery in Caenorhabditis elegans (C. elegans) in1993 [138], miRNAs have been found to be widespread amongmulti-cellular organisms, playing a central role in many physiolog-ical processes such as differentiation, proliferation and apoptosis[139] as well as having roles in many human diseases [140]. It ispredicted that miRNAs regulate the majority of protein-coding


genes, as 70% of mRNAs contain miRNA binding sites [141].Although individual microRNAs regulate many messengerRNAs,the effect upon protein levels is generally modest [142,143], indi-cating that the phenotypic effect a microRNA imposes is morelikely a cumulative result of the constellation of all the slightly al-tered proteins, rather than the result of large alterations of individ-ual proteins.

Currently there are over 1900 human microRNAs known [144].Considering the multitude of mRNA targets of each miRNA, it mayappear incomprehensible how miRNAs contribute to construc-tively coordinate cell-protein translation. However typically only200 miRNAs are significantly expressed above background in a gi-ven tissue or cell type [145,146], with many miRNAs being un-iquely expressed in specific tissues or cells [147,148]. Whencombined with the cell-specific mRNA repertoire, it is possible tounderstand the selectivity and specificity of certain miRNAs. Inan increasing number of diseases, expression analyses show manyderegulated miRNAs, and further investigation of these deregula-tions is necessary for understanding the role of miRNAs in theoccurrence and progression of diseases as well as for better todeveloping potential new treatments based on miRNA targeting.

In diabetes altered miRNA expression has also been docu-mented in various tissues such as adipose, liver, skeletal muscleand pancreatic islets [149,150], T regulatory cells [151] and serum[152,153]. However it is not yet known whether altered miRNAexpression is a consequence of diabetes, or a contributing factorto it.

Linking proinflammatory cytokine effects to microRNAs

The transcriptional regulation of miRNAs is as complex as forany protein-coding mRNA, since both are transcribed by RNA poly-merase II [154]. Therefore different trans- and cis-acting factorssuch as transcription factors, enhancers and silencing elementsdetermine the transcriptional activity, and thus epigenetic changessuch as DNA methylations can induce dynamic alteration in theexpressional level of miRNAs [155]. DNA methyltransferase 1(DNMT1) mediates epigenetic regulation of miRNA expression[156], and the pro-inflammatory cytokine IL-6 has been shown toinduce expression of DNMT1 in human erythroleukemia cells[157]. It may be interesting to investigate whether the detrimentaleffects of IL-6 upon the b-cell line INS-1 [158] could be mediatedthrough altered levels of the epigenetically regulated miRNAs.

Exposure of b cells to proinflammatory cytokines activatesNFjB, which is an important transcriptional activator for inflam-matory genes. A number of miRNAs expressed in response to NFjBactivation have been identified, such as miR-146a, miR-155, miR-147, miR-9, miR-301a, miR-21, miR-125a and b, miR-30b and sev-eral miRNA clusters (miR-17-92, miR-221/222 and miR-23b/miR-27b/miR-24-1) [159–167]. In pancreatic b cells and b-cell lines,miR-21 shows a 2-fold increase after two hours of cytokine expo-sure while miR-146a is increased 10- to 20-fold after 6 h [168,169],indicating a direct expression of these miRNAs upon NFjB activa-tion. When investigating miRNA expression 24–30 h after cytokineexposure in the MIN6 mouse b-cell line, additional miRNAs showaltered expression, among them miR-34a, miR-9, miR-96 and themiR-29 family (miR-29a/b/c) [168], possibly secondary to genes in-duced by the early activation of NFjB. In myeloid cells, the tran-scription factor PU.1 controls expression of miR-146a, while inprostate carcinoma and glioblastoma cells, c-Jun is necessary[160,171,172], showing that regulation of miRNA expressionthrough NFjB will depend on the ambient repertoire of transcrip-tional co-factors available in a specific cell type.

Besides alteration in transcription of miRNAs, changes in miRNAactivity may be imposed by pro-inflammatory cytokines on the

processional level as well. For example, class 1 KDACs (HDAC-1, -2, -3, and 8) are an integrated part of the Drosha/DGCR8 complex,contributing to regulation of the processing of primary miRNAtranscripts [173], and we have earlier shown that especially theClass 1 KDACs alter expression in b cells upon cytokine exposure[19]. This suggests that subtle changes in expression of a largenumber of miRNAs upon cytokine exposure may result from al-tered KDAC protein levels. In b cells, effects of miRNA expressionupon inhibiting KDAC function remains to be investigated; how-ever in other cell types, inactivation of KDACs using KDAC inhibi-tors does indeed result in altered expression of about 40% of theinvestigated miRNAs [174].

Many RNA binding proteins (hnRNP A1, KSRP, LIN-28, HuR,Dnd1, FXR1) have been shown to play important roles in miRNAbiogenesis and activity [175–180] and cytokines alter the expres-sion and function of RNA binding proteins [181,182]. In b cells, al-tered function of the RNA binding protein AUF1 mediates cytokineb-cell death [182], by destabilizing antiapoptotic mRNAs. As manyRNA binding proteins and miRNAs regulate mRNA stabilitythrough collaborating or competing for binding to target sites[177,183–186], it will be important to investigate whether cyto-kine mediated effects of RNA binding proteins in b cells are a resultof collaboration with, or competition of, miRNAs for binding tomRNA targets.

Transcriptional regulation mediated through microRNAs

The knowledge of miRNA regulation of gene transcription is stillin its infancy, and although it has long been known that overex-pression of miRNAs downregulates the level of target mRNAs,examples were also seen of increased expression of mRNAs[187]. However, as computational predictions could not find signif-icant miRNA target sequences in the 3’UTR of the upregulatedgenes, this was considered a secondary effect to the miRNA overex-pression [187], for example through downregulation of a transcrip-tional repressor. Major proteins involved in mediating RNAinterference (Dicer and Ago proteins) are also associated withand involved in heterochromatic silencing [188], and recently sev-eral laboratories have through their investigations of gene regula-tion at the transcriptional level discovered that miRNAs and othersmall RNA species also are directly involved in activating genetranscription, a mechanism now termed RNAa (RNA activation)and the small RNA molecules involved are named saRNA (smallactivating RNA) [189] to distinguish their effect from RNAi (RNAinterference). The activation of gene transcription is sequencedependent, i.e. the miRNA targets complementary promoter se-quences in a manner similar to that inhibiting mRNA translation,but now resulting in activation of mRNA transcription and a subse-quent increase in protein levels [189,190]. These examples of miR-NA mediated activation of gene transcription are yet few, possiblydue to lack of search rather than their rarity.microRNAs, e.g. miR-130a and miR-223, were recently found to repress the transcrip-tion of mRNA through binding and silencing promoter activity[191,192], confirming the ability of miRNAs to regulate genes atthe transcriptional level. Interestingly, miR-130a has previouslybeen shown to play a role in b-cell biology [193], indicating thatthis new uninvestigated field of miRNA regulation of genes at thetranscriptional level will be an important area of future researchwhen trying to understand b-cell biology and the cytokine medi-ated dysfunction.

Post transcriptional regulation by miRNAs in the b-cell

Although emerging evidence shows that microRNAs canfunction at the transcriptional level of gene regulation, the vast

Table 1Micrornas involved in b-cell function. microRNAs known to affect b-cell function are listed, together with the processes they are involved in and the mRNA targets, if known.microRNAs whose expression is induced upon cytokine exposure are written in bold, and microRNAs known to be expressed in part through NFkB activity are underlined.

miRNA Function Targets Reference

miR-9 Reduce insulin secretion Onecut2, Cdh1, Sirt1 [196–198]

miR-15a Increase insulin biosynthesis Ucp-2 [199]

miR-21 Reduce insulin secretionReduce apoptosis

Increase proliferation

Pdcd4, Tpm1, PTEN, TAp63, TGFBR2 [168,169,200–207]

miR-24 Increase insulin biosynthesis – [208]

miR-26 Increase insulin biosynthesis – [208]miR-29a/b/c Reduced glucose stimulated insulin

releaseIncreased apoptosis

Onecut2, Mcl1 [170]

miR-30d Increase insulin biosynthesis – [209]miR-34a Increase apoptosis

Reduce insulin secretionSirt1, CyclinD1, Bcl-2, c-Met, CDK4/6, Mycn VAMP2, Notch1, CCNE2, CCND1,E2F3.

[168,210–219]

miR-96 Reduce insulin secretion Noc2, GPC3, FOX1, FN1, ADCY6 [220,221]miR-124a Reduce insulin secretion FoxA2, Rab27A, Noc2, PTB, Mtpn [50,221–223]miR-130a Increase insulin secretion – [193]miR-133a Reduce insulin biosynthesis PTB [50]

miR-146a/ miR-146b

Increase apoptosisReduce insulin secretion

TRAF6, IRAK1, PTB, MAP3K8 (COT/Tpl2) [50,165,168]

miR-148 Increase insulin biosynthesis – [208]miR-182 Increase insulin biosynthesis – [208]miR-200a Increase insulin secretion – [193]miR-375 Reduce insulin biosynthesis

Reduce insulin secretionReduce/increase proliferation

Pdk1, Mtpn, Jak2. [223–229]

miR-410 Increase insulin secretion – [193]


majority of data currently shows miRNAs as mediators of post-transcriptional regulation. In the b cell, cytokine exposure resultsin alterations of miRNA expression, which in turn will result in reg-ulation of protein levels thus contributing to the cellular responseto cytokine-induced stress. Phenotypic effects of altered miRNAlevels can be investigated through knockdown and overexpressionexperiments; however the underlying mechanisms may be diffi-cult to determine due to the large amount of genes potentially reg-ulated by each miRNA, and their ability to act as both repressorsand activators at the levels of both transcription and translation[133,191,194,195]. Despite this, during the last several years theinterest in miRNAs and their importance for cellular functionsand disease development has resulted in the discovery of a numberof miRNAs that are important for b-cell function, and in severalcases some of the targets they regulate have been determined(Table 1).

miRNAs and b-cell dysfunction

The mechanisms by which miRNAs are regulated and contrib-ute to b-cell death in response to cytokines are not well under-stood, but may be elucidated by data derived from other celltypes. For example, miR-34a is well studied in the context of can-cer as a tumor suppressor. miR-34a is pro-apoptotic, and is directlytransactivated by the tumor suppressor protein p53. miR-34a pos-itively contributes to p53 activity by reducing Sirt1 levels, therebyincreasing p53 acetylation [210,215,219]. Other known targets ofmiR-34a include genes such as Cdk4, mycn, E2F3, and E2F5[217,218]. Furthermore, it has been found that induction of miR-34a in human fibroblasts downregulates the anti-apoptotic factorBcl-2 [230], and miR-34a targets Notch1 and inhibits cell prolifer-ation in glioblastoma multiforme cells [212].

The induction of miR-34a detected in b cells upon exposure tocytokines is slow, requiring 24–48 h [168], and therefore may notbe a direct result of cytokine signaling, but more likely a conse-quence of accumulating b-cell damage sufficient to induce a p53stress response. Upon upregulation of miR-34a in the b cell

[168], the consequence is increased cell death, while inhibition re-duces cell death in the presence of stress signals [214].

The miR-29 family is also slowly induced in b cells, doublingexpression over 30 h of cytokine exposure [170]. The increasedexpression both in MIN6 cells and dissociated human islets leadsto decreased levels of the antiapoptotic protein Mcl1, resulting inincreased cell death.

Expression of miR-21 is directly induced though NFkB and isalso well studied in the context of cancer. Interestingly, miR-21predominantly functions as an anti-apoptotic miRNA and targetsseveral tumor suppressor genes. In human and mouse glioblas-toma cells, loss of miR-21 leads to an increase in apoptosis [200],possibly through increased expression of its target, programmedcell death 4 (Pdcd4), a tumor-suppressor gene [201]. Loss of Pdcd4delays onset of diabetes in NOD as well as C57BL/6 mice, where thereduced levels of Pdcd4 protein protected pancreatic b-cells fromcytokine-induced cell death via down regulation of the apoptoticproteins Bax, Bad and Bid [169], indicating that induction of miR-21 expression upon cytokine exposure may be an attempt to pro-tect the b cell.

Expression of miR-146a is also directly induced through NFkB,and is known to downregulate IL-1 receptor-associated kinase 1(Irak1) and TNF receptor-associated factor 6 (Traf6) protein levels,resulting in a negative feedback reducing NFkB activity [165]. Inthe b cell however, the net effect of high miR-146a levels is in-creased cell death as seen when transfecting MIN6 cells withmiR-146a and correspondingly, reducing levels of miR-146a re-sults in protection of the cells from cytokine induced apoptosis[168]. The genes targeted by miR-146a resulting in increasedcell-death are not yet known; however the JNK pathway may beinvolved since knockdown of miR-146a attenuated cytokine in-duced c-Jun expression and phosphorylation [168].

Apart from the effects on viability of the b cell, altered mir-146a,miR-21, miR-34a and miR-29a/b/c levels affect b-cell function. In-creased miR-21, miR-34a and miR-29a/b/c levels were shown toreduce glucose-stimulated insulin secretion in pancreatic b cells[168,214], while inhibition of miR-34a, miR-21 or miR-146a allabrogated the reduction in glucose-induced insulin secretion when


exposed to cytokines [168]. So while functional evidence favors therole of cytokine-induced miRNAs (-146a, -34a) in cytokine-inducedb-cell death and dysfunction [168], the effect of increased miR-21expression is more ambiguous. Transient overexpression of miR-21in b-cell lines failed to reduce cytokine-induced cell-death [168];however in vivo data from mice indicate that increased miR-21can protect b cells through reducing levels of the pro-apoptoticPdcd4 [169]. This discrepancy could be due to the altered cell-death and -proliferation pathways which have occurred duringthe immortalization of the b-cell lines, or that the transient natureof the miR-21 overexpression experiments does not result in suffi-cient Pcdc4 downregulation. Interestingly, although lack of Pdcd4initially results in a substantial delay in development of diabetesin NOD mice, the animals catch up around 25 weeks of age, reach-ing the same level of incidence as wild type animals. This indicatesthat lack of Pcdc4 may eventually become detrimental to the b cell,as was recently shown in several cell types where loss of Pcdc4 re-sulted in apoptosis through increased translation of procaspase-3mRNA [231]. Alternatively, if the underlying reasons for b-cellstress are not alleviated, the accumulating b-cell dysfunctionmay result in diabetes over time despite inhibition of one deathpathway. The dual effects of overexpression of miR-21 upon b-cellviability and function will require further study to reveal whetherthe upregulation of miR-21 observed in b-cells exposed to cyto-kines is a protective response, or if it is a contributing factor tocytokine induced b-cell failure.

In summary, with the large numbers of miRNAs known to beexpressed in bcells, while few of their targets are known, it willbe an important future path of research to investigate the regula-tory roles of the individual miRNAs to determine their role in dys-function and death. Apart from miRNAs directly altered throughcytokine exposure, thereby directly mediating cell response tocytokines, miRNAs that have been preemptively altered in certainindividuals through environmental or genetic challenges may pre-dispose towards b-cell dysfunction, thereby increasing risk of dia-betes development. For example, environmental factors such asexposure to smoking and nicotine alters miRNA expression [232],while alcohol-induced oxidative stress also results in altered miR-NA levels [233]. Genetically, a number of microRNAs primarily orspecifically expressed in pancreatic islets [147,234] are located inan epigenetically regulated region on chromosome 14 (humans),which in stemcells alter expression upon KDAC inhibition [235].Many of the microRNAs present in this cluster were recentlyshown to be altered in the dysfunctional b cell of the type 2 diabe-tes model Goto-Kakizaki rat [236], and in humans a SNP(rs941576) present located upstream of the miRNA cluster alterssusceptibility to type 1 diabetes [237]. Future investigations ofthe contributions of these miRNAs towards b-cell function anddevelopment of diabetes will be interesting to perform.

Conclusions

As evident from this review, the biology and pathobiology ofROS, KDACs and miRNAs are interconnected, influencing gene tran-scription and translation to modulate cell function and viability.The ROS species superoxide radical and hydrogen peroxide haverecently been shown to induce miR-21 expression through activa-tion of NFkB in HELF cells [238] thereby linking NFkB regulatedmiRNAs to elevated ROS levels. And KDACs have also in a largenumber of reports been shown to regulate miRNA expression[174]; however studies involving b cells are generally lacking. Ascytokine-mediated b-cell death can be alleviated through inhibit-ing KDACs, investigations determining whether miRNA expressionalso is altered in b cells upon KDACi, and whether this contributesto the protective effect of KDAC inhibition, should be carried out.

Interestingly it was recently observed that KDACs of the class 1type are associated with the Drosha complex that processes theprimary miRNA transcripts, and alter pri-miRNA association withthe processing complex through deacytalation of DCRG8 [173].

In conclusion, this review illustrates how genetic, epigenetic,inflammatory and metabolic factors converge on gene transcrip-tion and protein translation to determine cell fate, and opensnew perspectives for interventional targets of potential use inmany chronic diseases, including diabetes.

Acknowledgments

Funding for the support of this work was as follows: G.W.N. wassupported by the European Communitys Seventh Framework Pro-gramme FP7/2007-2013-FP7-NMP-In vivo Imaging of Beta-cell byApplied Nano Technology (VIBRANT). M.L. was supported by a Uni-versity of Copenhagen Career PhD fellowship. M.B.B. was sup-ported by a SHARE (Synergy in Human and Animal Research)PhD fellowship. D.P.C was supported by JDRF (Juvenile DiabetesResearch Foundation) (# 26-2008-893) and the EFSD/JDRF/NovoNordisk European Programme in Type 1 Diabetes Research. J.B.H.was supported by a SHARE PhD fellowship. M.S.D. was supportedby a University of Copenhagen Career PhD fellowship. T.M-P. wassupported by Novo Nordisk and the Novo Nordisk Foundation.

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