# Vitamin D limits inflammation-linked microRNA expression in adipocytes in vitro and in vivo: A new mechanism for the regulation of inflammation by vitamin D

Esma Karkeni, Lauriane Bonnet, Julie Marcotorchino, Franck Tourniaire, Julien Astier, Jianping Ye, Jean-François Landrier

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Esma Karkeni, Lauriane Bonnet, Julie Marcotorchino, Franck Tourniaire, Julien Astier, et al.. Vitamin D limits inflammation-linked microRNA expression in adipocytes in vitro and in vivo: A new mechanism for the regulation of inflammation by vitamin D. Epigenetics, 2018, 13 (2), pp.156-162. [hal-01998541](https://hal HAL Id: hal-01998541)

Submitted on 29 Jan 2019

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
1 Vitamin D limits inflammation linked microRNA expression in adipocytes in vitro and in

2 vivo : A new mechanism of the regulation of inflammation by vitamin D

3
4 Esma Karkeni¹, Lauriane Bonnet¹, Julie Marcotorchino¹, Franck Tourniaire¹, Julien Astier¹,

5 Jianping Ye², Jean-François Landrier¹

6
7 ¹ NORT, Aix-Marseille Université, INRA, INSERM, 13000, Marseille, France
8 ² Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge,

9 Louisiana 70808, USA.

10
11 Abbreviated title : Vitamin D blunts inflammatory miRs

12 Keyterms : vitamin D, adipose tissue, adipocyte, anti-inflammatory strategy, miR, inflammation.

13
14 *Corresponding author and person to whom reprint requests should be addressed:*

15 Jean-François Landrier, UMR 1260 INRA/1062 INSERM/Université d'Aix-Marseille, 27 Bd Jean
16 Moulin, 13385 Marseille cedex 05, France. Phone: +33 4 91 29 41 17; Fax: +33 4 91 78 21 01; E-
17 mail: jean-francois.landrier@univ-amu.fr

18
19 Disclosure statement : The authors have nothing to disclose.

20
21 Abstract

22
23 Inflammation of adipose tissue is believed to be a contributing factor to many chronic diseases
24 associated with obesity. Vitamin D (VD) is now known to limit this metabolic inflammation by
25 decreasing inflammatory marker expression and leukocyte infiltration in adipose tissue. In this
26 study, we investigated the impact of VD on microRNA (miR) expression in inflammatory
27 conditions in human and mouse adipocytes, using high throughput methodology (miRNA PCR
28 arrays). Firstly, we identified three miRs (miR-146a, miR-150 and miR-155) positively regulated
29 by TNFα in human adipocytes. Interestingly, the expression of these miRs was strongly prevented
30 by 1,25(OH)2D preincubation. These results were partly confirmed in 3T3-L1 adipocytes (for miR-
31 146a and miR-150). The ability of VD to control the expression of these miRs was confirmed in
32 diet-induced obese mice: the levels of the three miRs were increased following High Fat (HF) diet
33 in epididymal white adipose tissue and reduced in HF diet fed mice supplemented with VD. The
34 involvement of NF-κB signaling in the induction of these miRs was confirmed in vitro and in vivo
35 using aP2-p65 transgenic mice. Finally, the ability of VD to deactivate NF-κB signaling, via p65
36 and IκB phosphorylation inhibition in murine adipocyte was observed, and could constitute a
37 driving molecular mechanism.

38 This study demonstrated for the first time that VD modulates the expression of miRs in adipocytes
39 in vitro and in adipose tissue in vivo through its impact on NF-κB signaling pathway, which could
40 represent a new mechanism of regulation of inflammation by VD.

41
42 **Keywords:** Inflammation; vitamin D; adipocyte; miRs; aP2-p65 transgenic mice, NF-κB

43
44 **Introduction**

45
46 Adipose tissue was originally considered as a passive reservoir for energy storage, mechanical and
47 heat insulation, and participating to the regulation of thermogenesis ¹. Now, this tissue is also
48 known to synthesize and secrete a large variety of pro-inflammatory and anti-inflammatory
49 adipokines, cytokines and chemokines ², that act at both autocrine/paracrine and endocrine level ³.
50 Other markers such as microRNAs (miRs) have been associated with inflammatory response in
51 adipose tissue during obesity ⁴⁻⁶. miRs are small non-coding RNAs that can modulate
52 transcriptional networks, influencing most of biological processes. miRs regulate gene expression
53 by degrading complementary mRNA targets or by inhibition of traduction, depending of the
54 complementarity of sequence with the target mRNA ⁷. Several miRs are involved in adipocyte
55 differentiation ⁸, fat cell behavior ⁹ and oxidative stress ⁴. The relevance of miRs in adipose tissue
56 biology is increasingly recognized, being intrinsically linked to different pathways, including
57 obesity-related inflammation. This is notably the case for miR-155 which was reported to be
58 increased in inflamed adipocytes ¹⁰, but also in subcutaneous adipose tissue biopsies of obese
59 subjects ¹¹, and which displays a strong impact on the regulation of inflammatory tone and
60 adipocyte differentiation ¹¹

61 Similarly to the increased pro-inflammatory markers, a low vitamin D (VD) status is common and
62 well documented in obesity ¹². Whether low VD status is the consequence of obesity, or whether
63 this factor accelerates the development of obesity in humans is presently unknown. However, the
64 VD insufficiency could influence the development of obesity-related diseases, including
65 inflammation and insulin resistance ¹²,¹⁶. Indeed, it was shown that 1,25(OH)₂D significantly
66 decreased the release of cytokines and chemokines by human preadipocytes ¹⁷ and by human
67 adipocytes ¹⁸. We also demonstrated the anti-inflammatory properties of 1,25(OH)₂D in murine
68 and human adipocytes, in both basal and TNFα-stimulated conditions ¹⁹. Similarly, 1,25(OH)₂D
69 reduced the expression of cytokines in human adipose tissue biopsies submitted to IL-1β
70 stimulation in vitro 20. The molecular mechanisms have been investigated, and VDR and NF-κB
71 signaling pathways and p38 MAP kinases were shown to be involved in 3T3-L1 adipocytes 19 and
72 in human adipocytes 21. Interestingly, Zoico et al. recently demonstrated that VD, similarly to
73 1,25(OH)2D, was able to blunt the LPS-mediated pro-inflammatory effect in human adipocytes 22.

74 Using a microarray approach, we recently demonstrated that 1,25(OH)₂D was able to downregulate
75 a large set of chemokines in human and murine adipocytes, and consequently reduced macrophage
76 migration mediated by adipocyte-conditioned medium 23. Finally, we demonstrated for the first
77 time this effect in vivo, in acute and chronic models of adipose inflammation 23.

78
79 In the present study, we aimed to explore the impact of VD on adipose inflammation associated
80 miRs. To this end, we evaluated the impact of 1,25(OH)₂D on miR expression in inflammatory
81 conditions in human and mouse adipocytes, using a high-throughput methodology (miRNA PCR
82 arrays). We also confirmed in vivo the anti-inflammatory effects of VD in a chronic mice model of
83 adipose tissue inflammation and identified molecular mechanisms involved in miR regulation by
84 VD.
85
## Results

### 1,25(OH)₂D modulates microRNA expression in human and murine adipocytes

To examine, in detail, the impact of 1,25(OH)₂D on the modulation of miRs in human adipocytes submitted to inflammatory stress, human adipocytes primocultures were preincubated with or without 1,25(OH)₂D for 24 h followed by incubation with TNFα. Using a miRNA PCR array approach, we quantified 84 miRs and 6 housekeeping genes. Results analysis showed that 76 miRs were expressed in adipocytes in basal condition. Among these miRs, only 3 were positively regulated by TNFα in our conditions. As shown in Figure 1A, the inflammatory response mediated by TNFα significantly increased the expression levels of miR-146a, miR-150 and miR-155 (4.3, 1.9 and 2.1-fold, respectively, Figure 1). Interestingly, their expression decreased in adipocytes preincubated with 1,25(OH)₂D in inflammatory conditions by 2.4, 2.1 and 1.6-fold, respectively compared with TNFα condition (Figure 1).

Our results were partly confirmed in 3T3-L1 adipocytes, where the levels of miR-146a and miR-155 increased in TNFα-treated cells (by 6.7 and 8-fold, respectively) and decreased in the adipocytes pretreated with 1,25(OH)₂D and incubated with TNFα (by 3.1 and 1.3-fold, respectively, Figure 2). No effect on miR-150 was observed in this cell model.

### Vitamin D₃ supplementation limits microRNA expression in epididymal white adipose tissue

To further examine the effect of VD on miR expression in vivo, a model of chronic inflammation induced by a HF diet was used. In this preclinical model, the induction of the miR-146a, miR-150 and miR-155 levels was observed after 10 weeks of HF diet (by 2.4, 1.4 and 1.5-fold, respectively) compared with control mice (Figure 3). Interestingly, VD supplementation for 10 weeks strongly limited the expression of these three miRs in epididymal adipose tissue (2.1, 1.3 and 1.3-fold, respectively, Figure 3).
111

1,25(OH)₂D limits microRNA expression through NF-κB signaling in 3T3-L1 adipocytes.

To identify signaling pathways involved in miR-146a and miR-155 regulation by TNFα, 3T3-L1 cells were incubated with specific inhibitors of MAP kinases (JNK, p38) and NF-κB signaling, and then stimulated or not with TNFα. NF-κB signaling inhibition resulted in a significant decrease in miR-146a and miR-155 expression (by 2.7- and 1.6-fold compared with TNFα condition) (Figure 4A) whereas no effect of JNK and p38 inhibitors were observed. To confirm the role of NF-κB in miR-146a and miR-155 regulation in vivo, we used a transgenic model overexpressing NF-κB p65 subunit in adipose tissue (aP2-p65 mice <sup>24</sup>). Results showed that miR-146a and miR-155 were significantly increased in epididymal adipose tissue of aP2-p65 mice compared with controls (by 2.2- and 10-fold, respectively; Figure 4B). Altogether these data strongly support the specific role of NF-κB on the regulation of these two miRs.

Thus, we examined the impact of 1,25(OH)₂D preincubation (10 nM and 100 nM) for 24 hours followed by a 24-hour incubation with TNFα on NF-κB signaling. To this end, the phosphorylation levels of p65 and IκB were quantified by ELISA. Expectedly, the phosphorylation levels of p65 and IκB were significantly increased into 3T3-L1 adipocytes incubated with TNFα (by 4.1- and 3.6-fold, respectively), whereas incubation with 1,25(OH)₂D strongly limited the phosphorylation of p65 and IκB, suggesting that 1,25(OH)₂D reduced NF-κB activation in 3T3-L1 adipocytes (Figure 4C).

130
131 **Discussion**
132
133 Our results indicate that obesity-related inflammation increased the production of miR-146a, miR-150 and miR-155 in adipocytes. We also reported a beneficial role of VD or its active form (1,25(OH)₂D) on these miR expression in human and murine adipocytes, but also in inflamed adipose tissue of mice. Molecular mechanisms for these effects were investigated and the NF-κB signaling pathway is suggested to be involved in miR regulation and to be targeted by 1,25(OH)₂D.
135
136
137
138 If the anti-inflammatory effect of VD has largely been reported based on cytokines and chemokines expression in adipocytes as described in the introduction, its impact regarding its ability to modulate inflammatory-linked miRs has never been reported.
139
140
141

142 In a preliminary study, we identified miR-146a, miR-150 and miR-155 as adipocyte inflammatory stimulus modulated miR. These miRs have been largely suspected to be involved in inflammatory tone regulation. Indeed, miR-155, which is described as a typical multifunctional microRNA 25, has been associated with the regulation of different immune-related processes, such as haematopoiesis 26, innate immunity 27, cancer 28 and B-cell and T-cell differentiation 29. In the context of obesity onset and/or obesity comorbidities, we have recently shown that miR-155 was induced in adipocytes submitted to inflammatory stress and in subcutaneous adipose tissue biopsies of obese subjects 11. We also showed that this miR induced inflammatory response, chemokine expression, and macrophage migration in 3T3-L1 adipocytes 11. A recent study also demonstrated that the deletion of miR-155 in female mice prevented diet-induced obesity, improved insulin sensitivity and energy uncoupling machinery and abrogated HF diet induced adipocyte hypertrophy and white adipose tissue inflammation 30. Together these data confirm the importance of miR-155 in adipose tissue function, notably in the control of inflammation.
143
144
145
146
147
148
149
150
151
152
153
154
To the best of our knowledge, no specific effect of miR-146a in adipocyte or adipose tissue biology has been reported. However, in inflammatory context, it has been reported that miR-146a is involved in the control of innate immunity. Indeed, miR-146a was found to be inducible upon stimulation with LPS in a NF-κB-dependent manner in human monocytes ³¹. Such induction of miR-146a could contribute to the establishment of endotoxin tolerance in monocytes and to the regulation of TNFα production ³². In addition, exposure of human lung alveolar epithelial cells resulted in a pronounced increase of miR-146a levels, that in turn induced a decrease of IL-8 and RANTES chemokines ³³. Finally, miR-146a deficiency during diabetes led to increased expression of M1 activation markers and pro-inflammatory cytokines and suppression of M2 markers in macrophages ³⁴. Together these data strongly suggest that miR-146a displays an anti-inflammatory effect. However, its role in adipose tissue will require further investigations.

Regarding miR-150, inflammatory or anti-inflammatory properties are less clear. A recent study demonstrated that miR-150 could physiologically modulate metabolic activities and inflammatory response both in cells and animals by regulating lipid metabolism and inflammatory response. Indeed, overexpression of miR-150 in macrophages resulted in an increase in lipid accumulation, associated with an elevated expression of several pro-inflammatory cytokines ³⁵. Conversely, when miR-150 knockout mice were challenged with a HF diet, these mice presented reduced whole body weight with less fat accumulation, improved systemic glucose tolerance and insulin sensitivity ³⁵.

In contrast, another study reported that miR-150 knockout mice showed exacerbated obesity-associated tissue inflammation and systemic insulin resistance ³⁶. Such versatility has already been suggested in human cancer, where the role of miR-150 is context-dependent, i.e. this miR can have either oncogenic or tumor suppressor activity in cells that originate from different tissues ³⁷. Thus its specific role in adipose tissue and notably in the control of inflammatory tone will require further investigations.
To identify molecular mechanisms involved in miR regulation several approaches were combined.
First, we demonstrated that miR-146a and miR-155 regulations were primarily NF-κB dependent
in 3T3-L1 adipocytes, through the use of specific NF-κB inhibitor that blunted TNFα-mediated
induction of these two miRs. The involvement of this signaling pathway was also confirmed in
vivo, through the use of aP2-p65 transgenic mice which display a specificadipose tissue
overexpression of p65<sup>24</sup> and that presented a strong induction of these two miRs. Based on this
putative role of NF-κB in the induction of miR-146a and miR-155, the effect of the VD on the
phosphorylation of two intermediates of the NF-κB signaling pathway (p65 and IκB) has been
investigated. As previously demonstrated<sup>19,23</sup>, we reported that 1,25(OH)<sub>2</sub>D has a strong limiting
effect on NF-κB signaling in 3T3-L1 adipocytes through the reduction of the phosphorylation
levels of p65 and IκB. This effect could be related to the ability of 1,25(OH)<sub>2</sub>D to bind VDR and
to interact with IKK<sup>38</sup> or could result from the induction of phosphatases involved in the
dephosphorylation of p65 and IκB. Altogether these data strongly suggested that the inhibition of
miRs by VD is largely mediated by its ability to reduce NF-κB signaling.
Interestingly, we observed both in vitro in human and murine adipocytes and in vivo in mice, a
global upregulation of the miRs in pro-inflammatory conditions, suggesting that these three miRs,
independently of their inflammatory effect (pro- or anti-), actively participated to inflammatory
process in adipocytes and in adipose tissue. We also observed that VD, through the down-
regulation of these three miRs, displayed an overall blocking effect of the inflammatory pathway
in adipocytes. If the modulation of miRs by VD is reported here for the first time in the context of
obesity associated comorbidities, similar ability has been widely described in cancer context.
Indeed, in prostate cancer cells<sup>39</sup>, in bladder cancer cells<sup>40</sup> or in lung cancer<sup>41</sup>, it has been shown
that 1,25(OH)<sub>2</sub>D differentially regulated miR expression profiles.
These original observations not only reinforce the role of VD as an anti-inflammatory agent in
adipose tissue and adipocytes, but it also demonstrates the relevance of the miR regulation as new
204 anti-inflammatory mechanism in adipose tissue / adipocytes, that could represent an interesting

205 new target to limit metabolic inflammation.

206
207 **Material and Methods**

208
209 **Reagents** - Dulbecco's modified Eagle's medium (DMEM) was purchased from Life
210 Technologies (Cergy Pontoise, France), and fetal bovine serum (FBS) was obtained from PAA
211 Laboratories (Les Mureaux, France). Isobutylmethylxanthine, dexamethasone and insulin were
212 purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). TRIzol reagent was obtained
213 from Life Technologies (Saint Aubin, France). QuantiTect SYBR Green PCR Master Mix,
214 miScript Universal Primer and miScript Primer assays were purchased from Qiagen (Courtabœuf,
215 France).

216
217 **Cell culture** - Adipocyte cells were grown at 37 C in a 5% CO₂ humidified atmosphere. The human
218 preadipocytes (3 independent cultures) were obtained from Promocell and cultured according to
219 the manufacturer's instructions. The mature adipocytes (day 12) were incubated with 1,25(OH)₂D
220 (100 nM, 24 h) followed by a 24-hour incubation with TNFα (15 ng/mL). Experiments were
221 performed in triplicate, on 3 independent cultures.

222 The 3T3-L1 preadipocytes (ATCC, Manassas, VA) were seeded in 3.5 cm-diameter dishes at a
223 density of 15 × 10⁴ cells/well, and grown in DMEM supplemented with 10% FBS, at 37 C, as
224 previously reported ⁴². To induce differentiation, two-day postconfluent 3T3-L1 preadipocytes
225 (day 0) were stimulated for 72 h with 0.5 mM isobutylmethylxanthine, 0.25 µmol/L
226 dexamethasone and 1 µg/mL insulin in DMEM supplemented with 10% FBS. The cultures were
227 then treated with DMEM supplemented with 10% FBS and 1 µg/mL insulin. The mature
228 adipocytes (d 8) were incubated with 1,25(OH)₂D (100 nM, 24 h) followed by a 24-hour incubation
229 with TNFα (15 ng/mL). Experiments were performed in triplicate, on 3 independent cultures.

230
231 **3T3-L1 adipocytes incubations** - To identify signaling pathways involved in microRNA regulation, 3T3-L1 cells were treated with specific inhibitors of MAP (Mitogen Activated Protein) kinases (JNK, p38) and NF-κB signaling (JNK inhibitor II (10 µM), SB 202190 (20 µM) and BAY 117082 (10 µM), respectively) for 1 h (all obtained from Calbiochem, Merck Millipore, Darmstadt, Germany) and then stimulated or not with TNFα (15 ng/mL) for 24 h. All the treatments were performed on day 8.

237
238 **NF-κB activation** - The levels of p65 (Ser536) and IκBα (Ser32/36) phosphorylation were quantified using the ELISA Instant One kit according to the manufacturer's instructions (eBiosciences SAS, Paris, France).

241
242 **miRNA PCR arrays** - The miRNA PCR arrays (Qiagen, Courtabœuf, France) were used to quantify miRs extracted from human culture adipocytes, according to the manufacturer's instructions. Reactions were performed in a 12.5 µL volume containing 6.25 µL of 2X QuantiTect SYBR Green PCR Master Mix (Qiagen, Courtabœuf, France), 1.25 µL of 10X miScript Universal Primer (Qiagen, Courtabœuf, France). After an initial incubation step of 15 min at 95°C, amplification reaction was performed in 40 cycles comprising 3 steps (94°C, 15 s; 55°C, 30 s and 70°C, 30 s). For each condition, the expression was quantified in duplicate and the SNORD68 was used as the endogenous control in the comparative cycle threshold (C<sub>T</sub>) method.

250
251 **RNA isolation and qPCR** - Total cellular RNA was extracted using TRIzol reagent according to the manufacturer's instructions. To quantify miR-146a, miR-150 and miR-155, cDNAs were first synthesized from 1 µg of total RNA in 20 µL using 5X miScript Hiflex Buffer, 10X nucleic mix and miScript reverse transcriptase according to the manufacturer's instructions (Qiagen). Real-time quantitative RT-PCR analyses were performed using the Mx3005P Real-Time PCR System
(Stratagene, La Jolla, CA) as previously described ⁴³. Reactions were performed in a 12.5 µL volume containing 6.25 µL of 2X QuantiTect SYBR Green PCR Master Mix (Qiagen), 1.25 µL of 10X miScript Universal Primer (Qiagen), 1.25 µL of 10X miScript Primer Assay (Mm_mir146_1 miScript Primer Assay, Mm_mir-150_1 miScript Primer Assay, Mm_mir-155_1 miScript Primer Assay and Hs_SNORD68 miScript Primer Assay (Qiagen)) and 2.5 µL of RNase-free water. For each condition, the expression was quantified in duplicate, and the SNORD68 was used as the endogenous control in the comparative cycle threshold (C<sub>T</sub>) method.

**Animal experiments** – The protocol was approved through the local ethics committee. Six-week-old male C57BL/6J mice were obtained from Janvier (Le Genest Saint Isle, France). The mice were fed ad libitum (chow diet A04, Safe, Augy, France), with full access to drinking water. The animals were maintained at 22°C under a 12 h light: 12 h dark cycle with a 50% humidity level. To assess the impact of vitamin D (cholecalciferol) on miRs expression, three experimental groups of mice were fed ad libitum with a low (10% of total energy as fat; TD06416; Harlan, Indianapolis, Indiana, USA) or HF diet, providing 45% of total energy as fat (TD06415, Harlan, Indianapolis, Indiana, USA), supplemented (3000 IU/kg of body weight, i.e. around 90 UI/j/mouse, Sigma-Aldrich, Saint Quentin Fallavier, France) or not (300 IU/kg of body weight, i.e. around 9 UI/j/mouse) with cholecalciferol. The mice (10 per group) were assigned into one of the three experimental groups depending on the diet, i.e., control, HF and HF plus vitamin D (HFVD), for 10 weeks as previously described ⁴⁴. Epidydimal adipose tissue was collected and stored at -80°C. aP2-p65 mice were generated on the C57BL/6J background as described elsewhere ²⁴, ⁴⁵. All of the mice were housed in the animal facility at the Pennington Biomedical Research Center with a 12:12-h light-dark cycle and constant temperature (22–24°C). The male mice were fed with a chow diet (MF 5001, 11% calorie in fat) and the epididymal fat tissue was collected at 20 weeks. The mice were housed at four per cage with free access to water and diet.
281
282 **Statistical analysis** - The data are expressed as the means ± SEM. Significant differences between
283 the control and treated group were determined using ANOVA, followed by the PLSD Fischer post
284 hoc test using Statview software, and p<0.05 was considered statistically significant. Significant
285 differences between two groups were determined using *t* test.
286
287 Acknowledgments

288

289 This work was partially funded through grants from the Fondation Coeur et Artères (FCA).

290

291

292 References

293 1. Trayhurn P, Beattie JH. Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ. Proc Nutr Soc 2001; 60:329-39.

295 2. Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. Annu Rev Immunol 2011; 29:415-45.

297 3. Proenca AR, Sertie RA, Oliveira AC, Campaaa AB, Caminhotto RO, Chimin P, Lima FB. New concepts in white adipose tissue physiology. Braz J Med Biol Res 2014; 0:0.

299 4. Arner P, Kulyte A. MicroRNA regulatory networks in human adipose tissue and obesity. Nat Rev Endocrinol 2015; 11:276-88.

301 5. Ge Q, Brichard S, Yi X, Li Q. microRNAs as a new mechanism regulating adipose tissue inflammation in obesity and as a novel therapeutic strategy in the metabolic syndrome. J Immunol Res 2014; 2014:987285.

304 6. Hulsmans M, De Keyzer D, Holvoet P. MicroRNAs regulating oxidative stress and inflammation in relation to obesity and atherosclerosis. Faseb J 2011; 25:2515-27.

306 7. Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 2010; 11:597-610.

308 8. Ortega FJ, Moreno-Navarrete JM, Pardo G, Sabater M, Hummel M, Ferrer A, Rodriguez-Hermosa JI, Ruiz B, Ricart W, Peral B, et al. MiRNA expression profile of human subcutaneous adipose and during adipocyte differentiation. PLoS One 2010; 5:e9022.

311 9. Oger F, Gheeraert C, Mogilenko D, Benomar Y, Molendi-Coste O, Bouchaert E, Caron S, Dombrowicz D, Pattou F, Duez H, et al. Cell-specific dysregulation of microRNA expression in obese white adipose tissue. J Clin Endocrinol Metab 2014; 99:2821-33.

314 10. Ortega FJ, Moreno M, Mercader JM, Moreno-Navarrete JM, Fuentes-Batllevell N, Sabater M, Ricart W, Fernandez-Real JM. Inflammation triggers specific microRNA profiles in human adipocytes and macrophages and in their supernatants. Clin Epigenetics 2015; 7:49.

317 11. Karkeni E, Astier J, Tourniaire F, El Abed M, Romier B, Gouranton E, Lin W, Borel P, Salles J, Walrand S, et al. Obesity-associated inflammation induces microRNA-155 expression in adipocytes and adipose tissue: outcome on adipocyte function. J Clin Endocrinol Metab 2016:jc20153410.

321 12. Landrier JF, Karkeni E, Marcotorchino J, Bonnet L, Tourniaire F. Vitamin D modulates adipose tissue biology: possible consequences for obesity? Proc Nutr Soc 2015;1-9.

323 13. Garcia OP, Long KZ, Rosado JL. Impact of micronutrient deficiencies on obesity. Nutr Rev 2009; 67:559-72.

325 14. Kaidar-Person O, Person B, Szomstein S, Rosenthal RJ. Nutritional deficiencies in morbidly obese patients: a new form of malnutrition? Part A: vitamins. Obes Surg 2008; 18:870-6.

328 15. Marcotorchino J, Tourniaire F, Landrier JF. Vitamin D, adipose tissue, and obesity. Horm Mol Biol Clin Investig 2013; 15:123-8.
330 16. Mezza T, Muscogiuri G, Sorice GP, Prioletta A, Salomone E, Pontecorvi A, Giaccari A.

331 Vitamin D deficiency: a new risk factor for type 2 diabetes? Ann Nutr Metab 2012; 61:337-48.

332 17. Gao D, Trayhurn P, Bing C. 1,25-Dihydroxyvitamin D3 inhibits the cytokine-induced

333 secretion of MCP-1 and reduces monocyte recruitment by human preadipocytes. Int J Obes (Lond)

334 2012; 37:357-65.

335 18. Lorente-Cebrian S, Eriksson A, Dunlop T, Mejhert N, Dahlman I, Astrom G, Sjolin E,

336 Wahlen K, Carlberg C, Laurencikiene J, et al. Differential effects of 1alpha,25-

337 dihydroxycholecalciferol on MCP-1 and adiponectin production in human white adipocytes. Eur

338 J Nutr 2012; 51:335-42.

339 19. Marcotorchino J, Gouranton E, Romier B, Tourniaire F, Astier J, Malezet C, Amiot MJ,

340 Landrier JF. Vitamin D reduces the inflammatory response and restores glucose uptake in

341 adipocytes. Mol Nutr Food Res 2012; 56:1771-82.

342 20. Wamberg L, Cullberg KB, Rejnmark L, Richelsen B, Pedersen SB. Investigations of the

343 anti-inflammatory effects of vitamin D in adipose tissue: results from an in vitro study and a

344 randomized controlled trial. Horm Metab Res 2013; 45:456-62.

345 21. Mutt SJ, Karhu T, Lehtonen S, Lehenkari P, Carlberg C, Saarnio J, Sebert S, Hypponen E,

346 Jarvelin MR, Herzig KH. Inhibition of cytokine secretion from adipocytes by 1,25-

347 dihydroxyvitamin D(3) via the NF-kappaB pathway. Faseb J 2012; 26:4400-7.

348 22. Zoico E, Franceschetti G, Chirumbolo S, Rossi AP, Mazzali G, Rizzatti V, Budui S,

349 Zamboni M. Phenotypic shift of adipocytes by cholecalciferol and 1alpha,25

350 dihydroxycholecalciferol in relation to inflammatory status and calcium content. Endocrinology

351 2014; 155:4178-88.

352 23. Karkeni E, Marcotorchino J, Tourniaire F, Astier J, Peiretti F, Darmon P, Landrier JF.

353 Vitamin D limits chemokine expression in adipocytes and macrophage migration in vitro and in

354 male mice. Endocrinology 2015; 156:1782-93.

355 24. Tang T, Zhang J, Yin J, Staszkiewicz J, Gawronska-Kozak B, Jung DY, Ko HJ, Ong H,

356 Kim JK, Mynatt R, et al. Uncoupling of inflammation and insulin resistance by NF-kappaB in

357 transgenic mice through elevated energy expenditure. J Biol Chem 2010; 285:4637-44.

358 25. Faraoni I, Antonetti FR, Cardone J, Bonmassar E. miR-155 gene: a typical multifunctional

359 microRNA. Biochim Biophys Acta 2009; 1792:497-505.

360 26. Masaki S, Ohtsuka R, Abe Y, Muta K, Umemura T. Expression patterns of microRNAs

361 155 and 451 during normal human erythropoiesis. Biochem Biophys Res Commun 2007; 364:509-

362 14.

363 27. O'Connell RM, Taganov KD, Boldin MP, Cheng G, Baltimore D. MicroRNA-155 is

364 induced during the macrophage inflammatory response. Proc Natl Acad Sci U S A 2007;

365 104:1604-9.

366 28. Gironella M, Seux M, Xie MJ, Cano C, Tomasini R, Gommeaux J, Garcia S, Nowak J,

367 Yeung ML, Jeang KT, et al. Tumor protein 53-induced nuclear protein 1 expression is repressed

368 by miR-155, and its restoration inhibits pancreatic tumor development. Proc Natl Acad Sci U S A

369 2007; 104:16170-5.

370 29. Kluiver J, Poppema S, de Jong D, Blokzijl T, Harms G, Jacobs S, Kroesen BJ, van den

371 Berg A. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large

372 B cell lymphomas. J Pathol 2005; 207:243-9.

373 30. Gaudet AD, Fonken LK, Gushchina LV, Aubrecht TG, Maurya SK, Periasamy M, Nelson

374 RJ, Popovich PG. miR-155 Deletion in Female Mice Prevents Diet-Induced Obesity. Sci Rep

375 2016; 6:22862.

376 31. Taganov KD, Boldin MP, Chang KJ, Baltimore D. NF-kappaB-dependent induction of

377 microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc

378 Natl Acad Sci U S A 2006; 103:12481-6.
379 32. Nahid MA, Pauley KM, Satoh M, Chan EK. miR-146a is critical for endotoxin-induced tolerance: IMPLICATION IN INNATE IMMUNITY. J Biol Chem 2009; 284:34590-9.

380 33. Perry MM, Williams AE, Tsitsiou E, Larner-Svensson HM, Lindsay MA. Divergent intracellular pathways regulate interleukin-1beta-induced miR-146a and miR-146b expression and chemokine release in human alveolar epithelial cells. FEBS Lett 2009; 583:3349-55.

381 34. Bhatt K, Lanting LL, Jia Y, Yadav S, Reddy MA, Magilnick N, Boldin M, Natarajan R. Anti-Inflammatory Role of MicroRNA-146a in the Pathogenesis of Diabetic Nephropathy. J Am Soc Nephrol 2015.

382 35. Luo N, Garvey WT, Wang D-Z, Fu Y. MicroRNA-150 Regulates Lipid Metabolism and Inflammatory Response. J Metabolic Synd 2013; 2.

383 36. Ying W, Tseng A, Chang RC, Wang H, Lin YL, Kanameni S, Brehm T, Morin A, Jones B, Splawn T, et al. miR-150 regulates obesity-associated insulin resistance by controlling B cell functions. Sci Rep 2016; 6:20176.

391 37. Moles R, Bellon M, Nicot C. STAT1: A Novel Target of miR-150 and miR-223 Is Involved in the Proliferation of HTLV-I-Transformed and ATL Cells. Neoplasia 2015; 17:449-62.

392 38. Chen Y, Zhang J, Ge X, Du J, Deb DK, Li YC. Vitamin D receptor inhibits nuclear factor kappaB activation by interacting with IkappaB kinase beta protein. J Biol Chem 2013; 288:19450-8.

393 39. Kutmon M, Coort SL, de Nooijer K, Lemmens C, Evelo CT. Integrative network-based analysis of mRNA and microRNA expression in 1,25-dihydroxyvitamin D3-treated cancer cells. Genes Nutr 2015; 10:484.

400 40. Ma Y, Hu Q, Luo W, Pratt RN, Glenn ST, Liu S, Trump DL, Johnson CS. 1alpha,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells. J Steroid Biochem Mol Biol 2015; 148:166-71.

403 41. Guan H, Liu C, Chen Z, Wang L, Li C, Zhao J, Yu Y, Zhang P, Chen W, Jiang A. 1,25-Dihydroxyvitamin D3 up-regulates expression of hsa-let-7a-2 through the interaction of VDR/VDRE in human lung cancer A549 cells. Gene 2013; 522:142-6.

406 42. Landrier JF, Gouranton E, El Yazidi C, Malezet C, Balaguer P, Borel P, Amiot MJ. Adiponectin expression is induced by vitamin E via a peroxisome proliferator-activated receptor gamma-dependent mechanism. Endocrinology 2009; 150:5318-25.

409 43. Landrier JF, Malezet-Desmoulins C, Reboul E, Marie Lorec A, Joseph Amiot M, Borel P. Comparison of different vehicles to study the effect of tocopherols on gene expression in intestinal cells. Free Radic Res 2008; 42:523-30.

412 44. Marcotorchino J, Tourniaire F, Astier J, Karkeni E, Canault M, Amiot MJ, Bendahan D, Bernard M, Martin JC, Giannesini B, et al. Vitamin D protects against diet-induced obesity by enhancing fatty acid oxidation. J Nutr Biochem 2014; 25:1077-83.

415 45. Tourniaire F, Romier-Crouzet B, Lee JH, Marcotorchino J, Gouranton E, Salles J, Malezet C, Astier J, Darmon P, Blouin E, et al. Chemokine Expression in Inflamed Adipose Tissue Is Mainly Mediated by NF-kappaB. PLoS One 2013; 8:e66515.

418
419
420 **Legends for figures**

421

**Figure 1.** 1,25(OH)₂D limits microRNA expression levels in human adipocytes. Cells were preincubated with 1,25(OH)₂D (100 nM) for 24 h and incubated with TNFα (15 ng/mL) for 24 h. The miR expression levels were quantified through miRNA PCR arrays approach. SNORD68 was used as the endogenous control. The values are presented as the means ± SEM. Bars not sharing the same letters are significantly different, *p* < 0.05. Experiments were reproduced in triplicate, at least 3 independent times.

422
423
424
425
426
427

428

**Figure 2.** 1,25(OH)₂D limits microRNA expression levels in murine adipocytes. 3T3-L1 adipocytes were preincubated with 1,25(OH)₂D (100 nM) for 24 h and incubated with TNFα (15 ng/mL) for 24 h. The expression levels of miR-146a and miR-155 were quantified through qPCR in 3T3-L1 adipocytes. SNORD68 was used as the endogenous control. The values are presented as the means ± SEM. Bars not sharing the same letters are significantly different, *p* < 0.05. Experiments were reproduced in triplicate, at least 3 independent times.

429
430
431
432
433
434

435

**Figure 3.** Vitamin D limits miR-146a, miR-150 and miR-155 expression in mice epididymal adipose tissue. The expression levels of miR-146a, miR-150 and miR-155 were quantified through qPCR in epididymal adipose tissue of mice fed standard, HF or VD-supplemented HF diets for 10 weeks (n=10) and expressed relative to SNORD68 in the white adipose tissue. The data are expressed as relative expression ratios. The values are presented as the means ± SEM. Bars not sharing the same letters are significantly different, *p* < 0.05.

436
437
438
439
440
441

442

**Figure 4.** 1,25(OH)₂D limits NF-κB activation. A. 3T3-L1 cells were treated with specific inhibitors of MAP (Mitogen Activated Protein) kinases (JNK, p38) or NF-κB signaling (JNK)

443
444
inhibitor II (10 µM), SB 202190 (20 µM) and BAY 117082 (10 µM), respectively) for 1 h and
then stimulated with TNFα (15 ng/mL) for 24 h, and miR-146a and miR-155 expression were
measured by qPCR. SNORD68 was used as the endogenous control. The data are expressed as
relative expression ratios. The values are presented as means ± SEM. Bars not sharing the same
letters are significantly different, *p* < 0.05. Experiments were reproduced in triplicate, at least 3
independent times. **B.** The expression of miR-146a and miR-155 were quantified by qPCR in
epididymal adipose tissue of aP2-p65 transgenic mice. SNORD68 was used as the endogenous
control. The data are expressed as relative expression ratios. The values are presented as means ±
SEM. Bars not sharing the same letters are significantly different, *p* < 0.05. **C.** The cells were
preincubated with 1,25(OH)₂D (10 and 100 nM) for 24 h in dose-dependent manner and incubated
with TNFα (15 ng/mL) for 5 min. The phosphorylation levels of the NF-κB subunits (p65 and IκB)
were evaluated using ELISA. The data are expressed as relative expression ratios. The values are
presented as the means ± SEM. Values not sharing the same letters are significantly different, *p* <
0.05. Experiments were reproduced in triplicate, at least 3 independent times.

459
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>Figure 3</figcaption></figure>
A

<figure><img src="image_10.png" /></figure>

B

<figure><img src="image_11.png" /></figure>

C

<table><thead><tr><th></th><th>Control</th><th>1,25(OH)<sub>2</sub>D (10 nM)</th><th>1,25(OH)<sub>2</sub>D (100 nM)</th><th>TNFα</th><th>1,25(OH)<sub>2</sub>D (10 nM) +TNFα</th><th>1,25(OH)<sub>2</sub>D (100 nM) +TNFα</th></tr></thead><tbody><tr><td>p65 phosphorylation</td><td>0.61±0.01<sup>a</sup></td><td>0.87±0.014<sup>a</sup></td><td>0.73±0.01<sup>a</sup></td><td>2.53±0.013<sup>b</sup></td><td>1.14±0.008<sup>a</sup></td><td>1.27±0.025<sup>a</sup></td></tr><tr><td>IκB phosphorylation</td><td>0.61±0.007<sup>a</sup></td><td>0.44±0.005<sup>a</sup></td><td>0.62±0.013<sup>a</sup></td><td>2.2±0.021<sup>b</sup></td><td>0.99±0.01<sup>a</sup></td><td>0.85±0.01<sup>a</sup></td></tr></tbody></table>