Hyperlipidemia-Induced MicroRNA-155-5p Improvesb-Cell Function by Targeting MafbMengyu Zhu,1 Yuanyuan Wei,1,2 Claudia Geißler,1 Kathrin Abschlag,1 Judit Corbalán Campos,1

Michael Hristov,1 Julia Möllmann,3 Michael Lehrke,3 Ela Karshovska,1,2 and Andreas Schober1,2

Diabetes 2017;66:3072–3084 | https://doi.org/10.2337/db17-0313

A high-fat diet increases bacterial lipopolysaccharide(LPS) in the circulation and thereby stimulates glucagon-like peptide 1 (GLP-1)–mediated insulin secretion by upre-gulating interleukin-6 (IL-6). Although microRNA-155-5p(miR-155-5p), which increases IL-6 expression, is upregu-lated by LPS and hyperlipidemia and patients with familialhypercholesterolemia less frequently develop diabe-tes, the role of miR-155-5p in the islet stress response tohyperlipidemia is unclear. In this study, we demonstratethat hyperlipidemia-associated endotoxemia upregulatesmiR-155-5p in murine pancreatic b-cells, which improvedglucosemetabolism and the adaptation of b-cells to obesity-induced insulin resistance. This effect of miR-155-5p isbecause of suppression of v-maf musculoaponeurotic fi-brosarcoma oncogene family, protein B, which promotesb-cell function through IL-6–induced GLP-1 production ina-cells. Moreover, reduced GLP-1 levels are associatedwith increased obesity progression, dyslipidemia, and ath-erosclerosis in hyperlipidemic Mir155 knockout mice.Hence, induction of miR-155-5p expression in b-cells byhyperlipidemia-associated endotoxemia improves the ad-aptation of b-cells to insulin resistance and represents aprotective mechanism in the islet stress response.

Failure of pancreatic b-cells to enhance insulin secretion inresponse to reduced systemic insulin sensitivity plays a keyrole in the development of hyperglycemia and type 2 diabe-tes (T2D) (1,2). Inflammatory macrophage recruitment intovisceral adipose tissue during obesity frequently contributesto insulin resistance and adipocyte dysfunction throughsecretion of inflammatory cytokines (3). Although b-cellscan maintain glucose homeostasis in insulin-resistant states

by increasing circulating insulin levels, chronically elevatedinsulin secretion may result in exhaustion of b-cell functionbecause of apoptosis or dedifferentiation (4,5). Obesity andT2D may promote b-cell failure by decreasing the secretionof glucagon-like peptide 1 (GLP-1), which enhances insulinsecretion and b-cell function, from intestinal L cells (6–11).In addition, pancreatic a-cells can be a source of GLP-1,for instance, in response to interleukin-6 (IL-6)–mediatedupregulation of proprotein convertase subtilisin/kexintype 1/3 (PC1/3; encoded by the proprotein convertasesubtilisin/kexin-type [Pcsk1] gene) and thereby improveb-cell function during obesity (7,12,13).

Lipopolysaccharide (LPS), which leaks into the circulationafter a high-fat meal because of increased intestinal perme-ability (14,15), also promotes insulin secretion by upregulat-ing GLP-1 production (16,17). In the circulation, LPS bindsprimarily to lipoproteins such as LDL and VLDL (18,19).Notably, patients with familial hypercholesterolemia have areduced risk for T2D and an increased LPS binding capac-ity because of the elevated lipoprotein levels (19,20). How-ever, chronically elevated LPS levels during high-fat dietfeeding also induce adipose tissue inflammation, insulin re-sistance, and obesity (21). In macrophages, many LPS ef-fects are mediated through the highly conserved vertebratemicroRNA-155-5p (miR-155-5p), which is preferentiallyupregulated upon Toll-like receptor 4 activation (22,23).Moreover, hyperlipidemia induces miR-155-5p expressionin macrophages and thereby changes its effect from inhib-iting macrophage proliferation in early atherosclerosis toimpairing efferocytosis and promoting inflammatory acti-vation in advanced lesions (23–28). In adipocytes, inflamma-tory cytokines, such as tumor necrosis factor-a, upregulate

1Institute for Cardiovascular Prevention, Ludwig-Maximilians-UniversitätMünchen, Munich, Germany2German Centre for Cardiovascular Research, Partner Site Munich Heart Alliance,Munich, Germany3Department of Internal Medicine I, University Hospital Aachen, Aachen, Germany

Corresponding author: Andreas Schober, aschober@med.lmu.de.

Received 13 March 2017 and accepted 19 September 2017.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0313/-/DC1.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

3072 Diabetes Volume 66, December 2017

PATHOPHYSIO

LOGY

miR-155-5p expression, which may contribute to obesityprogression in female mice by limiting brown adipose tissuedifferentiation (29,30). The role of miR-155-5p, however, inobesity and glucose homeostasis during hyperlipidemia-associated endotoxemia is unclear.

We found that endotoxemia induces miR-155-5pexpression in pancreatic b-cells, which increases insulin se-cretion by targeting v-maf musculoaponeurotic fibrosar-coma oncogene family, protein B (Mafb) in hyperlipidemicmice. MafB represses IL-6 expression in b-cells and therebyinhibits intraislet GLP-1 production. Through this mecha-nism, miR-155-5p improved the adaptation of b-cells tohyperlipidemic stress and the compensation for obesity-induced insulin resistance and likely limited the progressionof obesity and atherosclerosis.

RESEARCH DESIGN AND METHODS

For further details, refer to the Supplementary Data online.

AnimalsMir1552/2 mice were crossed with LDL receptor knockout(Ldlr2/2) or apolipoprotein E knockout (Apoe2/2) mice (allon a C57BL/6J background; The Jackson Laboratory)to obtain Mir1552/2Ldlr2/2 mice and Mir1552/2Apoe2/2

mice.Mir1552/2Ldlr2/2 mice andMir155+/+Ldlr2/2 mice at10–12 weeks of age were fed a diabetogenic diet supple-mented with cholesterol (DDC; 35.5% fat and 36.3% carbo-hydrates with 0.15% weight-for-weight total cholesterol;ssniff Spezialdiäten) or a normal diet (ND; 3.3% fat; ssniffSpezialdiäten).

Isolation of Pancreatic IsletsMurine pancreatic islets were isolated by collagenase di-gestion and density-gradient centrifugation. Briefly, collage-nase P solution (4 mL, 1 mg/mL; Roche Diagnostics) wasslowly injected into the common bile duct after occlusion ofthe ampulla in the duodenum. Islets were purified by gradi-ent separation using sodium diatrizoate (Histopaque 1119and Histopaque 1077; Sigma-Aldrich).

Cell Culture and TransfectionMIN6 cells (kindly provided by Dr. Ingo Rustenbeck, Uni-versity of Braunschweig, Braunschweig, Germany), humanislets (Pelobiotech), and GLUTag cells were transfectedwith locked nucleic acid (LNA)–miR-155-5p inhibitors (50nmol/L; Exiqon), miR-155-5p mimics (15 nmol/L; ThermoFisher Scientific), 155/Mafb target site blockers (TSBs; 50nmol/L; Exiqon), or scrambled controls using Lipofectamine2000 (Thermo Fisher Scientific).

miR Target Identification and Quantification SystemMIN6 cells and human islets were cotransfected with miR-155-5p mimics and the pMirTrap vector using the XfectmiR transfection reagent in combination with XfectPolymer (all from Clontech Laboratories). The pMirTrapvector expresses a DYKDDDDK-tagged GW182 protein.Cell lysates were incubated with anti-DYKDDDDK beads(Clontech Laboratories), and RNA was isolated from

input and immunoprecipitated samples and analyzed byquantitative real-time PCR (qPCR). Fold enrichment ofthe target genes in the GW182 immunoprecipitates wasnormalized to the enrichment of Gapdh.

In Vivo TSB TreatmentTen-week-old Ldlr2/2 mice fed an ND were injected intra-venously via the tail vein with 155/Mafb TSBs or controlTSBs (each 0.4 mg/20 g/injection; miRCURY LNA micro-RNA Target Site Blocker for in vivo use; Exiqon), as de-scribed in Supplementary Data.

Statistical AnalysisData represent the mean 6 SEM. Statistical analysis ofmicroarray data were performed by a modified t test usingGeneSpring software (GX13; Agilent Technologies). Studentt tests and one-way ANOVAs followed by the Newman-Keuls post hoc test were used for statistical comparisonsbetween groups using Prism 6 software (GraphPad Software).The variance is similar between the groups that are beingstatistically compared. A P value ,0.05 was considered sta-tistically significant.

RESULTS

Deletion of Mir155 Deteriorates Metabolic Diseasein Ldlr2/2 MiceTo study the effect of miR-155-5p on atherosclerosis in thecontext of obesity and T2D, we deleted the miR-155-5pcoding gene in hyperlipidemic Ldlr2/2 mice that developatherosclerosis, obesity, and diabetes after DDC feeding(31). After a 24-week DDC feeding period, the developmentof atherosclerosis and the necrotic core formation in thelesions were increased in Mir1552/2Ldlr2/2 mice comparedwithMir155+/+Ldlr2/2 mice (Fig. 1A). Lesions inMir1552/2

Ldlr2/2 mice contained less macrophages than inMir155+/+

Ldlr2/2 mice, whereas the lesional smooth muscle cell con-tent was similar in both groups (Supplementary Fig. 1A).Total cholesterol and triglyceride plasma levels werehigher in Mir1552/2Ldlr2/2 mice than those in Mir155+/+

Ldlr2/2 mice after the 24-week DDC feeding period (Sup-plementary Fig. 1B). InMir1552/2Ldlr2/2 mice, the choles-terol level was increased in the VLDL and LDL fractionand reduced in the HDL fraction (Fig. 1B). AlthoughMir1552/2Ldlr2/2 mice and Mir155+/+Ldlr2/2 mice gainedsimilar body weight in the first 20 weeks of DDC feeding,the body weight of Mir1552/2Ldlr2/2 mice increased morethan that of Mir155+/+Ldlr2/2 mice during the last 4 weeksof the 24-week DDC feeding period (Fig. 1C). This effect inMir1552/2Ldlr2/2 mice was associated with an increase inepididymal white adipose tissue (eWAT) weight (Fig. 1D),adipocyte size (Fig. 1E), and macrophage infiltration in ad-ipose tissue (Fig. 1F). Moreover, the expression of adipo-nectin (Adipoq) and leptin (Lep) was down- and upregulated,respectively, in the eWAT of Mir1552/2Ldlr2/2 mice (Sup-plementary Fig. 1C). The expression of the proinflammatorymacrophage-related gene nitric oxide synthase 2 (Nos2)and the anti-inflammatory macrophage marker mannose

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Figure 1—Effects ofMir155 knockout on atherosclerosis, obesity, and metabolism in Ldlr2/2 mice. MaleMir155+/+Ldlr2/2 andMir1552/2Ldlr2/2

mice were fed a DDC for 24 weeks. A: Lesion and necrotic core areas in aortic roots in mice after the 24-week DDC feeding period(n = 10 mice/group). Scale bars: 100 mm. B: Cholesterol levels in VLDL, LDL, and HDL fractions from mice after the 24-week DDC feedingperiod analyzed by high-performance liquid chromatography (n = 8 mice/group). C: Body weight gain of mice during the 24-week DDC feedingperiod (n = 10 mice/group). D and E: Quantitation of eWAT weight (D) and adipocyte size in the eWAT (E) from mice after the 24-week DDCfeeding period (n = 10 mice/group). Scale bars: 100 mm. F:Macrophage accumulation in eWAT frommice after the 24-week DDC feeding periodassessed by Mac2 immunostaining (n = 9 mice/group). The nuclei were counterstained with DAPI. Scale bars: 50 mm. G: FBG concentrations inmice during the 24-week DDC feeding period (n = 10 mice/group). Data are represented as mean 6 SEM. *P , 0.05; **P , 0.01; ***P , 0.001.

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receptor, C type 1 (Mrc1) was not different between thegroups (Supplementary Fig. 1C). Deletion of Mir155 didnot alter Il6mRNA expression, but increased Tnf expressionin eWAT (Supplementary Fig. 1C).

During the DDC feeding period, fasting blood glucose(FBG) levels increased constantly at a similar rate in bothgroups of mice, whereas FBG levels in Mir1552/2Ldlr2/2

mice were always higher than those in Mir155+/+Ldlr2/2

mice (Fig. 1G). Surprisingly, FBG levels were also higher inMir1552/2Ldlr2/2 mice compared with Mir155+/+Ldlr2/2

mice before feeding of the DDC (0 weeks) (Fig. 1G), indicatingthat the hyperglycemia in Mir1552/2Ldlr2/2 mice isnot because of the increased weight gain. These findingsindicate that miR-155-5p improves glucose homeostasisin hyperlipidemic mice and thereby limits obesity andatherosclerosis.

Mir155 Knockout Inhibits Insulin Production inHyperlipidemic MiceTo investigate the mechanism by which miR-155-5p affectsglucose homeostasis, we determined the effect of Mir155knockout on insulin and glucagon plasma levels. Fasting in-sulin levels were lower, whereas glucagon levels were higher inthe plasma from Mir1552/2Ldlr2/2 mice than in Mir155+/+

Ldlr2/2 mice fed the ND (0 weeks) and after the 24-weekDDC feeding period (Fig. 2A), indicating that loss of miR-155-5p compromises islet function. In islets from ND-fedMir1552/2Ldlr2/2 mice, the percentage of insulin-expressingcells and the insulin content were reduced compared withMir155+/+Ldlr2/2 mice (Fig. 2B). Conversely, the percent-age of glucagon-expressing cells and the glucagon proteincontent were higher in islets from Mir1552/2Ldlr2/2 mice(Fig. 2C).

Proglucagon is processed to GLP-1 and glucagon by PC1/3 (encoded by the Pcsk1 gene) and PC2 (encoded by thePcsk2 gene), respectively (6,8). GLP-1 can be generatedlocally in pancreatic a-cells and increases insulin and reducesglucagon secretion (6). Therefore, we studied the effect ofMir155 knockout on GLP-1 expression. The intraislet GLP-1protein content was reduced in ND-fed Mir1552/2Ldlr2/2

mice (Fig. 2D). Plasma GLP-1 levels were also lower inMir1552/2Ldlr2/2 mice than in Mir155+/+Ldlr2/2 micefed an ND (0 weeks) or the DDC for 24 weeks (Fig. 2E).These effects were associated with decreased insulin (Ins)expression in islets (Fig. 2F) and in b-cells (Fig. 2G)and upregulation of glucagon (Gcg) expression in islets(Fig. 2F) and in a- cells (Fig. 2G) from Mir1552/2Ldlr2/2

mice compared with those from Mir155+/+Ldlr2/2 mice.Moreover, like in whole islets, Pcsk1 expression wasdownregulated (Fig. 2G) in a- and b-cells from Mir1552/2

Ldlr2/2 mice. By contrast, the expression of somatostatin(Sst) and of the b-cell transcription factors ISL LIM homeo-box 1 (Isl1), aristaless-related homeobox (Arx), pancreaticand duodenal homeobox 1 (Pdx1), paired box 6 (Pax6),neurogenic differentiation 1 (Neurod1), and forkhead boxA1 (Foxa1) in islets was not different between the groups(Supplementary Fig. 2A). Islet cell apoptosis and the

accumulation of macrophages or T cells in islets were negligiblein both groups of mice (Supplementary Fig. 2B).

In vitro, miR-155-5p mimics treatment downregulatedGcg and Pcsk2 mRNA expression and upregulated Pcsk1mRNA expression in MIN6 cells (Fig. 2H and Supplemen-tary Fig. 2C). At the protein level, miR-155-5p mimic treat-ment increased the cellular insulin and GLP-1 content andreduced the glucagon level in MIN6 cells compared withcontrol mimics (Fig. 2H). Accordingly, Pcsk1 and Pcsk2 ex-pression was increased and reduced, respectively, in sorteda-cells after treatment with miR-155-5p mimics com-pared with control mimics (Supplementary Fig. 2E). In hu-man islets, miR-155-5p mimic decreased the expression ofGCG in a-cells and increased INS expression in b-cells,whereas PCSK1 expression was upregulated in a-cells(Supplementary Fig. 2F). Conversely, miR-155-5p inhibitortreatment increased Gcg and Pcsk2 expression and reducedPcsk1 expression in MIN6 cells (Supplementary Fig. 2Cand D), which resulted in decreased insulin and GLP-1content and increased glucagon content (SupplementaryFig. 2D).

In addition, overexpression of miR-155-5p promotedGLP-1 secretion from murine islets (Fig. 2I). By contrast,treatment of an enteroendocrine L-cell line with miR-155-5p mimics did not affect GLP-1 protein content and secretionand GCG and PCSK1 mRNA expression (SupplementaryFig. 2G and H). Hence, miR-155-5p promotes intraisletGLP-1 production by upregulating Pcsk1 expression andmay thereby improve glucose homeostasis.

Next, we studied glucose tolerance in Mir1552/2 mice inthe absence and presence of hyperlipidemia. Notably,Mir155 knockout increased blood glucose levels followingintraperitoneal glucose challenge in hyperlipidemic maleand female Ldlr2/2 (Fig. 2J and Supplementary Fig. 3A) orApoe2/2 mice (Supplementary Fig. 3B and C) fed an ND,whereas glucose tolerance was not affected by Mir155knockout in normal, lipidemic, male ND-fed Ldlr+/+ mice(Fig. 2K). Thus, miR-155-5p improved glucose homeostasisonly under hyperlipidemic conditions.

Hyperlipidemia-Associated Endotoxemia Induces IsletmiR-155-5p ExpressionNext, we studied the regulation of islet miR-155-5pexpression by hyperlipidemia and LPS. Feeding Ldlr2/2

mice the DDC for 24 weeks increased plasma endotoxinactivity and islet miR-155-5p expression compared withND feeding (Fig. 3A). In 10- to 12-week-old ND-fed mice,knockout of Ldlr increased plasma cholesterol and triglyc-eride levels (Supplementary Fig. 4A), circulating endotoxinactivity, and islet miR-155-5p expression (Fig. 3B and C).miR-155-5p was mainly detectable in glucagon2 cells inislets by combined immunostaining and in situ PCR (Fig.3C). In contrast to native LDL (nLDL), mildly oxidized LDL(moxLDL) upregulated miR-155-5p expression in MIN6cells compared with vehicle treatment (Fig. 3D). LPS stim-ulation increased miR-155-5p expression in MIN6 cells(Fig. 3E) and human islet cells (Fig. 3F). Moreover, mild

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oxidation increased the endotoxin activity in LDL (Fig. 3G).Knockout of Ldlr in ND-fed mice resulted in deposition ofoxidized LDL (oxLDL) in islets (Fig. 3H). Thus, enhancedendotoxin activity of oxLDL deposited in islets during hy-perlipidemia may induce miR-155-5p expression in b-cells.

LPS-induced insulin release from islets following glucosestimulation was decreased in islets from Mir1552/2Ldlr2/2

mice (Fig. 3I). Treatment of Ldlr2/2 mice with low-dose LPSupregulated islet miR-155-5p expression (Fig. 3J) and in-creased insulin and GLP-1 plasma levels (SupplementaryFig. 4B). The glucose-lowering effect of low-dose LPS follow-ing intraperitoneal glucose injection in Ldlr2/2 mice (Fig. 3K)was partially abolished by Mir155 knockout (Fig. 3K).Together, these data suggest that hyperlipidemia-induced

Figure 2—Effect of miR-155-5p on pancreatic islets. A: Fasting insulin and glucagon plasma concentrations in mice fed an ND (0-week DDC)and after the 24-week DDC feeding period (n = 6 mice/group). B: Quantitation of the percentage of insulin-expressing b-cells per total islet cells(n = 15 or 18 mice/group) and islet insulin concentrations (n = 6/group) by immunostaining and Luminex multiplex analysis, respectively, in 10- to12-week-old mice fed an ND. The nuclei were counterstained with DAPI. Scale bars: 50 mm. C: Quantitation of the percentage of glucagon-expressing a-cells per total islet cells (n = 15 or 18 mice/group) and islet glucagon concentrations (n = 6/group) by immunostaining and Luminexmultiplex analysis, respectively, in 10- to 12-week-old mice fed an ND. The nuclei were counterstained with DAPI. Scale bars: 50 mm. D: IsletGLP-1 protein concentration in 10- to 12-week-old mice fed an ND determined by Luminex multiplex analysis (n = 6/group). E: Fasting GLP-1plasma concentrations in mice fed an ND (0-week DDC) and after the 24-week DDC feeding period (n = 6 mice/group). F: Islet Ins, Gcg, Pcsk1,and Pcsk2 mRNA expression levels in 10- to 12-week-old mice fed an ND determined by qPCR (n = 6 or 8/group). G: Quantitation of geneexpression by qPCR in a- and b-cells sorted from islets of ND-fedMir1552/2Ldlr2/2 mice andMir155+/+Ldlr2/2 mice (10–12 weeks of age) (n =3 to 4/group). H: Ins, Gcg, Pcsk1, and Pcsk2mRNA expression levels (n = 4 or 6/group) determined by qPCR and insulin, glucagon, and GLP-1protein levels (n = 4/group) measured by Luminex multiplex analysis in MIN6 cells treated with miR-155-5p mimics or nontargeting oligonu-cleotides (control mimics). I: GLP-1 secretion from KCl-stimulated islets isolated from ND-fed Mir155+/+Ldlr2/2 mice (10–12 weeks of age) aftertreatment with miR-155-5p mimics or control mimics (n = 4/group). J: Intraperitoneal glucose tolerance test in male Mir1552/2Ldlr2/2 mice andMir155+/+Ldlr2/2 mice at 10–12 weeks of age fed an ND (n = 10 mice/group). K: Intraperitoneal glucose tolerance test in male Mir155+/+Ldlr+/+

mice (n = 6 mice/group) andMir1552/2Ldlr+/+ mice (n = 8 mice/group) at 10–12 weeks of age fed an ND. Data are represented as mean6 SEM.*P , 0.05; **P , 0.01; ***P , 0.001.

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miR-155-5p expression improves b-cell adaptation tohyperlipidemia-associated endotoxemia stress.

miR-155-5p Promotes IL-6 Expression in b-CellsTo determine how miR-155-5p regulates b-cell function, weanalyzed the effect ofMir155 knockout on islet gene expres-sion by microarray analysis. In ND-fed Mir1552/2Ldlr2/2

mice, 239 genes were upregulated (Supplementary Table 1),and 420 genes were downregulated (Supplementary Table 2)compared with Mir155+/+Ldlr2/2 mice (P , 0.05; abso-lute fold change $1.5; n = 3 samples/group). Differen-tially regulated genes were enriched in the carbohydrateand lipid metabolism pathways and in pathways related

to endocrine system function, cellular growth, DNA repli-cation, and cell survival, as determined by Ingenuity PathwayAnalysis software (Fig. 4A). Analysis of potential upstreamregulators of differential gene expression in islets indicatedCdkn1 activation and Cdk4 inhibition inMir1552/2Ldlr2/2

mice, which may reduce islet cell proliferation (Fig. 4B)(32). PTEN activation, which contributes to b-cell failure inmouse models of T2D (33), was increased in Mir1552/2

Ldlr2/2 mice (Fig. 4B). Moreover, Glut2-dependent pathwaysand pathways related to cyclic AMP, GLP-1, and glucose-dependent insulinotropic polypeptide signaling were inhibited,suggesting impaired glucose uptake and insulin secretion(34).

Figure 3—miR-155-5p mediates the effects of LPS and hyperlipidemia on glucose homeostasis. A: Serum endotoxin levels in mice fed an ND orthe DDC for 24 weeks (left; n = 6/group). Quantitation of miR-155-5p expression in laser-microdissected islets from mice fed the ND or the DDCfor 24 weeks (right; n = 4/group). **P , 0.01; ***P , 0.001. B: Serum endotoxin levels in 10- to 12-week-old mice fed an ND determined byLimulus amebocyte lysate test (left; n = 6 mice/group). Quantitation of miR-155-5p expression by qPCR in isolated murine islets from ND-fedmice (10–12 weeks of age) (right; n = 6 mice/group). *P , 0.05. C: Localization of miR-155-5p expression in islets from ND-fed Ldlr+/+ andLdlr2/2 mice (10–12 weeks of age) determined by in situ PCR and glucagon immunostaining. The nuclei were counterstained with DAPI. Scalebars: 50 mm. D: Quantitation of miR-155-5p expression in MIN6 cells treated with nLDL, moxLDL, or vehicle for 6 h (n = 5 to 6/group). *P, 0.05.Quantitation of miR-155-5p expression in MIN6 cells (E) and human islets (F) treated with LPS or vehicle for 6 h (n = 5 to 6/group). *P, 0.05. G:Endotoxin activity in nLDL or moxLDL determined by Limulus amebocyte lysate test (n = 3/group). ***P , 0.001. H: oxLDL immunostaining inislets from ND-fed mice (10–12 weeks of age). The nuclei were counterstained with DAPI. Scale bars: 50 mm. I: Glucose-induced insulinsecretion from islets isolated from ND-fed Mir1552/2Ldlr2/2 mice and Mir155+/+Ldlr2/2 mice (10–12 weeks of age) with or without LPS(50 ng/mL) stimulation. Insulin concentrations in the medium were measured by ELISA (n = 4/group). *P , 0.05. J: Quantitation of miR-155-5p expression by qPCR in islets isolated from ND-fed Ldlr2/2 mice 6 h after injection of LPS (2 mg/kg) or vehicle (n = 6mice/group). *P, 0.05. K:Intraperitoneal glucose tolerance test in Ldlr2/2 mice 6 h after injection of LPS (2 mg/kg) or vehicle (n = 6 mice/group). *P , 0.05; **P , 0.01between LPS Mir1552/2Ldlr2/2 and LPS Mir155+/+Ldlr2/2; #P , 0.01; ##P , 0.001 between LPS Mir155+/+Ldlr2/2 and vehicle Mir155+/+Ldlr2/2. Data are represented as mean 6 SEM. EU, endotoxin unit.

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Among the inflammatory pathways, IL-6 receptor acti-vation was reduced, and signaling pathways downstream ofthe IL-6 receptor, such as the Janus kinase/STAT andextracellular signal–regulated kinase 1/2 pathways, wereinhibited in Mir1552/2Ldlr2/2 mice (Fig. 4B). Accordingly,islet IL-6 mRNA and protein expression and the number ofIL-6–producing b-cells were reduced in Mir1552/2Ldlr2/2

mice compared withMir155+/+Ldlr2/2 mice (Fig. 4C and D).Whereas Il6 expression was unchanged in sorted a-cells, itwas downregulated in b-cells from Mir1552/2Ldlr2/2 micecompared with Mir155+/+Ldlr2/2 mice (Fig. 4E). Moreover,IL6 expression was upregulated by miR-155-5p mimictreatment in human b-cells, but was not affected ina-cells compared with control mimic (Fig. 4F). In vitro,gain- and loss-of-function experiments demonstrated thatmiR-155-5p upregulates IL-6 mRNA and protein expressionin MIN6 cells (Supplementary Fig. 5A and B). Inhibition ofIL-6 secreted from MIN6 cells using a blocking IL-6 anti-body reduced Ins and Pcsk1 expression and increased Pcsk2expression (Fig. 4G). In addition, treatment of sorted hu-man a-cells with conditioned medium from miR-155-5pmimic–treated human b-cells enhanced GLP-1 secretionand the cellular GLP-1 content (Supplementary Fig. 5C).Taken together, these results indicate that miR-155-5p inb-cells stimulates the expression and secretion of IL-6,

which in turn increases GLP-1 production by upregulatingPcsk1 expression in a-cells.

miR-155-5p Upregulates IL-6 by Targeting MafbTo determine the targets that mediate the effect of miR-155-5p on IL-6 expression in b-cells, we screened the 39-untranslated region (UTR) of the genes upregulated in isletsfromMir1552/2Ldlr2/2 mice for miR-155-5p binding sites.According to the TargetScan (v7.0) prediction algorithm,27 out of the 239 upregulated genes, including Mafb, sem-aphorin 5A (Sema5a), and mediator complex subunit 12-like(Med12l), contained miR-155-5p binding sites (Table 1).The miR-155-5p target sites in the Mafb and Sema5a 39-UTRs were conserved among species, whereas the other25 sites were poorly conserved. However, three of thepoorly conserved sites were also found in humans, includingthe site in the AU RNA-binding protein/enoyl-CoA hydra-tase (Auh), stathmin-like 2 (Stmn2), and Med12l mRNAs.Upregulation of islet Auh, Mafb, Med12l, Sema5a, andStmn2 expression inMir1552/2Ldlr2/2 mice was confirmedby qPCR (Fig. 5A). Treatment of MIN6 cells with miR-155-5p mimics (Fig. 5B) and inhibitors (Supplementary Fig. 6A)reduced and increased the expression of Auh,Mafb,Med12l,Sema5a, and Stmn2, respectively.

Next, we performed immunoprecipitation of the micro-RNA-induced silencing complex using extracts from MIN6

Figure 4—Mir155 deficiency reduces IL-6 expression in b-cells. A and B: Gene expression profiling by microarrays in islets isolated from ND-fedMir155+/+Ldlr2/2 and Mir1552/2Ldlr2/2 mice (10–12 weeks of age) (n = 3 samples/group). Biological processes enriched with differentiallyregulated genes (A) and upstream regulators (B) of differential gene expression predicted by Ingenuity Pathway Analysis software (P, 0.05; foldchange cutoff 1.5). C: Quantitation of IL-6 expression at the mRNA and protein level in islets isolated from ND-fed mice (10–12 weeks of age) byqPCR and ELISA, respectively (n = 6/group). D: Combined IL-6 and insulin immunostaining in pancreatic sections from ND-fed mice (10–12 weeks of age). Arrows indicate insulin+ cells expressing IL-6. Nuclei were counterstained with DAPI. Scale bars: 50 mm. E: Quantitation of Il6mRNA expression by qPCR in a- and b-cells sorted from islets of ND-fed Mir155+/+Ldlr2/2 mice andMir1552/2Ldlr2/2 mice (n = 3 to 4/group).F: Quantitation of Il6 mRNA expression by qPCR in sorted human a- and b-cells treated with miR-155-5p mimics or control mimics (n = 3 to4/group).G: Effect of anti–IL-6 antibody treatment on the expression levels of Ins,Gcg, Pcsk1, and Pcsk2 in MIN6 cells compared with treatmentwith isotype control antibodies (n = 4/group). Data are represented as mean 6 SEM. neg, negative. *P , 0.05; **P , 0.01; ***P , 0.001.

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cells and human islets overexpressing FLAG-tagged GW182(35). Among the potential target genes, miR-155-5p mimictreatment most strongly enriched Mafb in the miR-inducedsilencing complex of MIN6 cells and human islets. In con-trast to Auh, Med12l, and Sema5a, Stmn2 was also enrichedby miR-155-5p in both cell types (Fig. 5C and Supplemen-tary Fig. 6B). In Ldlr2/2 mice, Mir155 knockout increasedthe number of MafB-expressing cells in islets and, in con-trast to a-cells, upregulated Mafb expression in sortedb-cells (Fig. 5D and E). Moreover, miR-155-5p mimic treat-ment reduced MAFB expression in human b-cells, but notin a-cells (Supplementary Fig. 6C).

The miR-155-5p binding site in the MAFB 39-UTR hasbeen previously verified in B-cell lymphoma cells (Supplementary

Fig. 6D) (36). To test the function of this site, we designedLNA-modified oligonucleotides that selectively inhibit theinteraction between miR-155-5p and Mafb (155/Mafb TSB)(Supplementary Fig. 6D). In MIN6 cells, treatment with155/Mafb TSBs increased Mafb, Gcg, and Pcsk2 expressionand reduced Ins and Pcsk1 expression compared with controlTSBs (Fig. 5F). Notably, 155/Mafb TSB treatment reducedIL-6 expression at the mRNA and protein level (Fig. 5G).These data indicate that the effects of miR-155-5p onb-cells are mainly mediated by the targeting of Mafb.

To study howMafb regulates IL-6 expression, MIN6 cellswere transfected with a luciferase reporter vector contain-ing the wild-type Il6 promoter or the Il6 promoter contain-ing mutations in the predicted Mafb binding sites Mafb1and Mafb2 (Supplementary Fig. 6E). Treatment with miR-155-5p inhibitors reduced the luciferase activity in cellsexpressing the wild-type promoter (Fig. 5H and Supplemen-tary Fig. 6F), but not in cells expressing the promoter con-taining the mutated Mafb binding sites (Fig. 5H). Thesefindings suggest that reduced MafB-mediated transcrip-tional repression of IL-6 contributes to the effect of miR-155-5p on b-cell function.

Role of the miR-155-5p-Mafb Interaction in GlucoseHomeostasis In VivoTo study whether the effect of hyperlipidemia-induced miR-155-5p in b-cells on glucose homeostasis is mediated by thesuppression of Mafb, Ldlr2/2 mice were treated with 155/Mafb TSBs or nontargeting, LNA-modified oligonucleotides(control TSB). Body weights and differential blood countswere not different between the groups at 21 days after thetreatment (Supplementary Fig. 7A and B). Mafb mRNA ex-pression levels were increased in islets and spleen, but notin heart, liver, and eWAT in 155/Mafb TSB-treated mice(Fig. 6A). 155/Mafb TSB treatment did not affect isletAuh, Med12l, Sema5a, and Stmn2 expression levels (Supple-mentary Fig. 7C). The percentage of MafB-expressing cellsin islets was higher in 155/Mafb TSB-treated mice than inmice treated with control TSBs (Fig. 6B). Treatment with155/Mafb TSBs increased Gcg mRNA expression and thepercentage of a-cells and reduced Pcsk1 and Il6 expressionand the percentage of b-cells compared with control (Fig.6C and D). This effect in 155/Mafb TSB-treated mice wasassociated with reduced insulin and GLP-1 plasma levelsand increased glucagon plasma levels (Fig. 6E). Moreover,155/Mafb TSB treatment elevated FBG levels (Fig. 6F) andimpaired glucose tolerance following intraperitoneal glucoseinjection (Fig. 6G). These data indicate that hyperlipidemia-induced miR-155-5p expression improves b-cell adaptationand maintains glucose hemostasis by suppressing Mafb.

DISCUSSION

We found that hyperlipidemia and LPS upregulate miR-155-5p expression in b-cells, which improved glucosehomeostasis by targeting Mafb. In the absence of miR-155-5p, upregulation of MafB by hyperlipidemia inhibits IL-6 ex-pression and thereby reduces IL-6–mediated GLP-1 production

Table 1—Putative miR-155-5p target genes in pancreatic islets

Gene PCT Conservation

Mafb 0.39 Conserved

Sema5a 0.3 Conserved

Med12l 0.15 Poorly conserved*

Stmn2 0.13 Poorly conserved*

Auh 0.12 Poorly conserved*

F13a1 ,0.1 Poorly conserved#

Dhfr ,0.1 Poorly conserved#

Klhl42 ,0.1 Poorly conserved#

Ppp1r9a ,0.1 Poorly conserved#

Phf21a ,0.1 Poorly conserved#

Rab3c ,0.1 Poorly conserved#

Nedd4l ,0.1 Poorly conserved#

Homez ,0.1 Rodent-specific

Nrp1 ,0.1 Rodent-specific

Pde4d ,0.1 Rodent-specific

Zkscan3 ,0.1 Rodent-specific

Zfp14 ,0.1 Rodent-specific

Bik ,0.1 Mouse-specific

Camkk2 ,0.1 Mouse-specific

Clec1a ,0.1 Mouse-specific

Gpr179 ,0.1 Mouse-specific

Htra3 ,0.1 Mouse-specific

Myct1 ,0.1 Mouse-specific

Scai ,0.1 Mouse-specific

Zfp111 ,0.1 Mouse-specific

Zfp937 ,0.1 Mouse-specific

Zscan20 ,0.1 Mouse-specific

Among the genes significantly upregulated ($1.5-fold; P, 0.05) inpancreatic islets of Mir1552/2Ldlr2/2 mice compared withMir155+/+Ldlr2/2 mice (as determined by global gene expressionanalysis), miR-155-5p targets and the conservation of theputative miR-155-5p binding sites across species werepredicted by TargetScan software (http://www.targetscan.org/).PCT, probability of conserved targeting. *Conserved betweenhuman and rodent. #Seed sequences are different in mouse andhuman.

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Figure 5—Targeting ofMafb by miR-155-5p promotes IL-6 expression in islets. A: Quantitation of predicted miR-155-5p target gene expressionin islets isolated from ND-fedMir1552/2Ldlr2/2 mice andMir155+/+Ldlr2/2 mice (10–12 weeks of age) by qPCR (n = 6–8/group). B: Expression ofpredicted miR-155-5p targets in MIN6 cells after transfection with miR-155-5p mimics by qPCR (n = 6/group). Nontargeting oligonucleotideswere used as control. C: Enrichment of potential miR-155-5p targets in the Argonaute/RNA-induced silencing complexes from MIN6 cellsdetermined by GW182 immunoprecipitation (MirTrap-IP) and qPCR (n = 3/group). The results are expressed as fold enrichment of the transcriptsin miR-155-5p mimic–treated MIN6 cells compared with those treated with nontargeting control mimics. The fold enrichment of the AcGFP1control inmiR-132mimic–treated MIN6 cells was used as positive control. D:Quantitation of MafB-expressing cells in islets from ND-fedMir155+/+

Ldlr2/2 mice and Mir1552/2Ldlr2/2 mice (10–12 weeks of age) by combined MafB and insulin immunostaining (n = 10 mice/group). The nucleiwere counterstained with DAPI. Scale bars: 50 mm. E: Quantitation of Mafb mRNA expression by qPCR in a- and b-cells sorted from islets ofND-fed Mir155+/+Ldlr2/2 mice and Mir1552/2Ldlr2/2 mice (n = 3 to 4/group). F: Quantitation of Mafb, Ins, Gcg, Pcsk1, and Pcsk2 mRNAexpression in MIN6 cells treated with oligonucleotides that block the interaction between miR-155-5p and the 39-UTR ofMafb (155/Mafb TSB) ornontargeting TSBs (control TSB) by qPCR (n = 5/group). G: Quantitation of IL-6 expression at the mRNA and protein level in MIN6 cells treatedwith 155/Mafb TSB or control TSB by qPCR and ELISA, respectively (n = 5/group). H: Luciferase activity in MIN6 cells cotransfected with theempty luciferase reporter (control vector) or luciferase reporter constructs harboring the Il6 promoter region with or without site-directedmutations in the predicted Mafb binding sites (Il6 promoter vector, Il6 promoter-ΔMafb1, and Il6 promoter-ΔMafb2) and miR-155-5p LNAinhibitors or nontargeting LNA oligonucleotides (n = 4/group). The luminescence intensities of Gaussia luciferase (GLuc) were normalized tothe activity of secreted alkaline phosphatase (SEAP). Data are represented as mean 6 SEM. *P , 0.05; **P , 0.01; ***P , 0.001.

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in a-cells (Fig. 7). In obese mice, miR-155-5p–induced GLP-1 production may limit atherosclerosis, dyslipidemia, andthe progression of adiposity and improve the adaptation ofb-cells to insulin resistance.

Upregulation of miR-155-5p in response to LPS plays anessential role in inflammatory macrophage activation (37).In addition, moxLDL promotes inflammatory activation

and miR-155-5p expression in macrophages in a Toll-likereceptor 4–dependent manner (26). In line with these re-sults, we found that LPS and moxLDL increase miR-155-5pexpression in b-cells. LPS binds to LDL in the circulation,which reduces the biological activity of LPS and promotesendotoxin removal (18,38,39). However, mild oxidation ofLDL increased its endotoxin activity, presumably because

Figure 6—Effect of the interaction between miR-155-5p and Mafb on glucose homeostasis in Ldlr2/2 mice. A: Quantitation of Mafb mRNAexpression by qPCR in various tissues of ND-fed mice 21 days after the injection of 155/Mafb TSBs or control TSBs (n = 4/group). B:Quantitation of MafB-expressing cells in murine islets 21 days after the injection of 155/Mafb TSBs or control TSBs by combined MafB andinsulin immunostaining (n = 6 or 7 mice/group). The nuclei were counterstained with DAPI. Scale bars: 50 mm.C:Quantitation of gene expressionby qPCR in islets isolated from ND-fed mice 21 days after injection of 155/Mafb TSBs or control TSBs (n = 4/group). D: Quantitation of insulin-and glucagon-producing cells in islets from ND-fed mice 21 days after injection of 155/Mafb TSBs or control TSBs by immunostaining (n = 6 or7 mice/group). The nuclei were counterstained with DAPI. Scale bars: 50 mm. E: Fasting insulin, glucagon, GLP-1 plasma concentrations byLuminex multiplex analysis in ND-fed mice 21 days after treatment with 155/Mafb TSBs or control TSBs (n = 7 mice/group). FBG levels (F) andglucose tolerance (G) in ND-fed mice 21 days after injection of 155/Mafb TSBs or control TSBs (n = 7 mice/group). Data are represented asmean 6 SEM. *P , 0.05; **P , 0.01.

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of altered interactions between lipids from LDL and LPS,suggesting that LPS mediates the effect of moxLDL on miR-155-5p expression. Moreover, our finding that the deposi-tion of oxLDL in islets and endotoxemia were increased inLdlr2/2 mice indicates that LPS contributes to the upregu-lation of miR-155-5p in b-cells by hyperlipidemia. Low-doseLPS improves insulin secretion by upregulating GLP-1 pro-duction (17) and knockout of Mir155 reduced the effect ofLPS on glucose metabolism, suggesting that b-cell miR-155-5p contributes to LPS-induced insulin secretion. Accord-ingly, Mir155 knockout elevated plasma glucose levels inLdlr2/2 mice because of reduced insulin and increased glu-cagon production in islets. The effect of miR-155-5p on isletfunction is likely mediated by the upregulation of intraisletGLP-1, which improves b-cell function and inhibits gluca-gon expression (6,7).

The main mechanisms of b-cell failure in T2D develop-ment involve dedifferentiation, apoptosis, and impaired re-generation of b-cells (2,4,40). The members of the largeMaf protein transcription factor family, MafA and MafB,play critical roles in the development and function of a- andb-cells. In adult rodent islets, MafA is only expressed inb-cells and promotes insulin expression, whereas MafB isexclusively expressed in a-cells and induces Gcg transcrip-tion (41–44). In pregnant or obese mice, however, MafBexpression is upregulated in b-cells (45). Moreover, dere-pression of MafB in the absence of the b-cell–specific tran-scription factor pancreatic and duodenal homeobox 1 (Pdx1)leads to b-to-a-cell reprogramming, which may contributeto b-cell failure in T2D (40,43,46). Notably, high-fat diet

feeding induced re-expression of MafB in b-cells, suggestingthat hyperlipidemia promotes b-to-a-cell conversion (45).Accordingly, our findings indicate that hyperlipidemia-induced expression of miR-155-5p in b-cells limits the up-regulation of MafB and thereby improves b-cell function,probably because of enhanced GLP-1 production in a-cells.In mouse models of obesity and diabetes, IL-6 increases intra-islet GLP-1 expression in a-cell by upregulating Pcsk1 expres-sion (12). Notably, miR-155-5p mediates LPS-induced IL-6expression in macrophages (22,47), and high-fat diet feed-ing and inflammatory cytokines upregulate IL-6 in b-cells(48,49). Our findings indicate that increased LPS levelsduring high-fat diet feeding induces IL-6 in b-cells, whichcontributes to the autocrine stimulation of GLP-1 pro-duction by IL-6 in a-cells under normal conditions (50).Moreover, our results show that miR-155-5p increases IL-6expression in b-cells by targeting Mafb that acts as a re-pressor of IL-6 gene transcription. Taken together, ourdata indicate that hyperlipidemia-induced miR-155-5p ex-pression in b-cells reduces b-to-a-cell reprogrammingthrough suppression of MafB. Both GLP-1 and glucagonare processed from the proglucagon precursor throughthe PC1/3 and PC2, respectively (6,8). Although PC1/3 ex-pression in a-cells is low under normal conditions, lip-otoxic stress and glycemia upregulate Pcsk1 expressionand GLP-1 production, which enhances insulin secretion(12,13,51,52). Our data indicate that miR-155-5p increasesPcsk1 expression in a-cells by IL-6 secreted from b-cells andthereby shifts proglucagon processing from glucagon toGLP-1 production.

Figure 7—miR-155-5p improves islet adaptation to lipotoxic stress. Induction of miR-155-5p expression in b-cells by hyperlipidemia-associatedendotoxemia promotes a b-cell phenotype by targeting the transcription factorMafb, which results in derepression of IL-6 gene transcription andincreased production of intraislet GLP-1.

3082 MicroRNA-155-5p Improves b-Cell Adaptation Diabetes Volume 66, December 2017

In addition to reduced intraislet GLP-1 expression, wefound that Mir155 knockout decreased plasma GLP-1 levelsin Ldlr2/2 mice. Although postprandial increases of circulatingGLP-1 levels are because of its secretion by intestinal L cells,the source of fasting plasma GLP-1 is unclear. Insulin cantrigger GLP-1 secretion from L cells, and plasma GLP-1 levelsare elevated in hyperinsulinemic mice (53). Hence, reducedfasting insulin levels in Mir1552/2Ldlr2/2 mice may de-crease GLP-1 secretion from L cells and lower basal GLP-1plasma levels. Notably, GLP-1 receptor agonists and over-expression of GLP-1 reduce obesity in humans and adiposetissue inflammation in mice, respectively (54,55). Moreover,treatment with GLP-1 receptor agonists improves obesity-related dyslipidemia, probably by inhibiting hepatic VLDLproduction (54,56). Therefore, reduced GLP-1 plasma levelsmay contribute to adipose tissue inflammation, obesity pro-gression, and dyslipidemia in Mir1552/2Ldlr2/2 mice. Con-sequently, elevated LDL and VLDL levels can promote theprogression of atherosclerosis in obese Mir1552/2Ldlr2/2

mice. By contrast, Mir155 knockout in mice with normallipoprotein levels did not affect glucose tolerance, pre-sumably because of the low islet miR-155-5p expressionlevel in these mice. Accordingly, Mir155 knockout did notaffect obesity in Ldlr+/+ mice; however, female Mir155knockout mice were protected from obesity by increasedadipose tissue browning and reduced inflammatory macro-phage activation (30). Hence, the effect of miR-155-5p onobesity differs between mice with normal lipid levels andhyperlipidemia, likely because different cell types areaffected.

In conclusion, our results indicate a protective role ofoxLDL-associated LPS on b-cell function during hyperlipid-emia by inducing miR-155-5p, which prevents the upregu-lation of MafB and b-to-a-cell reprogramming. Hence,upregulation of miR-155-5p represents a self-protectivemechanism in the stress response of b-cells and improvesthe adaptation of b-cells to insulin resistance.

Acknowledgments. The authors thank Dr. Ingo Rustenbeck (University ofBraunschweig, Braunschweig, Germany) for providing the MIN6 cells.Funding. This work was supported by the German Federal Ministry of Educationand Research (01KU1213A), the German Research Foundation as part of theCollaborative Research Center 1123 (B04), and the German Centre for CardiovascularResearch (MHA VD1.2).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. M.Z. performed in vitro experiments and mouseexperiments, analyzed the results, performed statistical analysis, and wrote themanuscript. Y.W. assisted in the analysis of the data and discussed and interpretedthe results from the study. C.G. performed RNA extraction, qPCR assays, andluciferase reporter assays. K.A. and J.C.C. assisted in immunostainings and animalexperiments for high-fat diet study. M.H. assisted in the cell sorting and flowcytometric analyses. J.M. and M.L. provided GLUTag cells and assisted and advisedon GLUTag cell culture and GLP-1 secretion assay. E.K. discussed and revised themanuscript. A.S. designed the project, supervised the experiments, and wrote andrevised the manuscript. A.S. and M.Z. are the guarantors of this work and, as such,had full access to all of the data in the study and take responsibility for the integrity ofthe data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented orally at the 52ndAnnual Meeting of the European Association for the Study of Diabetes, Munich,Germany, 12–16 September 2016.

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