Sarah Kadiri

Martine Auclair

Jacqueline Capeau

Bénédicte Antoine
email: benedicte.antoine@inserm.fr

Depot-Specific Response of Adipose Tissue to Diet-Induced Inflammation: The Retinoid-Related Orphan Receptor α (RORα) Involved?

Keywords: Fat depots, inflammation, RORα, western diet, insulin sensitivity Fat depots, inflammation, RORα, western diet, insulin sensitivity

est destinée au dépôt

What is already known about this subject?

The increased mass of visceral fat depot is associated with metabolic risk, while the expansion of subcutaneous fat mass appears neutral or even protective regarding the metabolic risk, both in humans and rodents. Recent studies performed to understand this depot-specific behaviour of adipose tissue revealed differences in developmental lineage of adipocyte as well as in their micro-environment.

With regard to the metabolic risks, most studies have focused primarily on the visceral depot, while only few publications dealt with the elucidation of how increased amounts of subcutaneous depot could be neutral or even beneficial regarding the metabolic effects. Understanding the underlying mechanisms for these differences is of importance in the course of preventing the ongoing obesity and diabetes epidemic. The orphan nuclear factor RORα is involved in several processes that are dysregulated in expanding adipose tissue, such as inflammation, adipogenesis, circadian rhythms and lipid homeostasis. Its role of in the inflammation of adipose tissue thus remains controversial.

What does this study add?

RORα expression impacts the basal inflammation status of adipose tissue: when RORα is inefficient, the level of basal inflammation decreases. Such data confirm a "pro-inflammatory" role of RORα in physiological conditions, very likely because it was shown as a positive actor in the basal transcription of several cytokines.

All along the diet-induced fat expansion in wild-type mice, the inflammation of visceral fat increases (as generally observed in mice and human). Our data show here that, whenever the subcutaneous fat also hypertrophies, its inflammation level decreases. This differential behavior of the fat depots is associated with inverse effect on Insulin sensitivity, thus impacts on the functionality of the fat depot.

We then show that RORα is regulated in obesity in a depot-specific manner and has depot specific function. In response to a western diet (WD), RORα and its target-gene IBα, increases in the stroma vascular fraction (SVF) in the non-adipocyte fraction of subcutaneous fat and not of visceral fat. These findings suggest an anti-inflammatory role for RORα in response to WD, which occurs at the level of stroma vascular fraction of subcutaneous fat, thus possibly participating to the "healthy" expansion of subcutaneous fat.

Introduction

Obesity is characterised by hyperplasic and hypertrophic adipose tissue expansion, which is associated with a chronic low-grade inflammation. An emerging body of research suggests that inflammatory processes that occur within the intra-abdominal adipose tissue depot of humans and rodents are causatively related to many of the symptoms of the metabolic syndrome [START_REF] Nishimura | Adipose tissue inflammation in obesity and metabolic syndrome[END_REF]. This remodelling of adipose tissue inflammatory status is due to infiltration by several immune cell types including macrophages (2), T lymphocytes, natural killer cells and neutrophils [START_REF] Feuerer | Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters[END_REF]. The inflammatory adipose tissue releases pro-inflammatory cytokines, such as IL-6, TNFα, IL1β, which impair insulin sensitivity (4).

However, these modifications particularly concern the visceral fat depot, whose increased mass is associated with metabolic risk, while increased subcutaneous fat mass appears neutral or even protective regarding the metabolic risk, both in humans and rodents [START_REF] Porter | Abdominal subcutaneous adipose tissue: a protective fat depot?[END_REF][START_REF] Tran | Beneficial effects of subcutaneous fat transplantation on metabolism[END_REF]. Recent studies performed to understand this depot-specific behaviour of adipose tissue revealed differences in developmental lineage of adipocyte precursors [START_REF] Wang | Tracking adipogenesis during white adipose tissue development, expansion and regeneration[END_REF][START_REF] Chau | Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source[END_REF] as well as in their microenvironment [START_REF] Jeffery | The adipose tissue microenvironment regulates depot-specific adipogenesis in obesity[END_REF]. This implies that the immune cells, besides adipocytes and adipocyte precursors, could also differ according to the fat depot location. With regard to the metabolic risks, most studies have focused primarily on the visceral depot, while only few publications dealt with the elucidation of how increased amounts of subcutaneous depot could be neutral or even beneficial regarding the metabolic effects.

Members of the nuclear hormone receptor family are abundantly expressed in tissues with major metabolic activity, such as adipose tissue, liver and skeletal muscle. In vivo studies have revealed that some of the orphan nuclear receptors regulate the pathophysiology of obesity and insulin resistance (for a review see ref 10). The orphan nuclear factor RORα, for example, is involved in several processes that are deregulated in expanding adipose tissue, such as inflammation, adipogenesis, circadian rhythms and lipid homeostasis (see ref. [START_REF] Fitzsimmons | Retinoid-related orphan receptor alpha and the regulation of lipid homeostasis[END_REF] for a review). A mouse RORα loss-of-function mutantthe staggerer (sg/sg) mice -is protected against high fat diet-induced obesity [START_REF] Lau | The orphan nuclear receptor, RORalpha, regulates gene expression that controls lipid metabolism: staggerer (SG/SG) mice are resistant to diet-induced obesity[END_REF] and related systemic insulin resistance [START_REF] Kang | Transcriptional profiling reveals a role for RORalpha in regulating gene expression in obesity-associated inflammation and hepatic steatosis[END_REF]. The sg/sg mice have a global RORα defect that results in decreased and dysfunctional expression of the two mouse RORα isoforms, RORα 1 and RORα 4. The latter form is predominantly expressed in adipose tissue. These mice are lean and their pre-adipocytes better differentiate to adipocytes in vitro as compared to their wild-type (WT) mice counterparts [START_REF] Duez | Inhibition of adipocyte differentiation by RORalpha[END_REF][START_REF] Ohoka | The orphan nuclear receptor RORalpha restrains adipocyte differentiation through a reduction of C/EBPbeta activity and perilipin gene expression[END_REF]. We recently demonstrated that glyceroneogenesis, a pathway that controls fatty acids homeostasis, is a new target of RORα, thus limiting the storage of lipids into the adipocytes from sg/sg mice [START_REF] Kadiri | The nuclear ROR alpha regulates adipose tissue glyceroneogenesis in addition to hepatic gluconeogenesis[END_REF].

When fed a high-fat (HF) diet, sg/sg mice express a lower level of immune and inflammatory genes in their epididymal fat than WT mice on the same diet (13); a pro-inflammatory role for RORα was presumed in high fat diet-induced inflammation of visceral adipose tissue by these authors. To the contrary, others studies suggested that RORα may have a protective role during acute inflammation of muscle cells and macrophages [START_REF] Delerive | The orphan nuclear receptor ROR alpha is a negative regulator of the inflammatory response[END_REF][START_REF] Kopmels | Evidence for a hyperexcitability state of staggerer mutant mice macrophages[END_REF][START_REF] Kopmels | Interleukin-1 hyperproduction by in vitro activated peripheral macrophages from cerebellar mutant mice[END_REF][START_REF] Garcia | Disruption of the NFΚB/NLRP3 connection by melatonin requires retinoid receptor-related orphan recepto α and blocks the septic response in mice[END_REF]. The role of RORα in inflammation thus remains controversial.

Finally, sg/sg mice exhibited increased levels of insulin-stimulated phosphorylated Akt and of glucose uptake in muscle [START_REF] Lau | Homozygous staggerer (sg/sg) mice display improved insulin sensitivity and enhanced glucose uptake in skeletal muscle[END_REF], but the insulin sensitivity (IS) of their adipose tissues has not yet been investigated.

The purpose of the present work was to decipher the role of RORα in adipose tissue inflammation and IS under physiological condition, and in a context of excessive caloric intake. Here, we compared sg/sg mice with their WT littermates, first when fed a standard diet (SD), and after highcarbohydrate and high-fat western diet (WD). Only such an approach allowed a comparison of the diet related-effect on each genotype. We systematically analyzed both visceral (epididymal) and subcutaneous (inguinal) adipose tissues (EAT and IAT) because of their different impact on metabolism.

Material and methods

Animals and tissues. Dr J-L Danan gave us the heterozygous sg/+ mice, which came from Dr J Mariani's laboratory. Homozygous sg/sg mice were obtained by crossing heterozygous mice. All experimental procedures on mice were conducted in accordance with the guidelines of the Charles Darwin Ethics Committee (Ce5/2010/034). Three-month-old male mice were fed ad libitum either with a high-fat, high-carbohydrate 'western' diet (WD) (40% kcal fat and 40% kcal carbohydrates, TD96132, Harlan) or a standard diet (SD) for 12 weeks. After 4h fasting, mice were euthanized by cervical dislocation and adipose tissues removed.

Immunohistochemistry. Adipose tissue samples were fixed in formol and embedded in paraffin.

Sections were then immunostained for CD68 (MCA1957, AbD Serotec). For each mouse, 300-400 adipocytes/macrophages were counted in each fat depot.

RNA extraction, cDNA synthesis and quantitative PCR. Adipose tissue mRNA was extracted by using the RNeasy Lipid Tissue MiniKit (Qiagen), then reverse-transcribed by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR of the genes of interest was performed using a Light Cycler 480 Real-Time PCR System (Roche) and the specific primers listed as followed: F4/80: 5'-GCAAGGAGGACAGAGTTTATCGTG-3',5'-CTTTGGCTATGGGCTTCCAGTC-3'; CD68:5'-CCAATTCAGGGTGGAAGAAA-3',5'-CTCGGGCTCTGATGTAGGTC-3'; IL-6: 5'-AGAAGGAGTGGCTAAGGACCAA -3', 5'-ACGCACTAGGTTTGCCGAGTA-3' TNFα: 5'-TCTTTCTCGAACCCCGAGTGA-3', 5'-CCTCTGATGGCACCACCAG-3' IL-1β: 5'-ACACTCCTTAGTCCTCGGCCA-3', 5'-CCATCAGAGGCAAGGAGGAA-3' IL-10: 5'-ACGGCGCTGTCATCGATT-3', 5'-GGCATTCTTCACCTGCTCCA-3' PAI-1: 5'-CTATGCTGACATGACCACAGC-3', 5'-CTGAGCCATCATGGGCACAGAG-3' RORα: 5'-CGTTTGGCAAACTCCACC-3',5'-GATTGAAAGCTGACTCGTTCC -3' Expression of each gene was quantified by cycle threshold values and was normalized to HPRT (hypoxanthine phosphoribosyltransferase) mRNA.

Functional analysis in explants. Adipose tissue was minced into very small pieces (5-10 mg) and incubated overnight in DMEM containing 5mM glucose and 0.5% bovine serum albumin (BSA), then incubated (or not) with 100nM insulin in DMEM containing 17mM glucose for 10 min before western blot analysis or 2-deoxy-D-glucose uptake assay [START_REF] Lau | Homozygous staggerer (sg/sg) mice display improved insulin sensitivity and enhanced glucose uptake in skeletal muscle[END_REF] to evaluate insulin sensitivity. The 48h-culture media were used to measure cytokine secretion (MCP-1/CCL-2, TNF-α, IL-10) by ELISA (Prepotech).

Protein extraction and western blot analysis. Adipose tissue was extracted using Tissue Lyzer (Qiagen). Extracts were centrifuged first at 10,000 rpm and 4°C for 8 min to remove the fat coat, and then at 14,000 rpm and 4°C for 10 min to remove insoluble material. Supernatants were subjected to SDS-PAGE and western blotted with antibodies against pAkT (sc-7985), AkT (sc-8312), RORα (sc-6062), IκBα (sc-1643, Santa-Cruz) or IκBβ (#9248 Cell Signaling).

Adipose tissue sub-fractionation: Fat pads were excised and adipocytes were isolated from the SVF after collagenase digestion (Liberase TM, Roche) as previously described [START_REF] Dusaulcy | Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid[END_REF].

Statistical analysis.

The data are presented as means +/-SD. Statistical analysis was performed by one-way or two-way ANOVA or by unpaired Student T test; p< 0,05 was considered significant.

Results

Metabolic status and adipose tissue expansion according to the genotype and in response to WD

When fed the SD, the total mass of white adipose tissue in the sg/sg mice was 50% less than in the WT controls. Parallely, their mean adipocyte surface area in IAT and EAT sections was four-fold smaller than that in WT controls (Fig S1), as previously observed [START_REF] Lau | ROR α deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue[END_REF].

When fed the WD, WT and sg/sg mice gained 5g and 2g respectively. This was associated with a relative increase in the white adipose tissue mass of about four-fold in WT mice and two-fold in sg/sg mice, with the same impact on each fat depot. The mean adipocyte area in EAT and IAT from WT and sg/sg mice increased two-and four-fold respectively (Fig S1). WT mice became glucose intolerant and insulin-resistant whereas sg/sg mice were protected against the insulin-resistance induced by our "high-carbohydrate and high-fat" WD (not shown), as also described in response to a 60% high fat diet [START_REF] Kang | Transcriptional profiling reveals a role for RORalpha in regulating gene expression in obesity-associated inflammation and hepatic steatosis[END_REF].

The inflammatory profiles of epididymal and inguinal fat depots differ according to the genotype and in response to WD

To examine the inflammatory status of IAT and EAT in WT and sg/sg mice fed the SD or WD, we measured the mRNA levels of two macrophages markers (F4/80 and CD68), four proinflammatory cytokines (IL6, TNFα, IL1β, PAI-1) and one anti-inflammatory cytokine (IL10) (Fig 1). We evaluated the number of CD68+ cells by immunohistochemistry. We also measured the secretion of the chemokine MCP-1/CCL2 and of two cytokines (TNFα, IL10) by ELISA (Fig 2).

-Compared basal inflammatory status of adipose fat depots in WT and sg/sg mice fed the SD.

When compared to their corresponding WT depots, EAT and IAT of sg/sg mice fed the SD expressed lower amounts of most of the tested markers, when regarding the mRNA level (Fig 1 ) or the chemokine/cytokine secretion (Fig 2). When EAT and IAT were compared in WT mice fed the SD, we observed that the inflammatory status of IAT was globally higher than that of EAT (IAT had higher number of CD68+ macrophages and secretion of CCL2 and expressed higher mRNA levels of TNFα, IL1β and IL10 than the EAT) (Fig S2A ). By contrast, in sg/sg mice fed the SD, the inflammatory status of IAT was not higher than that of EAT (lower mRNA expression of IL1β and PAI-1) (Fig S2A ). Taken together, our data show for the first time that the absence of RORα has an impact on the basal inflammation status of fat depots, and particularly in IAT. These data show that, in WT mice fed the WD, the inflammatory markers increased in EAT while they decreased or plateaued in IAT, whereas the opposite occured in sg/sg mice fed the WD.

Insulin sensitivity of epididymal and inguinal fat depots differs according to genotype and to diet.

To investigate whether these differing inflammatory profiles of adipose tissues in WT and sg/sg mice correlated with differences in insulin sensitivity, we evaluated the ability of insulin to activate the insulin receptor signaling enzyme Akt. We measured the level of activated P-Akt in the absence or the presence of insulin, relative to the total Akt levels, in the two tissue types of both mouse strains fed either the SD or the WD. In sg/sg mice, the insulin sensitivity of both tissues was better than in WT mice and, in both strains fed the SD, we observed a higher level of P-Akt in EAT than in IAT (as illustrated by Fig. 3A), indicating that EAT was more sensitive to insulin than the IAT.

We further assessed these observations by analyzing 2-DOG incorporation in response to insulin as shown in Fig 3B .   When WT mice were fed the WD, the level of insulin-stimulated P-Akt in EAT was lower than in the WT mice on the SD, indicating a diet-increased insulin resistance in EAT (Fig 3C ), whereas in IAT, the response to insulin was improved by the WD regimen (Fig 3D). In the sg/sg mice on the WD, by contrast, insulin sensitivity was maintained in the EAT but decreased in the IAT (see histograms of Fig 3C andD). Thus, there is a direct correlation between insulin resistance and inflammation in the two strains in response to WD. However, it was EAT that became insulin resistant in the WT mice, whereas it was the IAT that exhibited decreased insulin sensitivity in sg/sg mice in response to the WD.

RORα α α

α protein increased in IAT of WT mice fed the WD as well as Iκ κ κ κB protein.

WD resulted in reduced inflammation in IAT of WT mice, this response being lost in IAT of sg/sg mice. We therefore suspected a role for RORα in these differences. To this end, we measured RORα expression in EAT and IAT in WT mice as well as in sg/sg mice maintained on either regimen (Fig. 4A). In SD-fed sg/sg mice, RORα mRNA levels were lower than in WT mice, as expected [START_REF] Lau | The orphan nuclear receptor, RORalpha, regulates gene expression that controls lipid metabolism: staggerer (SG/SG) mice are resistant to diet-induced obesity[END_REF], and they did not change in response to the WD. In SD-fed WT mice, RORα expression was similar in the two fat depots. However, because RORα is a member of the clock "machinery", we checked the circadian variation of the RORα protein. We observed a higher amount of the protein in EAT than in IAT of WT mice fed the SD, with a peak during the dark period ( To reveal where this WD-increased amount of RORα protein took place, we separated adipocytes from the SVF of both fat depots from WT mice fed the SD or the WD. Our data show that both the adipocytes and the SVF expressed RORα protein (Fig 5A). However, in response to WD, there was only an increase of RORα protein in the SVF of IAT, and not of EAT, whereas its amount decreased in adipocytes from both fat depots. Thus, our data show that there is a profound difference in the response of EAT and IAT to the WD, in terms of induction of RORα expression that only occurs in the SVF of IAT. This suggests a WD-induced response of the non-adipocyte fraction, and not of the adipocytes, that is depot-specific of IAT.

Finally, we confirmed that this WD-induced amount of RORα in the SVF of IAT was associated with a depot-specific increase of IκBα (Fig 5B). This confirms a depot-specific role for RORα that could contribute to the protection of diet-induced inflammation by providing an increased amount of an NF-κB inhibitor in the non-adipocyte fraction of IAT.

Discussion

Our investigation offers insights into a new mechanism underlying for the differences in dietinduced expansion of visceral and subcutaneous fat depots and their metabolic consequences. While expansion of the EAT results in its increased inflammation and insulin resistance, as generally observed, our data highlight that expansion of the IAT remarkably differs because it is not only metabolically neutral but associated with decreased inflammation and increased insulin sensitivity.

Elucidating the basis of such an "healthy" expansion of subcutaneous fat is of importance with regards to the prevention of diet-induced metabolic syndrome. In this study, we discover a depotspecific induction of RORα in the IAT of WT mice fed the WD that is associated with an increase of IκB protein, an inhibitor of NFκB-mediated transcriptional activation. Our data suggest that RORα could act as a negative regulator of the diet-induced inflammatory response in the subcutaneous fat depot, where it is expressed by non-adipocyte cells, perhaps contributing to the relative protection of IAT in diet-induced metabolic syndrome.

To mimic the cafeteria-type diet of humans, we choose to feed WT and sg/sg mice a diet that contains equal amounts of carbohydrates and lipids (40%) that we called "western" diet, rather than the often-used HF diet (60% of lipids and very few carbohydrates) that is not representative of any common "human" diet. Twelve weeks of this WD had the expected metabolic consequences for WT mice : increased insulin resistance at the systemic level and fat weight gain. Accordingly, in EAT, the inflammatory response was enhanced and the insulin sensitivity decreased compared to animals on the SD. Surprisingly, we observed that the IAT of WT mice responded inversely to WD than their EAT : the inflammatory status declined and insulin sensitivity increased.

Several reports already mentioned that IAT has a lower inflammatory status than EAT in mice having been fed a HF diet [START_REF] Kosteli | Weight loss and lipolysis promote a dynamic immune response in murine adipose tissue[END_REF][START_REF] Altintas | Mast cells, macrophages, and crown-like structures distinguish subcutaneous from visceral fat in mice[END_REF][START_REF] Strissel | Adipocyte death, adipose tissue remodeling, and obesity complications[END_REF] ; however, a reduced level of inflammation as compared to SD-fed mice was unexpected. For instance, when fat expansion was analyzed all along the HF diet [START_REF] Strissel | Adipocyte death, adipose tissue remodeling, and obesity complications[END_REF], EAT was shown to initiate adipocyte death sooner than IAT; such adipocyte death in EAT being timely linked to the recruitment and /or phenotypic switch of macrophages, AT inflammation and wholebody insulin resistance. However, none of these events were detectable in IAT before 12 weeks of HF diet [START_REF] Strissel | Adipocyte death, adipose tissue remodeling, and obesity complications[END_REF]. Our data confirm similar behavior -a distinct impact of WD diet on EAT and IAT inflammation whereas similarly hypertrophied, but they also bring the new notion that IAT can protect itself against the WD-induced inflammation. This new observation was perhaps allowed by the use, in our study, of a more balanced diet (WD instead of a HF diet).

This WD-induced decrease of inflammation in IAT was detectable because of the higher level of basal inflammatory status of IAT than EAT in WT mice fed the SD (as illustrated in Fig S2).

Accordingly, several investigations performed in mice and rats on chow diet have noticed a higher basal inflammatory status in IAT than in EAT [START_REF] Villena | Adipose tissues display differential phagocytic and microbicidal activities depending on their localization[END_REF][START_REF] Du | Depot-dependent effects of adipose tissue explants on co-cultured hepatocytes[END_REF][START_REF] Tsai | Decreased PPAR gamma expression compromises perigonadal specific fat deposition and insulin sensitivity[END_REF] as well as a higher content of cells not related to the adipocyte lineage in IAT [START_REF] Macotela | Intrinsic differences in adipocyte precursor cells from different white fat depots[END_REF]. For instance, mast cells, that can act as negative as well as positive regulators of immunity [START_REF] Galli | Immunomodulatory mast cells: negative, as well as positive, regulators of immunity[END_REF], are more prevalent in subcutaneous than visceral fat from lean mice [START_REF] Altintas | Mast cells, macrophages, and crown-like structures distinguish subcutaneous from visceral fat in mice[END_REF] as well as from humans [START_REF] Weidner | Evidence for morphologic diversity of human mast cells. An ultrastructural study of mast cells from multiple body sites[END_REF]. Our observation of higher amounts of RORα and IκBα and β protein in EAT than in IAT of SD-fed WT mice could alternatively participate to the lower basal inflammation status of EAT than IAT.

The next addressed question was how IAT actively protects itself against diet-induced inflammation and IR. The observation of an opposite phenomenon in the IAT of sg/sg mice fed the WD suggested us a role for RORα as a possible actor of an anti-inflammatory process in the IAT of WT mice. Accordingly, we found a depot-specific increase of RORα in the IAT of WT mice fed the WD, associated with an increase of a member of the IκB family, IκBβ, an inhibitor of NFκB-mediated transcriptional activation Thus, one can hypothesize that increased RORα expression may exert a protective role in IAT against diet-induced inflammation, as already shown in myocytes and macrophages during acute inflammation [START_REF] Delerive | The orphan nuclear receptor ROR alpha is a negative regulator of the inflammatory response[END_REF][START_REF] Kopmels | Evidence for a hyperexcitability state of staggerer mutant mice macrophages[END_REF][START_REF] Kopmels | Interleukin-1 hyperproduction by in vitro activated peripheral macrophages from cerebellar mutant mice[END_REF][START_REF] Garcia | Disruption of the NFΚB/NLRP3 connection by melatonin requires retinoid receptor-related orphan recepto α and blocks the septic response in mice[END_REF].

We further described that this increased expression of RORα did not happen into the adipocytes, but was located in the SVF, probably in immune cells, in parallel with the increase of its target-gene IκBα. If this is the case, the aim of further work will be to identify which cells are indeed dysfunctional in the IAT of sg/sg vs WT mice fed the WD. Mast cells could be such potential candidate because the production of TNFα and IL-6 was increased dramatically in RORα-/-mast cells after LPS stimulation [START_REF] Dzhagalov | Lymphocyte development and function in the absence of retinoid acid receptor-related orphan receptor α[END_REF]. In that field, our data are in accordance with investigations showing that RORα can negatively regulate cytokine production in response to an aggression by inhibiting the NF-κB pathway [START_REF] Delerive | The orphan nuclear receptor ROR alpha is a negative regulator of the inflammatory response[END_REF]. Very recently, in vitro overexpression of RORα was shown to promote macrophage M2 polarization [START_REF] Xiao | Retinoid acid receptor-related orphan receptor alpha (RORα) regulates macrophage M2 polarization via activation of AMPKα[END_REF]. Finally, our data also suggested that IκBβ, another member of the IκB family, was a new target gene of RORα.

However, we also confirmed the dual role of RORα in inflammatory processes. Indeed, RORα is expressed in resting macrophages and T lymphocytes [START_REF] Dzhagalov | Lymphocyte development and function in the absence of retinoid acid receptor-related orphan receptor α[END_REF] and involved in the basal transcription of several cytokines such as IL6 [START_REF] Journiac | The nuclear receptor ROR(alpha) exerts a bi-directional regulation of IL-6 in resting and reactive astrocytes[END_REF], IL17 [START_REF] Yang | T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ[END_REF], PAI-1 [START_REF] Wang | The orphan nuclear receptor Rev-erba regulates circadian expression of plasminogen activator inhibitor type 1[END_REF] and one chemokine, CCL2 [START_REF] Sato | A circadian clock gene, Rev-erbα, regulates the inflammatory function of macrophages through the negative regulation of ccl2 expression[END_REF] in different kind of immune cells. Accordingly, our analysis of several cytokines expression or amount show for the first time that the basal inflammatory status of each fat depot is greatly lowered in sg/sg mice as compared to WT mice on chow diet, and not only after HF feeding as previously shown [START_REF] Kang | Transcriptional profiling reveals a role for RORalpha in regulating gene expression in obesity-associated inflammation and hepatic steatosis[END_REF]. This decreased basal expression of cytokines is particularly pronounced in IAT and could be related to several missing or dysfunctional immune cells in sg/sg fat depot [START_REF] Dzhagalov | Lymphocyte development and function in the absence of retinoid acid receptor-related orphan receptor α[END_REF][START_REF] Yang | T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ[END_REF][START_REF] Halim | Retinoic-acidreceptor-related orphan nuclear receptorα is required for natural helper cell development and allergic inflammation[END_REF]. In that field, the high insulin sensitivity of adipocytes in sg/sg mice could be also related to their low inflammation environment, in addition to their small size.

In conclusion, the present study suggests a role for RORα in participating to the healthy expansion of inguinal subcutaneous fat in mice and opens a new perspective in the mechanisms underlying the different expansion way of fat depots according to their localization.

-

  Different inflammatory profiles in fat depots according to WD in the two mouse genotypes As expected, WD increased the inflammation of EAT in WT mice as compared to SD : higher amount of F4/80, IL6, IL10 and PAI-1 mRNAs (Fig 1A') and about a two-fold increase of the number of CD68+ macrophages (Fig 2A) and of CCL2 secretion (Fig 2B) together with increased secretion of TNFα and IL10 (not shown). However, in IAT, the expression of the majority of inflammatory markers was decreased by WD (Fig 1B'), as well as the number of CD68+ macrophages (Fig 2A) and the amount of secreted CCL2 (Fig2B). By contrast, in sg/sg mice, WD increased the inflammatory status of IAT (Fig 1B'), but not of EAT (Fig 1A'). These data were confirmed by the observation of an increased secretion of CCL2 (Fig2B) and of TNFα and IL10 (Fig 2C) by IAT from sg/sg mice fed the WD vs fed the SD.

  Fig 4B). When WT mice were fed the WD, RORα expression did not change in the EAT but, by contrast, markedly increased in the IAT when compared to the same tissue in SD-fed WT mice (Fig 4A). This was confirmed by checking the amount of RORα protein that increased in IAT of WT mice in response to WD (Fig 4C). IκBα protein, an inhibitor of NF-κB-mediated transcriptional activation, being shown to be a target gene of RORα (17), we first checked if this WD-induced amount of RORα in the SVF of IAT was associated with a depot-specific increase of the IκB family. Our data indeed show that WD increased IκBβ protein in the IAT of WT mice (in parallel with the increase of RORα) and not, in EAT (Fig 4D).

Figure 1 :

 1 Figure 1: The inflammatory profiles of epididymal and inguinal fat depots differ according to the

Figure 2 :

 2 Figure 2: Proportion of CD68-positive macrophages and cytokine secretion according to the

Figure 3 :

 3 Figure 3: The influence of diet on the insulin sensitivities of EAT and IAT in WT and sg/sg mice.

Figure 4 :

 4 Figure 4: RORα α α α increased in IAT of WT mice fed the WD in a depot-specific manner

Figure 5 :

 5 Figure 5: RORα α α α and Iκ κ κ κBα α α α increased in the stroma vascular fraction (SVF) of WT mice fed the WD.

  Fig 1 R1 Kadiri

Acknowledgments

We greatly thank Tatiana Ledent and Laetitia Dinard for their help in sg/sg mice housing facilities and Marie-Pauline Garcia for mice genotyping. We also thank the National Institute of Health for the free availability of the Image J software.

SK was the recipient of PhD grants from the Ministère de l'Education Nationale et de la Recherche and from CODDIM. BA is a CNRS researcher.

Funding: SK was the recipient of PhD grants from the Ministère de l'Education Nationale et de la Recherche and from CODDIM. BA is a CNRS researcher.

Depot-specific response of adipose tissue to diet-induced inflammation: the retinoid-related orphan receptor α involved?

Sarah Kadiri 1,2 , Martine Auclair 1,2 , Jacqueline Capeau 1,2 and Bénédicte Antoine

Legends of supplementary figures


Stéphane Ganassali

Justyna Matysiewicz

Assessing visual survey protocols to capture brand-related emotional insights

Keywords: Emotions, Brand attachment, Visual methods, Mixed methods, Collage, Quality of responses

Purpose -This paper aims to present the assessment of different self-reported approaches that can be used to identify and measure consumers' emotional responses towards brands. The goal is to determine whether visual and spontaneous protocols are able to generate deeper insights than only closed groups of scales, and to consider pictorial tools as innovative and challenging measurement techniques for brand value assessment.

Design/methodology/approach -Three versions of the same consumer online survey dedicated to identifying some brand-related consumer emotional insights were created to be compared in terms of quality of responses, interviewees' evaluation and richness of insights.

Findings -Visual protocols provide more specific emotional responses and are considered as useful for "capturing deep contextual meanings of consumer experience". They clearly provide deeper insights and better emotional granularity. It can be generally concluded that different emotions' self-report measurements are adapted to some diverse research questions or situations.

Research limitations/implications -This paper is based on research with a limited number of participants. It focuses on the use and consumer emotional insights delivered by three tested protocols rather than detailed analysis of the specific profiles of consumers.

Practical implications -The authors provide some recommendations of different research techniques, which can be used to identify and measure consumers' emotional reactions towards brands.

Social implications -Their paper encourages a critical reflection about research methods that are deployed for marketing and consumer behaviour purposes. Their work promotes a hybrid and not dogmatic approach, centred around the perception and motivation of the respondents more than on the expectations of the researcher only.

Originality/value -Research outcomes among different self-reported protocols using internet technologies are compared. Quality of responses and richness of insights are measured in a quite innovative and comprehensive way. The paper also gives detailed recommendations to researchers interested in consumers' emotional reactions towards brands measurements.

Introduction and review of literature Within a general context in which experiential aspects of consumption are considered as crucial [START_REF] Holbrook | The experiential aspects of consumption: consumer fantasies, feelings and fun[END_REF], various strong feelings can be observed in a consumer-brand relationship (CBR). Consumers can have a passion for the brand or hate it. Love is one of those strong feelings that marketers try to create between their brands and consumers [START_REF] Albert | When consumers love their brands: exploring the concept and its dimensions[END_REF]. Once customers turn into brand lovers, it is difficult for competitors to attract those loyal followers. They may also become the ambassadors of the brand and try sometimes to "evangelize" around them. At that point, emotional and experiential consumer insights become more important and crucial for marketing decisionmakers [START_REF] Ng | Insights into measuring emotional response in sensory and consumer research[END_REF], especially in some areas or sectors in which those aspects are very central, such as brand strategy, but also tourism, culture, entertainment or retailing. However, for certain consumers, brand-related emotions are difficult to capture [START_REF] Derbaix | La mesure des réactions affectives en marketing: évaluation des principaux outils[END_REF][START_REF] Thomson | The ties that bind: measuring the strength of consumers' emotional attachments to brands[END_REF]. Within the typology of affective reactions [START_REF] Derbaix | Affective reactions to consumption situations: a pilot investigation[END_REF], we consider brand-related "emotions" and "feelings", as the stimulus is specific (the brand) and the somatic intensity is moderate to strong. For emotions, the duration is brief, while for feelings it is moderate to long.

The aim of this study is therefore to evaluate some different instruments to capture brand-related consumer emotional insights designed under different approaches. There is a growing trend of promoting the joint adoption of both verbal and non-verbal approaches for better assessing emotional responses and providing additional or deeper insights regarding associations made by consumers about brands on large scale. Within a global context of a rising "image culture" [START_REF] Jansson | The mediatization of consumption: towards an analytical framework of image culture[END_REF], we tried to consider more illustrated and spontaneous instruments using pictures or collages and test them within an international environment.

Theoretical background Methods of assessing emotions

There are commonly three components of emotions: cognitive, physiological and behavioural [START_REF] Gil | Comment étudier les émotions en laboratoire?[END_REF]. Our research is focused on an understanding of the cognitive dimensions. Methods of assessing emotions can be generally divided according to the components of the emotional response as follows:

affective self-reports, including verbal self-report, visual self-report [START_REF] Bradley | Measuring emotion: the self-assessment manikin and the semantic differential[END_REF] and moment-to-moment rating [START_REF] Poels | How to capture the heart? Reviewing 20 years of emotion measurement in advertising[END_REF]; physiological measurements (e.g. skin conductance, pupillary responses and pulse rate); and behavioural changes (e.g. facial action coding system, e.g. [START_REF] Ekman | Facial signs of emotional experience[END_REF][START_REF] Kaiser | Facial expressions as indicators of appraisal processes[END_REF][START_REF] Förster | Do emotions pay off? Effects of media Brand emotions on cognitive relief, identification and prestige[END_REF][START_REF] Kim | Measures of emotion in interaction for health smart home[END_REF].

Physiological and behavioural measurements are difficult to use for a large sample and in a brand context based on several factors. First, physiological measurements allow detecting the occurrence of even mild emotions, but they cover the specific emotion attached to the brand. Second, the measurement of behavioural changes, e.g. by using facial action coding systems, does not yet consider marketing specific emotions. Those autonomic measurements are well-adapted to advertisement testing, for example [START_REF] Droulers | Apports de la psychophysiologie à l'étude des émotions en marketing[END_REF], when the stimulus is precise and temporized. However, brand-related emotions elicit more durable emotional states, and they could benefit from a more qualitative assessment, not just intensity or valence. Thus, we have determined that the cognitive methodology of self-reports is the most appropriate way to measure brand-related affective reactions. Selfreporting gauges subjective feelings requiring respondents to report them with the use of set rating scales or verbal protocols [START_REF] Förster | Do emotions pay off? Effects of media Brand emotions on cognitive relief, identification and prestige[END_REF]. We will assess some of these based on their ability to provide good-quality consumer answers and insights.

The power of pictures

More specifically, we consider protocols using pictures to properly capture consumers' emotional responses towards brands. The theory of "dual-coding" presupposes the primacy of images to capture the most emotional non-verbal reactions [START_REF] Paivio | Imagery and Verbal Processes[END_REF]. According to [START_REF] Harper | Talking about pictures: a case for photo elicitation[END_REF], the introduction of visual material in the interview gives access to "other parts of the human consciousness". Visual methods also tend to generate not only more but also an entirely different kind of data than those resulting from a purely verbal interview [START_REF] Harper | Talking about pictures: a case for photo elicitation[END_REF][START_REF] Warren | Show me how it feels to work here: using photography to research organizational aesthetics[END_REF]. This has a physical basis: the parts of the brain that process visual information are evolutionarily older than the parts that process verbal information. Thus, images evoke deeper elements of human consciousness than do words, whereas exchanges based on words alone use less of the brain's capacity than do exchanges in which the brain is processing images and words [START_REF] Harper | Talking about pictures: a case for photo elicitation[END_REF]. As explained by [START_REF] Anastas | Visuals stimulate richer response in focus groups and individuals inter-views[END_REF], visuals initiate respondents' imagination and verbal responsiveness. When asked a question such as "What are your feelings when you use this product?", some consumers become tense and anxious because they seldom have the opportunity to express feelings about anything, let alone a commonplace product. When asked, "Select a picture and describe how it shows your feelings when you use the product", they tend to relax and launch into complex stories [START_REF] Anastas | Visuals stimulate richer response in focus groups and individuals inter-views[END_REF]. [START_REF] Scott | Writing with pictures: toward a unifying theory of consumer response to images[END_REF] consider that pictures (in commercial communication) are beginning to function "in a manner analogous to a writing system". Then it would be possible to communicate brand information in a predictable way, sometimes more effectively, with pictures than with alphabetic writing. The basis for what Scott and Vargas refer to as "writing-with-pictures" is located in the contemporary diffusion of mass media, which makes it possible to learn picture meanings in a way that was not feasible in earlier eras. [START_REF] Scott | Images in advertising: the need for a theory of visual rhetoric[END_REF] sees a convention-based system in which "viewers and makers share certain expectations, schemata, and implicit rules".

Methods of collecting images (known as "collages") for capturing consumer behaviours and attitudes have recently attracted increasing levels of interest. The Zaltman metaphor elicitation technique method [START_REF] Zaltman | Rethinking marketing research: putting people back in[END_REF], which has been popular for decades, invites respondents to produce a composition using several chosen images to express their point of view on the theme being studied. The selected images lead to a discussion with an analyst, who records and interprets the explanations given by the respondent in accordance with a clearly defined procedure [START_REF] Zaltman | Seeing the voice of the customer: metaphor-based advertising research[END_REF]. The "photo-language" technique is also quite well-known in the social sciences. Such techniques involve asking participants (in a group or individually) to express themselves in relation to a collection of images prepared by the researchers, for example, teenagers on the topic of sexuality [START_REF] Baptiste | Photolangage. Une méthode Pour Communiquer en Groupe par la Photo[END_REF]. Another example is the "album on line" (AOL) method [START_REF] Vernette | Une nouvelle méthode pour interpréter le sens d'une expérience de consommation: l'Album on line (AOL)[END_REF] in which following individual reflection on a given scenario, participants agree on a selected "album" of images (obtained online) to express their representations of the theme being studied. Keywords and brief narratives are also generated. On this basis, the AOL protocol has been clearly designed for capturing consumption experiential contents.

The use of pictures as a means of expression for emotional reactions is sometimes considered as less biased than verbal instruments [START_REF] Derbaix | L'affectif dans les Comportements d'achat et de Consummation[END_REF], which was recently discussed and validated through research by [START_REF] Yoon | Embodied typology of positive emotions: the development of a tool to facilitate emotional granularity in design[END_REF]. In the field of design studies, they used pictures to facilitate "emotional granularity" for a finer and deeper expression of emotional reactions. Some recent research projects have begun to investigate the efficiency of such protocols such as the "online multi-image elicitation" (OMIE) [START_REF] Ganassali | Le mur d'images en ligne: présentation et apports d'un protocole hybride[END_REF] or the "quali-quant synaesthesia" [START_REF] Pawle | How does your cappuccino feel?[END_REF], for example. In the field of brand image management, the use of pictures has provided helpful insights in many research problems, such as recently to explain brand meaning gaps and dynamics [START_REF] Wilson | Brand meaning gaps and dynamics: theory, research, and practice[END_REF].

Mixed-methods

Parallel to the use of pictures, researchers need to consider some new "mixed" protocols [START_REF] Spanjaard | Emotions in supermarket brand choice: a multimethod approach[END_REF] to combine the interests of both qualitative techniques (spontaneity and depth of analysis) and quantitative techniques (volume and objectification). There is a clear and recent growing trend of interest in mixed methods in the social sciences [START_REF] Tashakkori | Handbook of Mixed Methods in Social & Behavioral Research[END_REF] to bridge the traditional gap between extreme positivist and interpretivist approaches. More specifically, it is envisaged to develop a number of electronic versions of traditional qualitative visual methods (e.g. collages), to update them [START_REF] Koller | Mixed methods in online researchconceptualisation and future research agenda[END_REF] and take advantage of the power of dissemination of the internet.

The general concept is to keep the natural advantages of classic qualitative techniques like depth and subtlety of analysis while increasing the number of observations possible because of available multimedia internet protocols. This would result in a more significant number of answers, which allows the researcher to analyse and discuss the observed outcomes on a more objective basis.

Those mixed protocols, some of which are presented later in this paper, are also based on images collected from the internet and are then shown to respondents as part of an online survey, which is comparable to a more recent methodological approach described as "digital visual anthropology" [START_REF] Pink | Digital visual anthropology: potential and challenges[END_REF]. It is important to draw a connection between this type of method and the rise in certain contemporary Western societies of what some authors have called the "image culture". In this culture, media images (e.g. via Facebook, Instagram or Snapchat) are being increasingly used as sources and expressions of cultural identity [START_REF] Jansson | The mediatization of consumption: towards an analytical framework of image culture[END_REF] and are a reflectionor indeed the objectof several modern consumer phenomena, especially for teenagers and young adults.

Objectives and methodology of the study

Our objective is to compare certain traditional closed scales, in which a list of items is proposed and evaluated through Likert measurements, to other more illustrated and spontaneous self-report instruments using pictures or collages. We want to determine whether visual and spontaneous protocols are able to generate better and deeper insights than the groups of verbal scales only.

Criteria to assess the quality of responses

According to the possible consequences of satisficing responding behaviours [START_REF] Krosnick | The threat of satisficing in surveys: the shortcuts respondents take in answering questions[END_REF], we consider the response and completion rates [START_REF] Ganassali | The influence of the design of web survey questionnaires on the quality of responses[END_REF]. We also are interested in the evaluation of the instrument by the respondent, in their capacities of favouring the respondent's ability and motivation and to reduce the task difficulty. Then, we can obtain a double assessment of satisficing behaviours and the related quality of responses: a declarative one via the final evaluative questions and a factual one through the actual response behaviours. Additionally, we can also consider the "evocative richness" of responses, which measures the volume of relevant emotional contents formulated by the respondents in their reactions. For example, via a systematic content analysis, it is possible [START_REF] Mossholder | Measuring emotion in open-ended survey responses: an application of textual data analysis)[END_REF] to quantify the number of experiential or emotional insights included in the textual responses to the open-ended questions of the survey.

Methodology: visual and scales protocols

We have designed three versions of the same consumer survey dedicated to identifying a number of brand-related consumer emotional insights. The three survey protocols share the same introductory stagein which respondents are asked to state their current favourite brand, the same final section dedicated to the evaluation of the survey (ability, motivation and task difficulty) and some socio-demographics. The first protocol is simply based on the emotional attachment scale developed by [START_REF] Thomson | The ties that bind: measuring the strength of consumers' emotional attachments to brands[END_REF] and comprised ten items evaluated through a Likert scale that can be defined as "assisted declarative". Likert scales are currently and by far the dominant measuring instruments for quantitative studies about CBRs [1]. The second is "assisted associative" and consists of an OMIE elaborated according to the principles defined by [START_REF] Ganassali | Le mur d'images en ligne: présentation et apports d'un protocole hybride[END_REF]. The respondents are asked to first choose three pictures to describein that casetheir feelings towards their favourite brand [2]. In the next screen, the three chosen pictures are presented again, and respondents are asked to explain why they chose them and what they represent. It was proved that those kinds of mixed protocols were promising as a means to stimulate the abundance and diversity of responses in consumer behaviour studies. The third scenario is "spontaneous associative", and, like a collage, the consumer can choose the pictures they want to express their feelings. This is referred to as the "on-line collage" and originates from the results of some first experiments (AOL, for example -Vernette, 2007) combining the advantages of several existing techniques. In the AOL process, participants extract keywords from stories associated with the studied experience and select representative images on the internet. A collective album is subsequently created by a moderator or by a process of collective selection. In the on-line collage protocol, respondents are requested to search for pictures on the internet and upload them on the survey questionnaire as an individual expression of their emotions towards any relationship with a brand or consumer experience. Of course, those three protocols do not exactly follow the same objective. The Likert scales are supposed to be used for confirmatory research, while the visual protocols (especially n°3) are more exploratory by nature.

The three tested protocols

The following screen shots comprising Figure 1 illustrate the three protocols and the way they are concretely presented to the survey target respondents.

Sample

For that methodological piece of research, we used a convenience sample. In November 2015, the surveys were first circulated amongst two groups of bachelor and master degree students in two countries: France and Poland. They were also distributed to a panel of company managers in France to obtain a larger variety of profiles in terms of age groups. The three versions of the survey were randomly assigned to target contacts. In total, we received 532 responses. The average age of the respondents was 31 years. The sample was composed of 60 per cent of female respondents, and the level of education was high with on average between three and four years of higher education.

Findings and results

We begin introducing the results with behavioural and factual measurements and then look at the evaluation of the respondents. Finally, we consider the very challenging evocative richness of the protocols.

Dropouts, time and completion rates

As can be seen from Table I, the dropout rates are similar for Protocols 1 and 2 (between 5 and 6 per cent) but higher for Protocol 3, which reaches to almost 20 per cent. The completion times for the three different protocols are difficult to compare because the tasks For the completion rate, we are interested in the proportion of missing values in the various situations considering that only the very first question related to the favourite brand is programmed as mandatory and cannot be skipped. The highest completion rate is obtained for the OMIE protocol: more than 91 per cent of the respondents completed the full task by choosing two pictures and justifying their choices with free text inputs. For Protocol 1, 87 per cent of the participants completed all ten scales. Finally, only 82 per cent of the respondents uploaded one or more pictures for Protocol 3 and justified their choices with free text inputs. On average, respondents uploaded two or three pictures.

Ability, motivation and task difficulty

As the likelihood to satisfice (and then to answer in an inaccurate way) is linked to respondent ability, respondent motivation and task difficulty [START_REF] Krosnick | The threat of satisficing in surveys: the shortcuts respondents take in answering questions[END_REF], we also compare the perception of the respondents on those dimensions for the three different protocols. At the end of the survey, respondents were asked to assess the questionnaire with eight related questions and an OMIE. As we can see from Table II, Protocol 1 (the emotional attachment scale) is considered as easy and quick but not very interesting and enjoyable. In addition to that, its score for expressiveness is low. Protocol 2, OMIE is well evaluated: quite easy and quick, exciting, enjoyable and helpful for the expression of feelings. Protocol 3 (the online collage) is rated higher for feelings expressiveness and lower for quickness and easiness.

Evocative richness

Comparing the evocative richness of such different protocols is very challenging. As richness may also be related to the nature of the indicated favourite brands, we decided to compare the insights produced by the three protocols mainly for the two most frequently chosen brands, specifically Apple and Ikea, and to have them assessed through their results' summaries by a group of experts.

First protocol: emotional attachment scale In total, 10 per cent of the evaluations state that the proposed scales poorly describe the emotions. That figure increases to 24 per cent if we take the precedent grade (just before "describes poorly"). From a traditional Likert scale's protocol, we can clearly compare the level of emotions elicited by the two brands. Generally, Apple is rated emotionally higher than Ikea. For Apple, the level of connection is specifically strong (5.58 out of 7). However, Ikea is considered more friendly and peaceful than Apple (Figure 2).

Second protocol: online multi-image elicitation

As an outcome of the analyses performed within the OMIE protocol [START_REF] Ganassali | Le mur d'images en ligne: présentation et apports d'un protocole hybride[END_REF], brands, selected pictures and textual justifications[3] can be represented together on a factorial map so that associations may appear. In our example, it is quite clear that Apple is associated with emotions such as not only surprise, impressiveness or sharing but also relaxation. In those results, Ikea relates to conviviality, joy, happiness and friendship. The other plotted elements on the factorial map may help to possibly identify the major dimensions differentiating brand-related emotional responses in our study: sophistication/ distance on the left versus ordinariness/familiarity on the right of the horizontal axis; collective pleasure on the bottom versus individual care on the top of the vertical axis.

Third protocol: online collage with uploaded pictures

Based on approximately 20 respondents per brand, from Figure 5, we can analyse the uploaded pictures for the two brands in a systematic way. Three topics are coded by a procedure of content analysis [START_REF] Bell | Content analysis of visual images[END_REF]: colours, genders and of course leading emotions.

As shown in Table III, we can clearly see that evocations are quite different for the two brands. Apple is black and white (and a little grey too), whereas Ikea is whiter and more multicolour. Additionally, Ikea is more feminine. As far as emotions and feelings are concerned, our coding shows that the associations for the two brands are efficiency, polyvalence and touch for Apple, with some aspects of innovation, simplicity and aesthetics; for Ikea, mainly a profusion of colours and aesthetics, with some minor dimensions of sharing, creativity/inspiration, joy and comfort.

Evaluation of insights through results' summaries

As a final stage of our work, we conducted an experiment to derive a coherent assessment of the insights produced by the results of our three protocols. As a first step, we asked international postgraduate students to write summaries of results on the basis of the outcomes of the three protocols (Figures 3, 4 and5). Two groups of three or four individuals were randomly assigned to one of the three cases and were asked to write a short summary (half to one page) formulating some comments and conclusions to describe the emotions consumers feel towards Apple and Ikea. Each group was given one hour to perform the task. As a second step, coordinators of the two groups assigned to the same protocol were asked to put their works together so that we could have one summary per protocol in the end. The third step was an online survey [4] in which we asked experts to evaluate those three summaries in the way they would be useful and relevant for Apple and Ikea marketing managers to properly capture and understand the emotions that consumers associate with their brands. They were required to read and rate two summaries of results (randomly assigned to them among the three), on a list of six criteria designed to assess the depth and relevance of insights. They also had to choose the best one from the perspective of Apple and Ikea marketing managers. We also collected 62 expert evaluations from 19 different countries (mainly France, Poland and Germany). Of this group of experts, 60 per cent are academics in the field of marketing or more specifically brand management or marketing research and 40 per cent are research analysts or marketing managers. The group comprised 55 per cent of men and 45 per cent of women with a median age of 44 years.

As we can see from Figure 5, Protocol 2 (OMIE) gets the highest evaluations[5] on the six criteria we defined, and Protocol 1 was the lowest on five of the six criteria.

Based on the summaries chosen by our experts for being globally the best taking the perspective of Apple and Ikea's marketing managers, we can confirm that the visual protocols statistically give better results. OMIE is considered as the best in almost 70 per cent of the situations versus almost 55 per cent for the on-line collage. The scale is only chosen in 28 per cent of the cases (Table IV). Efficiency/reliability (8), Touch ( 5) Polyvalence (4), Innovation (3), Simplicity (3) and Aesthetics/design (3) Multicolour ( 10), Aesthetics/design (9), Profusion (7), Sharing (6), Creativity/ inspiration (6), Joy (5) and Comfort (4)

Conclusions

This study contributes to existing literature through advancing our understanding of the potential use of visual and spontaneous mixed protocols for consumer behaviour research. For exploratory purposes, our results suggest that those techniques are able to generate deeper insights than only using a closed group of scales.

As they do not follow exactly the same methodological objective, it is not surprising to see that the three protocols provide researchers with different levels of quality and different types of insights. Regarding the depth of the insights, the Likert scales are very standardised and therefore easy to analyse for comparing consumers' perceptions about different brands. However, the quality of the insights is strongly related to the accuracy of the tool for capturing the studied phenomenon. In the case of emotions, it remains quite challenging to find the right instrument able to capture a wide variety of emotions. In our example, the emotional attachment scale [START_REF] Thomson | The ties that bind: measuring the strength of consumers' emotional attachments to brands[END_REF] first shows that one brand ranks globally higher than the other in terms of global emotional intensity. It specifies the dimensions on which the "competing" brands are better: connection for Apple, friendliness and peacefulness for Ikea in our case. It is also difficult to ensure that some important brand emotions are not missing because they could not be properly captured by the standardised instrument used. Like all positivist tools [START_REF] Mukherji | Research Methods in Early Childhood: An Introductory Guide[END_REF], those types of scales may lack in accuracy and may produce superficial information. In particular, for emotions, it is admitted [START_REF] Paivio | Imagery and Verbal Processes[END_REF]) that their verbalisation is not always easy or possible. Our respondents rate the scale protocol quite low in terms of expressiveness. In addition to that, 40 per cent of consumers are choosing only three points or fewer within the whole set of seven modalities. In the lists of scale questions, the respondents sometimes tend only to choose a very narrow range of responses from all the possibilities. This behaviour pattern referred to as "nondifferentiation" where respondents satisfice shows a lack of interest and a weak level of effort for responding [START_REF] Ray | Des limites de l'échelle 1-10: Caractérisation des sous-échelles utilisées par les répondants[END_REF]. In terms of memorisation, according to the dual coding theory, it has been recognized that the verbal system is activated when verbal stimuli are present. It dominates in the case of tasks that require verbal, more rational responses, whereas the non-verbal system is activated when non-verbal stimuli are present and dominates in the case of tasks that require non-verbal, more affective responses [START_REF] Paivio | Imagery and Verbal Processes[END_REF]. It was recently demonstrated (Herz and Diamantopoulos, 2013, p. 111) in a mixed study of country-specific associations that visual methods (collages in this case) perform better when it comes to detecting consumption-related affective associations. The authors point out that "the adoption of both verbal-and nonverbal-based approaches provides complementary insights on consumer brand associations". This provides confirmation that mixed visual protocols such as OMIE would be highly relevant for research where the affective dimension is important. Recently, the use of images as a means of expressing emotional reactions was discussed and validated in a study by [START_REF] Yoon | Embodied typology of positive emotions: the development of a tool to facilitate emotional granularity in design[END_REF]. The authors used images to facilitate "affective granularity" that is a more refined and in-depth expression of affective responses. [START_REF] Derbaix | Pour un développement des mesures de l'affectif en marketing: synthèse des prérequis[END_REF] proposed a classification of affects with seven categories: appreciation (the most cognitive of all reactions), attitude, preference, temperament, humour, sentiment and shock (the most affective). One can therefore say that techniques like OMIE are particularly useful for detecting the most intense affects such as humour, sentiments or even strong experiences, which verbal accounts cannot always accurately render [START_REF] Derbaix | Pour un développement des mesures de l'affectif en marketing: synthèse des prérequis[END_REF]. This is mainly why visual protocols may be specifically promising: they are considered generally as useful for "capturing deep contextual meanings of consumer experience" [START_REF] Rohani | One step closer to the field: visual methods in marketing and consumer research[END_REF]. We have tested two of them and they clearly seem to provide researchers with deeper insights, in particular a better emotional granularity [START_REF] Yoon | Embodied typology of positive emotions: the development of a tool to facilitate emotional granularity in design[END_REF]. The respondents definitively rate those visual instruments as more interesting, enjoyable and expressive. The online-collageas a very spontaneous instrumentprovides researchers with more specific emotional responses. For example, the multicolour feeling associated with Ikea cannot be easily elicited through pre-defined scales of emotional attributes. However, as shown in our experiment, that protocol may increase significantly the number of dropouts because it may be perceived sometimes as difficult and demanding, even if in the end, a 20 per cent dropout rate is not that high for such a task. If we look at the respondents' evaluations, the OMIE seems to be a good compromise between convenience and expressiveness. Looking at the insights we obtained from the responses, they are more specific than the Likert scales' dimensions, but clearly, less rich than the ones obtained by the online collage. However, the quality of data, the respondents' assessment and the experts' insights evaluation are higher with OMIE.

More than promoting one protocol instead of another, we may learn from our experiment that those different emotional self-report measurements can be adapted to certain diverse research situations. Likert scales are useful for very confirmatory or comparative analyses, when researchers are clear about the nature of the emotions they want to assess. In that case, for example, they may resort to the related instruments like the ones specifically developed for brand love [START_REF] Albert | When consumers love their brands: exploring the concept and its dimensions[END_REF] or brand trust [START_REF] Delgado-Ballester | Development and validation of a brand trust scale[END_REF]. For a more exploratory approach, the online collage may be well adapted. In fact, as a spontaneous, open and projective protocol disseminated in large scale through the internet, that instrument is likely able to identify more precisely the various dimensions of the emotional relationship between consumers and their favourite brands. The combination of visual and textual responses provides researchers with a wide set of complementary information which they naturally have to interpret and recode cautiously. The same applies for the OMIE. As the respondent is proposed a selection of 20 to 30 pictures, that instrument is less open than the collage. However, the textual justifications in the second stage of the protocol may also elicit more specific and precise emotional states.

The results of our study lead us to question the systematic use of Likert scales to assess or capture consumers' emotional responses to brands regardless of the situation. Essentially, we do not have to really consider the three protocols we assessed as competitive ways to collect data. We could actually recognise that they could help CBR researchers in various research situations. Online collage is certainly the most adapted to a very exploratory context, when the nature of the CBR is to be discovered. Likert scales are adapted to a confirmatory approach, when the characteristics of the relationship are already well-known, and when researchers want, for example, to compare the level of that sentiment across various profiles of consumers or correlate it with some other concepts. The OMIE seems to be suitable to some intermediate situations, when the CBR is estimated, but should be specified in a more refined way. Additionally, OMIE mixes different types of measurements [START_REF] Ganassali | Le mur d'images en ligne: présentation et apports d'un protocole hybride[END_REF]: selection of pictures, textual justifications of choice and possibly, traditional scales for complementary evaluations, which lead to triangulation opportunities. As we can see, the range of possible effective methods for measuring consumers' responses towards brands is finally quite wide but it seems like CBR researchers do not yet use it enough.

In many social science surveysand particularly in marketingthe researcher or research analyst expects active (and even "productive") participation from the subjects solicited. They hope for their involvement and commitment so as to obtain sufficient breadth and depth of expression in their "insights", which are supposed to be useful when making decisions in the future. In this respect, mixed visual protocols like OMIE online collages are promising when it comes to stimulating the abundance and diversity of responses. Such approaches now offer many opportunities for research questions relating to experiential consumption, advertising persuasion or the detection and evaluation of the emotional [START_REF] Albert | When consumers love their brands: exploring the concept and its dimensions[END_REF] or symbolic values of a brand. This is particularly relevant for certain areas such as tourism, leisure activities, culture, luxury products, hygiene and beauty products, where it is sometimes difficult to express brand attributes verbally. From a managerial perspective, those visual mixed techniques are some concrete examples of the twofold quali-quant approach through which practitioners are generally convinced [START_REF] Macer | Cracking the code: what customers say in their own words[END_REF]. Furthermore, it represents excellent value for money as it generates a large volume of insights that are relatively rich and objective for relatively low implementation costs. Finally, this type of visual protocol carries good aesthetic appeal, thus contributing to a more modern and enhancing image of the focal organisation, which is not a negligible advantage when the research targets customers or staff performance.

As a future research, we could try to identify whether there are some specific profiles of consumers who would be more sensitive or reactive to online visual protocols than others, for example, young people or technophiles. Perhaps there could be some cultural differences as well in the way we use pictures and words to express our feelings, and this could be investigated in the future within a larger intercultural study.

Notes 1. We accessed a database of 520 international papers published from 1999 to the presentall related to consumer-brand relationship, used by Noël Albert for a forthcoming article. A lexical analysis of the full texts showed the following results: "scale" is quoted 1531 times in 260 articles (50 per cent of the papers), "item" is quoted 2128 times in 268 papers (52 per cent of the papers). "Picture" is quoted 357 times in 118 articles (26 per cent) and "open-ended question" (or equivalent) only three times in three articles (0.006 per cent of the papers).

2. The set of pictures proposed to the respondents is developed according to a procedure described by the authors. The images are selected by a group of researchers to cover all of the theoretical dimensions identified in the literature of the focal concept. The multi-image presentation is designed so that at least three images are related to each dimension. An inter-coder reliability test is recommended to verify that the right images have been associated with the different themes.

For further details and examples, see [START_REF] Ganassali | Le mur d'images en ligne: présentation et apports d'un protocole hybride[END_REF].

3. Textual justifications were coded via lexical analysis [START_REF] Bolden | Bridging the quantitative-qualitative divide: the lexical approach to textual data analysis[END_REF].

4. See www.sphinxonline.com/SurveyServer/s/etudiup/eval3prots/questionnaire.htm 5. P2 is higher than P1 for the five first criteria -P3 is different from P1 for criteria 1 and 2 -P2 is higher than P3 for criterion 4 -t test statistically significant at p = 0.05

Figure 1 .

 1 Figure 1. Screenshots of the three different tested protocols

Figure 2 .

 2 Figure 2. Emotional differences between Apple and Ikea captured by Protocol 1

Figure 3 .

 3 Figure 3. Emotions captured by Protocol 2 (OMIE): results of correspondence analysis

Figure 4 .Figure 5 .

 45 Figure 4. Pictures uploaded to express feelings towards Apple (back) and Ikea (front)

Table I .

 I

			Dropout	Average completion time
		Protocols	rate (%)	(excl. 5% lower and higher)	Protocol completion rate (%)
		P1 (emotional attachment scale)	5,7		4 m 58 s		87,2
	Dropout, completion rates and completion	P2 (OMIE) P3 (online collage)	5,4 19,8 (*)		5 m 48 s 9 m 25 s		91,4 82,3 (**)
	time for the three	Notes: *Significantly different from P1 and P2 at p < 0.05; **significantly different from P2 at p < 0.05
	tested protocols	(Exact Fisher tests)
			P1 (emotional	P2	P3 (online	Significant difference at
		Protocols	attachment scale)	(OMIE)	collage)	p < 0.05 (t test)
		It is well-designed		3,71	3,99	3,76	None
		It helped me to express my feelings		2,76	3,27	3,35	P1=P2 and P1=P3
		It motivated me to answer		3,31	3,71	3,18	P1=P2 and P2=P3
	Table II. Respondents'	It was easy to use It was easy to answer It was interesting		4,89 4,55 3,49	4,97 4,65 3,81	3,72 3,73 3,78	P1=P3 and P2=P3 P1=P3 and P2=P3 None
	evaluations for the	I enjoyed it		3,55	3,92	3,73	P1=P2
	three tested protocols	It was quick		5,19	5,20	4,09	P1=P3 and P2=P3

Table IV .

 IV Success rates for the three protocols

	Protocols	No. of evals	No. of bests	% of success
	P1 Scales	46	13	28,3
	P2 OMIE	43	30	69,8 (*)
	P3 Online	35	19	54,3 (*)
	collage

Note: (*) P2 and P3 percentages are significantly different from P1, at p = 0.05 (t test)

The authors would like to acknowledge European Master in Business Studies students for their participation in the experiment and Sphinx Company for technological support.