FXR activation normalizes insulin sensitivity in visceral preadipocytes of a rabbit model of MetS

in Journal of Endocrinology

Insulin resistance is the putative key underlying mechanism linking adipose tissue (AT) dysfunction with liver inflammation and steatosis in metabolic syndrome (MetS). We have recently demonstrated that the selective farnesoid X receptor (FXR) agonist obeticholic acid (OCA) ameliorates insulin resistance and the metabolic profile with a marked reduction in the amount of visceral AT (VAT) in a high-fat diet (HFD)-induced rabbit model of MetS. These effects were mediated by the activation of FXR, since treatment with the selective TGR5 agonist INT-777 was not able to ameliorate the metabolic parameters evaluated. Herein, we report the effects of in vivo OCA dosing on the liver, the VAT, and the adipogenic capacity of VAT preadipocytes (rPADs) isolated from rabbits on a HFD compared with those on a control diet. VAT and liver were studied by immunohistochemistry, Western blot analysis, and RT-PCR. rPADs were exposed to a differentiating mixture to evaluate adipogenesis. Adipocyte size, hypoxia, and the expression of perilipin and cytosolic insulin-regulated glucose transporter GLUT4 (SLC2A4) were significantly increased in VAT isolated from the HFD rabbits, and normalized by OCA. The expression of steatosis and inflammation markers was increased in the liver of the HFD rabbits and normalized by OCA. rPADs isolated from the HFD rabbits were less sensitive to insulin, as demonstrated by the decreased insulin-induced glucose uptake, triglyceride synthesis, and adipogenic capacity, as well as by the impaired fusion of lipid droplets. OCA treatment preserved all the aforementioned metabolic functions. In conclusion, OCA dosing in a MetS rabbit model ameliorates liver and VAT functions. This could reflect the ability of OCA to restore insulin sensitivity in AT unable to finalize its storage function, counteracting MetS-induced metabolic alterations and pathological AT deposition.

Abstract

Insulin resistance is the putative key underlying mechanism linking adipose tissue (AT) dysfunction with liver inflammation and steatosis in metabolic syndrome (MetS). We have recently demonstrated that the selective farnesoid X receptor (FXR) agonist obeticholic acid (OCA) ameliorates insulin resistance and the metabolic profile with a marked reduction in the amount of visceral AT (VAT) in a high-fat diet (HFD)-induced rabbit model of MetS. These effects were mediated by the activation of FXR, since treatment with the selective TGR5 agonist INT-777 was not able to ameliorate the metabolic parameters evaluated. Herein, we report the effects of in vivo OCA dosing on the liver, the VAT, and the adipogenic capacity of VAT preadipocytes (rPADs) isolated from rabbits on a HFD compared with those on a control diet. VAT and liver were studied by immunohistochemistry, Western blot analysis, and RT-PCR. rPADs were exposed to a differentiating mixture to evaluate adipogenesis. Adipocyte size, hypoxia, and the expression of perilipin and cytosolic insulin-regulated glucose transporter GLUT4 (SLC2A4) were significantly increased in VAT isolated from the HFD rabbits, and normalized by OCA. The expression of steatosis and inflammation markers was increased in the liver of the HFD rabbits and normalized by OCA. rPADs isolated from the HFD rabbits were less sensitive to insulin, as demonstrated by the decreased insulin-induced glucose uptake, triglyceride synthesis, and adipogenic capacity, as well as by the impaired fusion of lipid droplets. OCA treatment preserved all the aforementioned metabolic functions. In conclusion, OCA dosing in a MetS rabbit model ameliorates liver and VAT functions. This could reflect the ability of OCA to restore insulin sensitivity in AT unable to finalize its storage function, counteracting MetS-induced metabolic alterations and pathological AT deposition.

Introduction

The metabolic syndrome (MetS) is a constellation of metabolic abnormalities centered on insulin resistance and visceral fat accumulation. In addition to insulin resistance and visceral obesity, MetS is accompanied by dyslipidemia with high triglyceride levels and low HDL cholesterol concentrations and hypertension (Després & Lemieux 2006). Non-alcoholic fatty liver disease (NAFLD), a pathophysiological accumulation of lipids in the liver, is considered the hepatic hallmark of MetS (Farrell 2009). NAFLD, when associated with inflammation, evolves into non-alcoholic steatohepatitis (NASH), which can lead to cirrhosis and hepatocarcinoma. Insulin resistance has been implicated in both the initiation of NAFLD and its transition into NASH (Larter et al. 2010).

In MetS, adipose tissue (AT) is not only increased in mass, but also dysfunctional and characterized by hypertrophic insulin-resistant adipocytes, fulfilling their storage function but unable to take up any more triglycerides. The excess of circulating triglycerides ultimately leads to fat accumulation in ectopic areas, such as the liver, heart, and skeletal muscle. This ectopic fat deposition amplifies insulin resistance and can interfere with cellular functions (Després & Lemieux 2006, Virtue & Vidal-Puig 2010, Snel et al. 2012). In response to triglyceride overload, naive preadipocytes might be prompted to differentiate into mature adipocytes, thus serving as a buffer against lipid accumulation in non-adipose cells. However, insulin resistance may impair the differentiation of preadipocytes, with a consequent inability to store excess lipids by enlarged mature adipocytes (Gustafson et al. 2009). Finally, the presence of insulin-resistant adipocytes is considered as the key distinguishing feature between ‘metabolically healthy’ and ‘metabolically abnormal’ obesity (Samocha-Bonet et al. 2012).

During the past few years, bile acids (BAs) have emerged as important modulators of metabolic homeostasis and insulin resistance (Thomas et al. 2008). BAs through dedicated receptors – in particular the nuclear hormone receptor farnesoid X receptor (FXR, also known as NR1H4) and the G protein-coupled receptor TGR5 – modulate several metabolic pathways regulating glucose, triglyceride, and cholesterol levels and energy homeostasis. Interestingly, Fxr-deficient (Fxr−/−) mice display elevated plasma and hepatic cholesterol and triglyceride levels, along with an accelerated hepatic response on being fed a high-carbohydrate diet, and develop peripheral insulin resistance (Sinal et al. 2000). Three independent reports have linked FXR deficiency to impaired insulin sensitivity (Cariou et al. 2006, Ma et al. 2006, Zhang et al. 2006). In addition, Tgr5 (Gpbar1)-deficient mice exhibit impaired glucose tolerance (Thomas et al. 2009). In mice fed a high-fat diet (HFD), INT-777, a specific TGR5 agonist without FXR agonist activity (Thomas et al. 2009) lowers serum glucose and insulin levels and improves glucose tolerance (Sato et al. 2007).

The most clinically advanced FXR agonist is the semi-synthetic BA derivative obeticholic acid (OCA, 6-ethyl-chenodeoxycholic acid or INT-747), which is able to improve insulin sensitivity in patients with type 2 diabetes and NAFLD (Adorini et al. 2012, Mudaliar et al. 2013). OCA is a first-in-class FXR agonist, endowed with high binding affinity and potency for FXR (EC50 0.1 μM), with a 200-fold lower activity for TGR5 (Rizzo et al. 2010).

We have recently developed a non-genomic model of MetS, by feeding rabbits a HFD. Such a model recapitulates the human MetS phenotype (hypertension, hyperglycemia, dyslipidemia, VAT accumulation, and glucose intolerance), including a condition of hypogonadotrophic hypogonadism, as we have demonstrated in several previous studies (Filippi et al. 2009, Vignozzi et al. 2011, 2012, Maneschi et al. 2012, Morelli et al. 2012, 2013). Interestingly, VAT isolated from MetS animals was also dysfunctional, being characterized by insulin-resistant preadipocytes with impaired triglyceride synthesis and adipogenesis (Maneschi et al. 2012). OCA dosing in MetS rabbits not only prevents HFD-induced VAT expansion, but also reduces fasting glucose levels and glucose intolerance (Vignozzi et al. 2011, Morelli et al. 2012). In addition, OCA treatment ameliorates MetS-associated dysfunctions in corpora cavernosa (Vignozzi et al. 2011) and bladder (Morelli et al. 2012).

The aim of the present study was to investigate the role played by OCA in VAT dysfunction and steatohepatitis not only by evaluating the morphological and functional features of the liver and VAT, but also by analyzing the insulin sensitivity of VAT preadipocytes. In particular, the insulin response of rabbit preadipocytes, isolated from the different experimental groups, was investigated in terms of triglyceride synthesis and lipid droplet formation and mRNA expression of adipogenesis-specific genes, as well as glucose uptake. We also report the effect of treatment with the selective TGR5 agonist INT-777 to discriminate between FXR- and TGR5-mediated metabolic activities. The rabbit model of HFD-induced MetS allowed us to analyze in detail AT function, providing sufficient amount of visceral fat for all the different experimental purposes.

Our results indicate that OCA treatment ameliorates, via FXR activation, liver and VAT functions, most probably by restoring insulin sensitivity in VAT.

Materials and methods

MetS rabbit model

The HFD-induced rabbit model of MetS was obtained as described previously (Filippi et al. 2009). Male New Zealand White rabbits (Charles River, Calco, Lecco, Italy), weighing about 3 kg, were randomly numbered and assigned to two different groups: untreated group (n=38), fed a control diet (CON), or treated group (n=60), fed a HFD (0.5% cholesterol and 4% peanut oil), for 12 weeks. The composition of the CON and HFD is reported in Table 1. A subgroup of HFD rabbits was planned to be treated with the FXR and TGR5 agonist OCA (10 mg/kg, daily 5 days a week for 12 weeks, by oral gavage; n=18), as described previously (Vignozzi et al. 2011, Morelli et al. 2012), or with the selective TGR5 agonist INT-777 (30 mg/kg, daily 5 days a week for 12 weeks, by oral gavage; n=6; Pellicciari et al. 2009). The dose of OCA used was selected based on the efficacy and pharmacokinetics analysis carried out in rodents (Pellicciari et al. 2002). After a 3-month chronic feeding at the dose of 10 mg/day per kg BW to the rabbits, OCA was mainly present in the plasma as a glycine conjugate (20% of the total BAs) and in lower amounts (15%) as the unconjugated parent compound. No other major metabolites resulting from a 7-dehydroxylation process, glucuronides, and other polar metabolites were identified (Intercept Pharmaceuticals (New York, NY, USA), Internal Report 2011). Structure, potency, and selectivity toward other nuclear hormone receptors have been described previously (Pellicciari et al. 2002, Rizzo et al. 2010). Blood samples were obtained from marginal ear vein at baseline and at week 12 in all the groups. Mean arterial pressure measurements and oral glucose tolerance test were carried out before killing, as described previously (Filippi et al. 2009). After 12 weeks of treatment, the rabbits were killed using a lethal dose of pentobarbital (100 mg/kg), and the specimens of the liver, VAT (accumulated between the intestinal loops and mesentery), and gallbladder were carefully excised, weighed, collected, and processed for the subsequent analyses. VAT samples from all the rabbit groups were also processed for the isolation of preadipocytes. Biochemical and hormonal serum analyses were performed as described previously (Filippi et al. 2009, Morelli et al. 2012, Vignozzi et al. 2012). Based on an interim analysis, due to the lack of an effect of INT-777 on hyperglycemia and on overall MetS parameters (see below) and to an unexpected gallbladder hypertrophy, experiments with INT-777 were stopped, and therefore available data are limited to six rabbits.

Table 1

Composition of the control diet and high-fat diet

AnalysisControl diet (CON) (%)High-fat diet (HFD) (%)
Water1212
Protein16.512.6
Fat
 Vegetable derived3.56
 Animal derived00.5
Fiber15.521.2
Ash8.59.2

To evaluate the effects of MetS, we designed an algorithm taking into account the presence, as a dummy variable, of one or more of the following factors: hyperglycemia, high triglyceride levels, high cholesterol levels, increased blood pressure, and visceral fat accumulation. Cut-offs for each factor were derived by the mean±2 s.d. of the analyzed parameter, as measured in the CON rabbits. Positivity for three or more factors indicates MetS.

Ethics statement

This study was carried out in strict accordance with the recommendations in the Italian Ministerial Law #116/92 for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Florence (protocol number: 4/III). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

Sample size

Assuming a probability of the occurrence of MetS of 2.5% in the CON group and a probability of 60% in the HFD group (data derived from our previous publications on the same model (Filippi et al. 2009, Vignozzi et al. 2011, 2012, Maneschi et al. 2012, Morelli et al. 2012, 2013)), the use of 74 rabbits with an allocation ratio of 1:1 between the groups allows a power close to 100% in distinguishing a difference in the rate of development of MetS between the two treatment groups. Assuming a probability of the occurrence of MetS equal to 60% in the group fed the HFD and a probability of 10% in the group fed the HFD+OCA (data derived from our previous publications on the same model (Vignozzi et al. 2011, Morelli et al. 2012)), the use of 54 rabbits with an allocation ratio of 2:1 allows a power of about 95% in distinguishing a difference in the rate of development of MetS between the two treatment groups.

Histomorphometric analysis of VAT

VAT specimens were analyzed by hematoxylin and eosin staining to measure adipocyte diameter, as described previously (Maneschi et al. 2012), using the Nikon Microphot-FXA microscope (Nikon, Tokyo, Japan) equipped with the free software program ImageJ (NIH, Bethesda, MD, USA), considering adipocytes to be regularly spherical.

Hypoxia detection and immunohistochemistry

VAT oxygenation was analyzed using the bio-reductive drug pimonidazole hydrochloride (hypoxyprobe-1, 60 mg/kg), injected i.p. 1 h before killing, as described previously (Maneschi et al. 2012, Morelli et al. 2012, 2013, Vignozzi et al. 2012).

Preparation of total and membrane/cytosolic fractions for western blot analysis

For protein extraction from the VAT samples, the frozen tissues were ground in liquid nitrogen and divided into two aliquots: one for total protein extraction and the other for membrane/cytosolic preparations. Membrane and cytosolic fractions were prepared using the ProteoExtract subcellular proteome extraction kit (Calbiochem-Merck KGaA, Darmstadt, Germany), according to the manufacturer's instructions. Protein extracts were quantified with the BCA reagent (Pierce, Rockford, IL, USA), and 15 μg of each sample were resolved by 10% SDS–PAGE. Western blot analysis with an anti-glucose transporter type 4 (GLUT4) antibody (Upstate Biotechnology, Lake Placid, NY, USA) and anti-perilipin antibody (Santa Cruz Biotechnology, Inc.) was performed as described previously (Maneschi et al. 2012). Equal protein loading was verified by reprobing the membrane with an anti-actin antibody (Santa Cruz Biotechnology, Inc.). Densitometry analysis of band intensity was performed using the Photoshop 5.5 Software (Adobe Systems, Inc. Italia srl).

Liver histology

Liver steatosis was assessed by Oil Red O staining of the liver sections. Frozen sections were cut in a cryostat and fixed in 4% paraformaldehyde for 20 min at room temperature (RT). Then, the sections were treated for 2–5 min with isopropanol and stained with Oil Red O for 20 min. Oil Red O was prepared by diluting a stock solution (0.3 g of Oil Red O in 100 ml of isopropanol) with water (3:2) followed by filtration. After Oil Red O staining, the sections were washed several times in water and stained with hematoxylin and eosin to highlight the hepatocyte nuclei. Finally, the sections were photographed, and computer-assisted quantification of Oil Red O positivity was done after background subtraction using the Adobe Photoshop 6.0 Software (Adobe Systems).

Immunohistochemistry for TNFα (TNF) in the liver sections

Liver sections were incubated overnight at 4°C with a primary anti-TNFα (TNF) antibody (infliximab 1:100 vol/vol, DakoCytomation, Copenhagen, Denmark). The sections were rinsed in PBS and incubated with a biotinylated secondary antibody and then with a streptavidin–biotin–peroxidase complex (Ultravision large volume detection system anti-polyvalent, Lab Vision, Fremont, CA, USA). The reaction product was developed with 3′,3′-diaminobenzidine tetrahydrochloride as the chromogen (Sigma–Aldrich). Control experiments were performed by omitting the primary antibody. The slides were evaluated and photographed using a Nikon Microphot-FXA microscope. Computer-assisted quantification of the staining of TNFα was done after background subtraction using the Adobe Photoshop 6.0 Software (Adobe Systems).

Isolation, characterization, and differentiation of rabbit visceral fat preadipocytes

The isolation of rabbit preadipocytes (rPADs) from VAT was carried out as described previously (Maneschi et al. 2012). Briefly, VAT samples were digested with 1 mg/ml collagenase type 2 (Sigma–Aldrich) for 1 h, treated with red blood cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA; 10 min at RT), then centrifuged at 2000 g for 10 min at RT, resuspended in a complete medium (DMEM containing 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM l-glutamine, and 1 μg/ml amphotericin-B; Sigma–Aldrich), and filtered through a 150 μm mesh filter to remove debris. Finally, the cells were cultured in a complete culture medium at 37°C in a humidified atmosphere of 95% air–5% CO2. A subconfluent (90% of the cell culture dish) and homogeneous fibroblast-like cell population at passage 0 (P0) was obtained after 4–5 days of culture. The subconfluent cells were trypsinized and plated in cell culture dishes (P1). For all the experiments, only P1 cultures were used, and the experiments were repeated using at least three different rPAD preparations for each experimental group. rPADs were characterized by flow cytometry with the following conjugated monoclonal antibodies: CD34-PE, CD45-FITC, CD31-FITC, CD14-PE, CD90-PE, CD106-FITC (BD Pharmingen, San Diego, CA, USA), and CD105 PE (Ancell, Bayport, MN, USA), as described previously (Maneschi et al. 2012). The differentiation of rPADs, 2 days after confluence (time 0), was induced by exposing them to a differentiation mixture (DIM) containing 5 μg/ml insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) in 5% stripped FBS-supplemented DMEM for 8 days (Student et al. 1980). The culture medium was replaced every 48 h, and then the cells were shifted into a medium containing 5 μg/ml insulin for 48 h.

Qualitative and quantitative estimation of triglyceride accumulation

Qualitative and quantitative analyses of intracellular lipids were carried out using Oil Red O staining (Sigma) and AdipoRed Assay (Cambrex BioScience, Walkersville, MD, USA) respectively, as described previously (Maneschi et al. 2012). Briefly, both untreated and DIM-induced rPADs were washed in PBS and fixed in 10% formalin for 1 h at RT, followed by staining with Oil Red O for 5 min. After staining, the plates were washed twice in water and photographed. For the AdipoRed Assay, the medium was removed in both untreated and DIM-induced rPADs, and each well was carefully rinsed with 200 ml PBS. Then, the rPADs were incubated with 200 μl PBS and 5 μl of AdipoRed at RT for 10–15 min and immediately placed in a fluorimeter for fluorescence measurement (excitation at 485 nm and emission at 572 nm). Triglyceride content was normalized on protein content. Both untreated and DIM-treated rPADs, AdipoRed stained, were imaged immediately using a Leica DMI6000 microscope equipped with a DFC350FX camera. The images were acquired using the Leica N3 filter set and a Fluotar 20× 0.4NA long-working distance objective with a correction collar. AdipoRed-positive cells, identified as those clearly exhibiting lipid droplet staining, were counted using the ImageJ software and expressed as the percentage of total cells.

Confocal microscopy

DIM-treated rPADs, following AdipoRed staining, were immediately imaged using a Leica SP2-AOBS confocal microscope, as described previously (Maneschi et al. 2012). The images were collected as z-stacks through a 63× 1.2NA water-immersion objective, taking care to minimize spherical aberration, and then deconvolved with the Huygens Professional Software (Scientific Volume Imaging (SVI), Hilversum, The Netherlands) using the Classic Maximum Likelihood Estimation (CMLE) algorithm and a theoretical Point Spread Function (PSF). Finally, these images were quantitatively analyzed using the Volocity 5 Software (Perkin-Elmer, Foster City, CA, USA) to measure the number and volume of the lipid droplets.

Glucose uptake

Glucose uptake by rPADs was measured as described previously (Maneschi et al. 2012). DIM-exposed rPADs were cultured for 24 h in a serum-free medium, followed by incubation in increasing concentrations of insulin (1, 5, 10, and 50 nM) diluted in glucose-free Krebs phosphate buffer (2.5 mmol Ca2+ and 1 mg/ml BSA), to evaluate insulin-dependent stimulation. At the end of the incubation period, rPADs were further incubated with 3H-2-deoxy-d-glucose (16 μM (1 μCi/μl); ICN Pharmaceuticals, Costa Mesa, CA, USA) for 5 min. The cells were then washed with PBS and lysed with NaOH 0.5 M, and the incorporated radioactivity was measured by scintillation spectrometry using a β-counter (Perkin-Elmer). Data were normalized on protein content.

RNA extraction and quantitative RT-PCR analysis

The isolation of RNA from the tissue and cells was performed as described previously (Morelli et al. 2012, 2013). Specific primers for all the target genes have been described previously (Filippi et al. 2009, Maneschi et al. 2012, Morelli et al. 2012, 2013, Vignozzi et al. 2012). The expression of the 18S rRNA subunit was quantified with a predeveloped assay (Applied Biosystems).

Statistical analysis

Results are expressed as means±s.e.m. for n experiments as specified. The statistical analysis was performed with a one-way ANOVA test followed by the Tukey–Kramer post hoc analysis in order to evaluate differences between the groups, and P<0.05 was considered significant. Correlations were assessed using Spearman's method, and the statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS, Inc.) for Windows 15.0. Stepwise multiple linear regressions were applied for the multivariate analysis, whenever appropriate. Half-maximal response effective concentration (EC50) values and maximal effect (Emax) values were calculated using the computer program ALLFIT (De Lean et al. 1978).

Results

OCA ameliorates HFD-induced metabolic alterations and VAT dysfunction

Feeding rabbits the HFD enhanced all the components of MetS, including insulin resistance and visceral fat accumulation. The prevalence of three or more MetS factors, identifying the MetS condition, according to the human definition (Alberti et al. 2009), was verified in 62.9% of the HFD rabbits (P<0.001 vs CON). Table 2 reports in detail the effects of the HFD on the metabolic and hormonal parameters. Treatment with the selective FXR agonist OCA for 12 weeks significantly reduced fasting blood glucose levels and glucose intolerance (both P<0.01 vs HFD). In addition, VAT weight was markedly decreased, even after normalization for total body weight (P<0.001 vs HFD). VAT amount in the OCA-treated HFD rabbits was even below the CON level (P<0.001; Table 2). In particular, OCA treatment was able to significantly reduce the prevalence of MetS (P<0.01 vs HFD, see Table 2). Conversely, treatment with the specific TGR5 agonist INT-777 did not exert any significant effect either on the prevalence of MetS (P=0.09 vs HFD) or on glycemia and glucose intolerance. INT-777 induced only a significant reduction in the amount of VAT (even after normalization for body weight; P<0.001 vs HFD). In addition, a peculiar increase in gallbladder weight was observed in the HFD rabbits treated with INT-777 (P<0.001 vs HFD and P<0.001 vs CON). The parameters of the relative transcripts of FXR and TGR5 in VAT isolated from the different experimental groups are also reported for comparison. In the CON rabbits, the parameters of the transcripts of FXR were fivefold higher (P<0.001) than that of the transcripts of TGR5, which were both unaffected by the HFD (see Table 2). OCA treatment but not INT-777 treatment further increased FXR mRNA expression (P<0.01 vs HFD). Conversely, INT-777 significantly upregulated TGR5 gene expression (P<0.05 vs HFD), but not FXR gene expression. Because no significant changes were observed regarding HFD-induced hyperglycemia and glucose intolerance in the INT-777-treated HFD rabbits, no further studies were performed on this particular group.

Table 2

Metabolic and hormonal parameters in the experimental rabbits. Relative mRNA expression of FXR and TGR5 was evaluated using quantitative RT-PCR in VAT samples of the CON (n=38), HFD (n=36), HFD+OCA (n=18), and HFD+INT-777 (n=6) groups. Data were calculated according to the comparative Ct method using 18S rRNA subunit as the reference gene for normalization

CON (n=38)HFD (n=36)HFD+OCA (n=18)HFD+INT-777 (n=6)
Total body weight (g)
 Baseline3258.6±63.03290.4±42.03361.7±52.43266.6±107.5
 Week 123909.9±38.33745.3±34.83663.1±79.3*3404±106.4
Blood glucose (g/l)
 Baseline1.18±0.041.29±0.031.25±0.061.24±0.2
 Week 121.25±0.031.94±0.07‡,¶1.40±0.06b2.03±0.3*,∥
OGTT (iAUC)
 Week 12157.0±5.0224.8±7.4181.6±9.6b217.1±15.7
Cholesterol (mg/dl)
 Baseline36.9±2.144.1±2.036.8±2.031.2±1.6
 Week 12 42.8±3.01447.6±64.7‡,¶1242.1±91.5‡,¶1711±134.6‡,¶
Triglycerides (mg/ml)
 Baseline81.5±4.486.6±4.176.8±5.793.83±9.9
 Week 1296.5±4.5304.5±25.1‡,¶230.7±36.6†,∥156.8±30.5
AST (U/l)
 Baseline30.9±3.126.2±2.225.8±2.337.8±8.6
 Week 1235.9±3.079.7±6.9‡,¶74.0±7.9‡,¶55.8±6.9§
ALT (U/l)
 Baseline25.7±2.223.0±1.627.3±3.134.3±4.6
 Week 1228.5±1.946.1±3.1‡,¶35.6±4.333.3±6.3
Liver weight (g, % of total body weight)
 Week 122.9±0.14.24±0.14.21±0.23.72±0.2§
MAP (mmHg)
 Week 1291.5±2.2133.4±3.5129.2±4.3143.2±3.2
VAT weight (g, % of total body weight)
 Week 120.92±0.051.09±0.040.41±0.06¶,c0.51±0.09∥,c
Gallbladder (mg, % of total body weight)
 Week 1227.6±4.631.1±9.218.7±6143.4±35.1¶,c
17β-Estradiol (pmol/l)
 Week 12168.7±91307.6±243135.5±50c107±9.2§,c
Presence of MetS (%)062.98.3b100
FXR expression in VAT (mRNA/18S)55.62±9.2160.99±12.7697.28±6.3¶,b66.69±15.52
TGR5 expression in VAT (mRNA/18S)11.42±1.329.97±1.9211.20±1.8917.25±0.87∥,a

iAUC, incremental area under the curve of glucose blood level during oral glucose tolerance test (OGTT); AST, aspartate aminotransferase; ALT, alanine aminotransferase; MAP, mean arterial pressure; VAT, visceral adipose tissue. *P<0.05, P<0.01, and P<0.001 vs baseline; §P<0.05, P<0.01, and P<0.001 vs CON week 12; and aP<0.05, bP<0.01, and cP<0.001 vs HFD week 12.

We next analyzed the correlation between the expression of the FXR gene and that of several genes related to inflammation, adipogenesis, glucose transport, and insulin signaling in the VAT of rabbits fed the CON or HFD. As shown in Table 3, a significant positive relationship was found between the expression of the FXR gene and that of the genes related to adipogenesis (CCAAT enhancer binding protein-α (c/EBPα (CEBPA)), peroxisome proliferator-activated receptor γ (PPARγ (PPARG)), fatty acid binding protein 4 (FABP4), leptin, adiponectin, PPARα (PPARA), and phospholipase A2 (PLPA2)), glucose transport (GLUT4 (SLC2A4), ras homolog gene family, member A (RHOA), Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), and ROCK2), and inflammation (interleukin 6 (IL6) and monocyte chemoattractant protein-1 (MCP1 (CCL2))).

Table 3

Association between FXR mRNA and VAT-specific genes in VAT

rP valuen
Adipogenesis
 c/EBPα0.4440.00539
 PPARγ0.449<0.000159
 FABP40.3780.00456
 Adiponectin0.4050.00258
 Leptin0.3150.01757
 PPARα0.481<0.000159
 PLPA20.3590.01248
Glucose transport and insulin signaling
 GLUT40.3470.00760
 RHOA0.2800.03060
 ROCK10.2750.03460
 ROCK20.3750.00360
Inflammation
 IL60.4280.00156
 MCP10.3340.01355

Correlations coefficients (r) and level of significance (P value) were derived from the univariate analysis.

The histomorphometric analysis of adipocytes, and the hypoxic state, along with perilipin expression, in the three experimental groups are shown in Fig. 1. The HFD induced a significant increase in adipocyte diameter (P<0.01 vs CON; Fig. 1A, B and D), hypoxyprobe staining (P<0.05 vs CON; Fig. 1E, F and H), and perilipin expression (P<0.01 vs CON; Fig. 1I). All the parameters were reduced by OCA treatment when compared with not only the HFD (P<0.0001), but also the CON (P<0.0001) (see bar graphs in Fig. 1D, H and I). In addition, GLUT4 expression in the adipocyte membrane fraction was decreased and GLUT4 was stacked in the cytosol of the HFD rabbits (P<0.01 vs CON), while OCA completely normalized GLUT4 membrane translocation (P<0.05 vs HFD; Fig. 1J).

Figure 1
Figure 1

Effects of OCA treatment on the morphological and functional features of VAT in the experimental rabbits. (A, B and C) Representative images of the hematoxylin and eosin-stained VAT sections showing different adipocyte sizes among the experimental groups (magnification 20×, scale bar=50 μm). Adipocyte size was significantly increased in the HFD rabbits when compared with that in the CON and OCA-treated HFD rabbits. (D) Histomorphometric analysis of adipocyte diameter (μm) in the different experimental groups (n=3 for each group). (E, F and G) Immunohistochemical staining of hypoxyprobe adducts in VAT sections. Hypoxyprobe adducts were revealed in hypoxic cells (PO2 <10 mmHg) of VAT transverse sections by a MAB (magnification 10×, scale bar=50 μm). An intense hypoxyprobe positivity was detected in VAT isolated from the HFD rabbits (F), while only scanty positive labeling was present in VAT isolated from the CON (E) and OCA-treated HFD (G) rabbits. (H) Computer-assisted quantitative image analysis of three independent experiments (n=3 for each group). (I) Protein expression of perilipin in VAT extracts isolated from the experimental rabbits. Representative immunoblots with anti-perilipin and anti-actin primary antibodies and the corresponding graphical representation of optical density (OD) analysis of perilipin band intensity normalized over actin are shown (n=5 for each group). (J) Analysis of GLUT4 membrane translocation in VAT. The lower panel shows representative immunoblots with anti-GLUT4 primary antibody on the membrane (m) and cytosolic (c) fractions of VAT isolated from the CON, HFD, and OCA-treated HFD rabbits. The bar graph shows the optical density analysis of membrane:cytosolic GLUT4 ratio (n=5 for each group). Data are expressed as the percentage of CON values. *P<0.05, **P<0.01, and ***P<0.0001 vs CON; °P<0.05 and °°°P<0.0001 vs HFD. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-13-0109.

Citation: Journal of Endocrinology 218, 2; 10.1530/JOE-13-0109

The correlation of visceral fat weight and several of the aforementioned VAT genes is reported in Table 4. Essentially, increased visceral fat accumulation was positively associated with the genes related to adipogenesis (c/EBPα, FABP4, and leptin), lipogenesis (diacylglycerol O-acyltransferase (DGAT2) and lipoprotein lipase (LPL)), NO signaling (endothelial nitric oxide synthase (eNOS (NOS3)) and protein kinase G1 (PKG1)), glucose transport (GLUT4, RHOA, ROCK1, ROCK2, and vimentin (VIM)), inflammation (MCP1), steroid sensitivity ((estrogen receptor α (ERα (ESR1))), and cytoskeleton remodeling (α smooth muscle actin (αSMA)). As shown in Table 5, in vivo OCA dosing induced a downregulation of the expression of most of these genes, including the progesterone receptor, indicating a decreased estrogen action (see Table 2). Conversely, OCA dosing upregulated the expression of the FXR downstream gene small heterodimer partner (SHP, Table 5).

Table 4

Association between visceral fat weight and VAT-specific genes in VAT

rP valuen
Adipogenesis
 c/EBPα0.2960.0552
 FABP40.392<0.000170
 Leptin0.396<0.000170
Lipogenesis
 DGAT20.3790.00168
 LPL0.2400.0571
NO signaling
 ENOS0.2910.0174
 PKG10.2490.0574
Glucose transport
 GLUT40.378<0.000174
 RHOA0.373<0.000174
 ROCK10.3250.0172
 ROCK20.2890.0171
 VIM0.2580.0568
Inflammation
 MCP10.3740.00174
Steroid sensitivity
 ERα0.3620.0157
Cytoskeleton remodeling
 αSMA0.3160.0561

Correlations coefficients (r) and level of significance (P value) were derived from the univariate analysis.

Table 5

Effect of OCA treatment on the mRNA expression of VAT-specific genes. Data are expressed as the percentage of variation vs HFD

GenesPercentage of variation (HFD+OCA vs HFD)
SHP274.3±92.6
FABP4−47±11.3
c/EBPα−61.2±12.3
LPL−49.6±7.9*
Leptin−58.2±23*
GLUT4−31.7±8.7*
IRS1−32±3.9
RHOA−37±8.2
ROCK1−34.8±7.8
ROCK2−56±16.1
DGAT2−63.5±17.3*
PR (PGR)−42.3±8.1*
VIM−17.7±2.3
αSMA−48.8±15.8
MCP1−13.7±5.1
eNOS−4.8±1
ERα−22±5.8
PKG1−21.4±4.7

*P<0.05, P<0.01, and P<0.001 vs HFD.

OCA ameliorates HFD-induced liver steatosis and inflammation

As reported in Table 2, the HFD also induced a significant increase in liver weight, as well as aspartate aminotransferase serum levels, which were not normalized by OCA dosing. We then evaluated FXR and TGR5 relative mRNA expression in the liver. We found that TGR5 was 2-log unit less expressed than FXR (29.51±6.6 and 6177.92±851.16 respectively). The correlation between the expression of liver FXR and that of several genes related to steatosis, inflammation, fibrosis, and metabolism in the CON and HFD rabbits is reported in Table 6. As shown in Table 7, the mRNA expression of FXR and the FXR primary response gene cholesterol 7α-hydroxylase (CYP7A1) was significantly increased in the liver of the HFD rabbits (P<0.05 and P<0.01 respectively). OCA dosing upregulated the expression of the FXR (P<0.01 vs CON) and SHP (P<0.001 vs CON and P<0.01 vs HFD) genes, while the expression of the CYP7A1 gene was downregulated (P<0.05 vs CON and P<0.0001 vs HFD; see also Table 7). Immunohistochemical studies using Oil Red O staining revealed a homogeneous and abundant hepatic lipid deposition in the HFD rabbits when compared with the CON rabbits (P<0.0001; Fig. 2A and B). OCA dosing was able to markedly counteract lipid accumulation, which was mainly limited to the perilobular region occupied by the portal system (Fig. 2C). The quantitative computer-assisted analysis of Oil Red O staining is shown in Fig. 2D. Gene expression of PPARγ, a specific steatosis marker, was significantly increased in the HFD rabbits (P<0.001 vs CON) and normalized by OCA dosing (P<0.01 vs HFD; Fig. 2E). Similar results were obtained for adiponectin mRNA (P<0.01 vs CON and P<0.01 vs HFD; data not shown). The livers isolated from the HFD rabbits also exhibited an intense intrahepatocyte immunopositivity for anti-TNFα antibody (P<0.01 vs CON; Fig. 2F and G), which was significantly blunted by OCA dosing (P<0.01 vs HFD; Fig. 2H). The quantitative computer-assisted analysis of anti-TNFα staining is shown in Fig. 2I. The expression of inflammation genes, TNFα (P<0.001; Fig. 2J), IL6 (data not shown; P<0.05), and IL10 (data not shown; P<0.001), was significantly increased in the liver of the HFD rabbits when compared with the CON rabbits. OCA dosing normalized the expression of both TNFα (Fig. 2J) and IL6 (data not shown), while it significantly increased that of IL10 (P<0.05, data not shown).

Table 6

Association between the expression of FXR mRNA and that of other genes related to steatosis, metabolism, inflammation, and fibrosis in the liver

rP valuen
Steatosis
 PPARγ0.623<0.000169
 Adiponectin0.3240.00767
Metabolism
 PPARα0.419<0.000169
 PLPA20.4290.00652
Inflammation
 TNFα0.3770.00267
 IL60.2910.01767
 MCP10.3820.00169
 COX2 (PTGS2)0.3880.00168
 IL80.509<0.000161
 IL100.455<0.000161
 CD4 0.2480.04665
 CD80.3950.00353
 CD680.445<0.000165
Fibrosis
 αSMA0.563<0.000162
 RHOA0.636<0.000166
 ROCK10.569<0.000166
 ROCK20.4210.00164
 TGFβ (TGFB1)0.496<0.000163
 COL1A10.4120.00253
 COL3A10.505<0.000153
 TIMP10.530<0.000163
 TIMP20.672<0.000150
 MMP20.641<0.000150
 MMP90.551<0.000149

Correlation coefficients (r) and level of significance (P value) were derived from the univariate analysis.

Table 7

Expression of genes involved in FXR activation in livers isolated from all the rabbit groups. Expression of genes involved in FXR activation (FXR, SHP, and CYP7A1) was detected by qRT-PCR in livers isolated from all the rabbit groups

FXRSHPCYP7A1
CON (n=31)100±6.6100±12.05100±14.35
HFD (n=36)131.4±11*188.4±42.1300.4±75
HFD+OCA (n=18)145.1±10.6336.8±58.7‡,§81±34.7*,∥

Data are expressed as the percentage of CON *P<0.05, P<0.01, and P<0.0001 vs CON; §P<0.01 and P<0.0001 vs HFD.

Figure 2
Figure 2

Amelioration of HFD-induced liver steatosis and inflammation by OCA. (A, B and C) Lipid accumulation was revealed in liver sections of the experimental rabbits by Oil Red O staining (magnification 10×, scale bar=50 μm). An abundant hepatic lipid deposition was found in the HFD rabbits (B) when compared with the CON rabbits (A). OCA dosing was able to markedly counteract lipid accumulation, mainly limited to the perilobular region occupied by the portal system (C). The quantitative computer-assisted analysis of Oil Red O staining is shown in (D). (E) Relative mRNA expression of steatosis marker (PPARγ) was evaluated using quantitative RT-PCR in liver samples of the CON (n=38), HFD (n=36), and HFD+OCA (n=18) rabbits. Data were calculated according to the comparative Ct method using the 18S rRNA subunit as the reference gene for normalization. Results are expressed as percentage over the CON. (F, G and H) Immunohistochemistry for TNFα in liver sections of the experimental rabbits (magnification 20×, scale bar=50 μm). Livers isolated from the HFD rabbits (G) exhibited an intense intrahepatocyte immunopositivity for anti-TNFα antibody, when compared with those isolated from the CON rabbits (F), which was significantly blunted by OCA dosing (H). The quantitative computer-assisted analysis of anti-TNFα staining is shown in (I). (J) Relative mRNA expression of inflammation marker (TNFα) was evaluated using quantitative RT-PCR in the liver samples of the CON (n=38), HFD (n=36), and HFD+OCA (n=18) rabbits. Data were calculated according to the comparative Ct method using 18S rRNA subunit as the reference gene for normalization. Results are expressed as percentage over the CON. *P<0.01, **P<0.001, and *** P<0.0001 vs CON; °P<0.05, °°P<0.01, and °°°P<0.0001 vs HFD. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-13-0109.

Citation: Journal of Endocrinology 218, 2; 10.1530/JOE-13-0109

OCA ameliorates spontaneous adipogenic differentiation in rabbit preadipocytes

We next investigated the adipogenic capacity of rPADs isolated from VAT. Each cell preparation was characterized by flow cytometry for the expression of mesenchymal stem cell (MSC) markers and hematopoietic–monocytic contamination. The percentage of positive cells expressing the MSC markers CD90, CD105, and CD106 was not different among the groups (data not shown). All rPADs were negative for endothelial (CD31), hematopoietic (CD34 and CD45), and monocytic (CD14) markers (data not shown). Expression analysis by qRT-PCR showed that the expression of CD90 (THY1) and the specific marker of adipocyte commitment dickkopf 1 (DKK1) was not different among the groups (data not shown). Interestingly, preadipocytes isolated from the OCA-treated rabbits exhibited a significantly increased expression of adipogenic-specific genes, such as FABP4 (P<0.001), c/EBPα (P<0.01), and PPARγ (P<0.01), compared with those isolated from both the CON and HFD groups (data not shown).

The spontaneous adipogenic potential was investigated in rPADs cultured for 10 days (Fig. 3A, B and C). The qualitative (Oil Red O staining; Fig. 3A) and quantitative (AdipoRed assay; Fig. 3B) estimation of triglyceride accumulation showed an increased lipid content in the cytosol of rPADs isolated from the OCA-treated HFD rabbits when compared with those isolated from both the HFD and CON groups. The HFD-reduced percentage of the AdipoRed-positive cells was also completely normalized by OCA dosing (P<0.01), even significantly higher than that in the CON group (P<0.01; Fig. 3C).

Figure 3
Figure 3

Amelioration of both spontaneous and DIM-induced adipogenic differentiation in rPADs by in vivo OCA dosing. (A) Lipid content (white arrows) in untreated rPADs isolated from each experimental group, as evaluated by Oil Red O staining. (B) Quantitative assessment of lipid content in untreated rPADs isolated from each experimental group, as evaluated by the AdipoRed assay. Results are expressed as relative fluorescence unit (RFU)/μg of protein (n=6 for each group). (C) Analysis of the lipid droplet content in untreated, AdipoRed-stained, rPADs isolated from all the rabbit groups. AdipoRed-positive cells were counted using the ImageJ Software and are expressed as the percentage of total cells. (D) Lipid content in DIM-induced rPADs isolated from each experimental group, as evaluated by Oil Red O staining. (E) Quantitative assessment of lipid content in DIM-induced rPADs isolated from each experimental group, as evaluated by the AdipoRed assay. Results are expressed as RFU/μg of protein (n=6 for each group). (F) Analysis of the lipid droplet content in DIM-exposed, AdipoRed-stained, rPADs isolated from all the rabbit groups. AdipoRed-positive cells were counted using the ImageJ Software and are expressed as the percentage of total cells. ^P<0.01 and ^^P<0.001 vs all the other groups; *P<0.01 vs CON; and °P<0.01 vs HFD. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-13-0109

Citation: Journal of Endocrinology 218, 2; 10.1530/JOE-13-0109

OCA ameliorates DIM-induced adipogenic differentiation in rPADs

We next evaluated the adipogenic potential by exposing in vitro rPADs to a DIM for 10 days (Fig. 3D, E and F). Oil Red O staining and AdipoRed assay showed a reduced adipogenic differentiation, characterized by a reduced triglyceride content (Fig. 3E) and a reduced percentage of AdipoRed-positive cells (Fig. 3F), in rPADs isolated from the HFD rabbits when compared with those isolated from the CON rabbits (both P<0.01). OCA treatment of the HFD rabbits completely normalized the percentage of AdipoRed-positive cells (Fig. 3F) and triglyceride content (both P<0.01 vs HFD), with the latter being even higher than that in the CON rabbits (P<0.01; Fig. 3D and E).

The responsiveness of rPADs to the DIM was also investigated in terms of the expression of adipocyte-related genes (DKK1, c/EBPα, PPARγ, FABP4, adiponectin, and leptin). As reported in Table 8, after 10 days of exposure to the DIM, there was a significant induction of the expression of all the investigated genes in rPADs isolated from the CON rabbits (all genes P<0.01 vs relative time 0). Conversely, in rPADs isolated from the HFD rabbits, DIM exposure was unable to significantly induce the expression of the investigated genes, with the exception of FABP4 mRNA. OCA treatment normalized the DIM-induced expression of all these adipocyte-specific genes (Table 8). Similarly, cyclin D3 (CCND3) mRNA expression was significantly induced in all the DIM-treated rPADs, with the exception of rPADs isolated from the HFD rabbits (Table 8). Conversely, CCND1 mRNA expression was significantly increased only in DIM-treated rPADs isolated from the HFD group and not in those isolated from the other groups (P<0.05, Table 8).

Table 8

Effect of DIM on the mRNA expression of adipocyte-related genes in rPADs. Relative mRNA expression of adipocyte-related genes was evaluated using quantitative RT-PCR in untreated (time 0) and DIM-exposed rPADs from the CON, HFD, and HFD+OCA groups (five different experiments, each performed in triplicate using a different cell preparation per group). Data were calculated according to the comparative Ct method using 18S rRNA subunit as the reference gene for normalization. Results are expressed as fold change over time 0

CONHFDHFD+OCA
Adipocyte-related genes
 DKK16.4±21.5±0.313.6±2.1
 c/EBPα2.3±0.51.2±0.32.5±0.5
 PPARγ2.5±0.51.1±0.31.7±0.1
 FABP420.6±75.3±1.110.9±3.6
 Adiponectin9.5±4.30.9±0.12.6±0.7
 Leptin8.7±2.6†,‡0.7±0.21.8±0.4
 CCND10.8±0.32.6±0.7*1.1±0.1
 CCND32.3±0.5*1.2±0.31.9±0.3†,∥

*P<0.05 and P<0.01 vs relative time 0; P<0.01 vs all the other groups; and §P<0.05 and P<0.01 vs relative CCND1.

Lipid droplets in rPADs isolated from the HFD rabbits exhibited a reduction in the average number and an increase in the average volume per cell when compared with those in rPADs isolated from the CON rabbits (P<0.05 and P<0.0001 respectively; Fig. 4A, B, D and E). In vivo OCA dosing induced both an increase in the number (P<0.0001; Fig. 4C and D) and a reduction in the volume (P<0.0001; Fig. 4C and E) of lipid droplets when compared with those in rPADs isolated from the HFD rabbits.

Figure 4
Figure 4

Positive effect of OCA on lipid droplet fusion. rPADs isolated from the CON (A), HFD (B), and OCA-treated HFD (C) rabbits were imaged by confocal microscopy (scale bar=10 μm). Images were quantitatively analyzed using the Volocity 5 Software (Perkin-Elmer, Foster City, CA, USA) to measure the number (D) and volume (μm3; E) of lipid droplets within single cells. At least eight cells were analyzed for each group. (F and G) Relationship between the lipid droplet volume (expressed as μm3, ordinate) and the SNAP23 or SYNT5 mRNA expression (abscissa) in both untreated and DIM-induced rPADs as derived from univariate Spearman's regression analysis. *P<0.05, **P<0.01, and ***P<0.0001 vs CON; °°°P<0.0001 vs HFD.

Citation: Journal of Endocrinology 218, 2; 10.1530/JOE-13-0109

Using qRT-PCR, we observed a significant upregulation of the expression of genes of the SNARE complex involved in lipid droplet handling, synaptosomal-associated protein 23 (SNAP23) and syntaxin 5 (SYNT5), in both untreated and DIM-induced rPADs isolated from the HFD rabbits when compared with those isolated from the CON rabbits (Table 9). In vivo OCA dosing normalized the expression of these genes (Table 9). The expression of SNAP23 and SYNT5 in both untreated and DIM-induced rPADs isolated from all the groups, expressed as a function of lipid droplet volume, is shown in Fig. 4F and G. A significant positive relationship was found between the lipid droplet volume and SNAP23 (r=0.928, P=0.008; Fig. 4F) and SYNT5 (r=0.829, P=0.04; Fig. 4G). Conversely, in vivo OCA dosing had effects comparable to those of the CON.

Table 9

Effect of in vivo OCA on the mRNA expression of genes involved in lipid droplet fusion (SNAP23 and SYNT5). Relative mRNA expression of the genes of the SNARE complex involved in lipid droplet handling (SNAP23 and SYNT5) was evaluated using quantitative RT-PCR in untreated (time 0) and DIM-exposed rPADs isolated from the CON, HFD, and HFD+OCA rabbits (six different rPAD preparations from each experimental group)

SNAP23SYNT5
CON
 Untreated1.90±0.204.22±0.62
 DIM2.09±0.233.88±0.36
HFD
 Untreated2.46±0.21*9.90±2.44
 DIM2.95±0.44*12.02±3.33
HFD+OCA
 Untreated1.70±0.133.52±0.39
 DIM2.09±0.19§4.38±1.09§

*P<0.05, P<0.01 vs CON; §P<0.05 and P<0.01 vs HFD.

OCA ameliorates glucose uptake in rPADs

The effect of OCA on insulin sensitivity was investigated by measuring 3H-2-deoxy-d-glucose uptake in DIM-induced rPADs, after exposure to increasing concentrations of insulin. As shown in Fig. 5, insulin dose dependently stimulated glucose uptake in rPADs isolated from the three experimental groups with significant differences for both EC50 and Emax (P<0.0001). In vivo OCA dosing restored the normal sensitivity to insulin (CON and HFD+OCA shared EC50=2.96±0.51 nM; HFD EC50=13.5±6.09 nM). The Emax of the HFD rabbits was dramatically decreased (128±4%) when compared with that of both the CON (CON Emax=273±3%, P=0.001) and OCA-treated HFD (HFD+OCA Emax=205±3%, P=0.004) groups, although the Emax of the latter group was still lower than that of the CON rabbits (P=0.006).

Figure 5
Figure 5

Insulin sensitivity of DIM-exposed rPADs. Dose–response curves of radiolabeled 3H-glucose uptake in DIM-treated rPADs after exposure to increasing concentrations of insulin are shown. Results are expressed as percentage over 0 nM insulin (five different experiments, each performed in duplicate and using a different cell preparation per group). The relative EC50s and Emax values are reported in the text. *P<0.01 and **P<0.001 vs CON; °P<0.01 vs HFD+OCA.

Citation: Journal of Endocrinology 218, 2; 10.1530/JOE-13-0109

Discussion

In this study, we demonstrate that pharmacological activation of FXR by OCA treatment prevents several HFD-induced alterations in the liver, while normalizing hyperglycemia and glucose intolerance as well as all the MetS-related VAT dysfunctions, including preadipocyte differentiation toward a mature phenotype and lipid droplet handling.

This study, in addition to confirming previous results (Maneschi et al. 2012), highlights several novel aspects in the relationship between AT and MetS. Our studies were carried out using a non-genomic, rabbit model of MetS, which essentially recapitulates the human phenotype (Filippi et al. 2009, Vignozzi et al. 2011, 2012, Maneschi et al. 2012, Morelli et al. 2012, 2013). Feeding a HFD for 12 weeks induces a sharp increase in fasting glycemia, glucose intolerance, and VAT amounts, as well as hypertension and dyslipidemia. In the HFD-induced rabbit model of MetS, VAT is not only increased in mass but also dysfunctional, with an impaired triglyceride synthesis and insulin-stimulated adipogenesis. We demonstrate that this animal model of MetS is also characterized by liver inflammation and steatosis, the main features of NASH. There is a close relationship between VAT dysfunction and NASH in MetS. Insulin resistance is the putative key underlying mechanism linking these two clinical entities (Cusi 2012, Targher & Byrne 2013).

In the present model of MetS, HFD induced a significant increase in liver weight and an abundant lipid accumulation, which were associated with an increased expression of steatosis markers, such as PPARγ and adiponectin. Livers isolated from MetS rabbits were also severely inflamed, as demonstrated by an increased expression of TNFα and IL-6 – pro-inflammatory cytokines involved in the transition from NAFLD to NASH. Indeed, the activation of inflammatory pathways in NASH is related to hepatic toxicity resulting from intrahepatic triglyceride overload (Cusi 2012). The major contributor to an increased triglyceride accumulation is dysfunctional AT (Donnelly et al. 2005). Interestingly, as described previously (Maneschi et al. 2012), we confirmed that VAT adipocytes isolated from MetS animals are dysfunctional. An increase in size and hypoxia, along with a reduced membrane translocation of GLUT4 and an increased expression of perilipin, was observed in VAT adipocytes isolated from the HFD rabbits. Indeed, not only the total mass of AT conveys a metabolic risk, but the size of adipocytes is also important, being positively associated with insulin resistance (Jacobsson & Smith 1972, Salans et al. 1974). Findings regarding the normalization of insulin resistance after weight loss, associated with a reduction in adipose cell size (Salans et al. 1968), further corroborate this concept. Interestingly, a putative mechanism by which insulin resistance could develop in hypertrophic fat cells may originate from hypoxia. Previous studies have indicated that hypoxia develops in VAT, as adipocyte size and tissue mass increase, leading to – via different mechanisms, including reduction in the expression of GLUT4 – an insulin-resistant phenotype (O'Rourke et al. 2011, Trayhurn 2013). Accordingly, a reduced GLUT4 translocation to the plasma membrane was observed in hypertrophic fat cells, when compared with the smaller ones (Salans et al. 1968, Salans & Dougherty 1971, Smith 1971, Jacobsson & Smith 1972, Olefsky 1976, Franck et al. 2007, Goossens 2007). Moreover, enrichment of perilipin 1 in large vs small adipocytes has also been associated with reduced insulin sensitivity in hypertrophic fat cells (Laurencikiene et al. 2011). Perilipin, a reliable marker of adipogenesis, is a major anti-lipolytic protein, coating the cytosolic surface of intracellular lipid droplets, protecting or exposing the triacylglycerol core of the droplets to lipases (Brasaemle 2007), thus controlling access to the adipocyte triglyceride stores that supply energy to most tissues. As a regulator of lipid storage and lipolysis, perilipin 1 is thus positioned to modify not only the risk of obesity but also its complications (Smith & Ordovás 2012).

In the present study, we extensively investigated insulin sensitivity and lipid droplet remodeling in adipocytes. rPADs isolated from VAT of the HFD rabbits exhibited a lower capacity to respond to insulin in terms of triglyceride synthesis and glucose uptake. Insulin resistance in rPADs was also demonstrated by the failure to upregulate the expression of adipogenesis-specific genes such as DKK1, c/EBPα, PPARγ, FABP4, adiponectin, and leptin. In addition, DIM-exposed rPADs from VAT of the HFD rabbits exhibited a prevalent expression of CCND1 when compared with the expression of CCND3 (Fu et al. 2004, Sarruf et al. 2005). Cyclins function as key components of the cell-cycle core machinery in adipocytes. Indeed it has been reported that CCND1 inhibits adipocyte differentiation through the repression of the expression and transactivation of PPARγ, while CCND3 promotes adipocyte differentiation as the coactivator of PPARγ. Accordingly, a lower percentage of AdipoRed-positive cells was also observed in the HFD rabbits. These findings thus further support the view of impaired adipocyte maturation in VAT isolated from the HFD rabbits.

In addition, lipid droplets of rPADs isolated from the MetS rabbits were reduced in number and increased in volume, with an increased expression of factors involved in lipid droplet fusion, namely the SNARE complex. Lipid droplets are formed as primordial droplets and increase in volume by a fusion process that requires the SNARE complex, including SNAP23 and SYNT5 (Boström et al. 2007). Accordingly, in the present study, we found that the expression of both SNAP23 and SYNT5 was increased in rPADs isolated from the HFD rabbits. In addition, in both untreated and DIM-induced rPADs, we found a positive association between lipid droplet volume and SNAP23 or SYNT5 mRNA expression. SNAP23 is also required for insulin-stimulated translocation of GLUT4 to the plasma membrane (Foster et al. 1999, Kawanishi et al. 2000), and it may play a role in the development of insulin resistance. Indeed, when SNAP23 is diverted from the plasma membrane, and thus away from the mechanism involved in insulin-stimulated GLUT4 translocation and glucose uptake, it is instrumental in the processes of lipid droplet fusion. This could represent a putative mechanism by which the development of insulin resistance is associated with the enhanced fusion of lipid droplets.

The most striking feature of the present study is that OCA treatment restores the differentiation of MetS preadipocytes toward a more mature and efficient metabolic phenotype, documented by their higher content of small-volume lipid droplets, associated with a decreased expression of factors known to orchestrate their fusion, such as the SNARE complex, including SNAP23. Consistent with the positive effect of OCA on HFD-induced VAT dysfunction, DIM-exposed rPADs isolated from the OCA-treated MetS rabbits exhibited an increased ability to respond to insulin, in terms of glucose uptake and adipocyte differentiation capacity, when compared with rPADs isolated from the HFD rabbits. In addition, in rPADs isolated from the OCA-treated HFD rabbits, all the other DIM-induced adipocyte features, including triglyceride synthesis, adipogenesis-specific gene expression (DKK1, c/EBPα, PPARγ, FABP4, adiponectin, and leptin), preadipocyte maturation (CCND1 and CCND3), and the number of differentiating cells (AdipoRed staining), were also normalized. Interestingly, OCA exerts its pro-adipogenic effects even in the earlier stage of adipocyte differentiation, as DIM-untreated preadipocytes isolated from the OCA-treated HFD rabbits exhibited an increased expression of adipogenesis-specific genes (such as c/EBPα, PPARγ, and FABP4, even when compared with those in the CON rabbits) as well as a significant increase in both triglyceride accumulation and percentage of differentiating cells. Overall, these findings are in line with previous observations showing the effect of OCA on the promotion of insulin sensitivity and adipocyte differentiation, both in vivo (Cariou et al. 2006, Ma et al. 2006, Zhang et al. 2006) and in vitro, as in the preadipocyte cell line 3T3-L1 (Cariou et al. 2006, Rizzo et al. 2006). In the present study, we demonstrated that this VAT weight reduction is associated with adipocytes that are smaller in size. In vitro, we found that preadipocytes isolated from the OCA-treated HFD rabbits were able to differentiate into adipocytes with multilocular lipid droplets and increased insulin sensitivity. Interestingly, these phenotypic features have been recognized to characterize the metabolically healthy adipocytes, with increased energy consumption through free fatty acid oxidation and consequently reduced fat mass and insulin resistance (Timmons et al. 2007). An increased free fatty acid oxidation could be the underlying mechanism of the reduced visceral fat mass observed in the OCA-treated HFD rabbits. A major limitation of the present study is the lack of investigation on energy consumption in preadipocytes isolated from the different experimental groups. However, several recent studies have demonstrated that the activation of FXR enhances energy expenditure, reducing circulating levels of free fatty acids and insulin resistance (Fiorucci et al. 2010).

The present study also indicates that FXR could be a target for treating MetS-induced VAT alterations. In homogenates of visceral fat, indeed, we found that the expression of FXR is positively associated with the expression of genes involved in insulin signaling and glucose transport (GLUT4, RHOA, ROCK1, and ROCK2), adipogenesis (c/EBPα, PPARγ, FABP4, adiponectin, leptin, PPARα, and PLPA2), and inflammation (IL6 and MCP1). Moreover, OCA dosing completely normalized GLUT4 membrane translocation and VAT oxygenation, as well as perilipin expression, and drastically reduced adipocyte size, which was significantly reduced even when compared with that observed in the CON rabbits. OCA dosing also reduced the expression of several genes associated with visceral fat accumulation, including those related to inflammation (MCP1), steroid sensitivity (ERα), adipogenesis (c/EBPα, FABP4, and leptin), lipogenesis (DGAT2 and LPL), NO signaling (eNOS and PKG), glucose transport (GLUT4, RHOA, ROCK1, ROCK2, and VIM), and cytoskeleton remodeling (αSMA).

Concomitantly, OCA also ameliorates HFD-induced glucose intolerance and fasting hyperglycemia. The increased insulin sensitivity may be responsible for the preservation of ‘metabolically healthy’ VAT phenotype as well as the amelioration of liver abnormalities. The present data, showing that OCA dosing can reduce HFD-induced liver steatosis and inflammation, as well as ALT serum levels, are in line with previous results obtained in insulin-resistant Zucker fa/fa rat model (Cipriani et al. 2010). The reduced hepatic lipid levels correlate with the increased insulin sensitivity in adipocytes, as reported previously (Renga et al. 2010). Interestingly, OCA has been evaluated in three phase II clinical trials, including one in patients with type 2 diabetes and NAFLD (Adorini et al. 2012, Mudaliar et al. 2013). In this trial, OCA was demonstrated to induce a systemic improvement of insulin sensitivity and an improvement in both hepatic and peripheral glucose uptake. Interestingly, a significant decrease in the levels of liver fibrosis biomarkers was also observed following OCA treatment (Adorini et al. 2012, Mudaliar et al. 2013).

Our results indicate that the beneficial effect of OCA on HFD-induced insulin resistance is mediated by the specific activation of FXR, rather than TGR5, at both VAT and hepatic levels. Indeed, we found that i) the treatment of the HFD rabbits with the selective TGR5 agonist INT-777 does not affect HFD-induced glucose intolerance and increased fasting glycemia; ii) the expression of TGR5 in the liver and VAT is markedly lower compared to FXR; iii) the expression of FXR primary response genes, SHP and CYP7A1, is respectively upregulated and downregulated by OCA treatment, as expected following FXR activation (Rizzo et al. 2006). These data, together with the known 200-fold greater agonistic activity of OCA for FXR when compared with TGR5 (Rizzo et al. 2010), support the view that all the observed OCA effects on HFD-induced MetS are selectively mediated by FXR activation.

In conclusion, in an animal model of HFD-induced MetS, OCA dosing not only ameliorates liver steatosis and inflammation but also counteracts all the HFD-induced VAT alterations, restoring preadipocyte differentiation through a positive and persistent effect on insulin sensitivity. The present preclinical evidence and the clinical experience with this first-in-class FXR agonist support the potential of OCA to counteract diet-related metabolic disorders.

Declaration of interest

E M, L V, A M, T M, S F, I C, P C, E S, A C, B M, R V, and G B V have nothing to declare. L A is an employee of Intercept Pharmaceuticals (18 Desbrosses Street, New York, New York 10013, USA). M M is a scientific consultant for Bayer Pharma AG (Germany) and Eli Lilly (Indianapolis, Indiana, USA).

Funding

This work was supported by PRIN (Programmi di ricerca di Rilevante Interesse Nazionale, protocol no. 20099BXMJH 002) and FIRB (Fondo per gli investimenti alla ricerca di base, protocol no. 2010RBFR10VJ56 002), both funds from the Italian Minister of University, Research and Instruction, by Under40-Young Investigators funds from the Italian Minister of Health (grant no. GR2008-1137632), and by a scientific grant from Intercept Pharmaceuticals (18 Desbrosses Street, New York, New York 10013, USA).

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(E Maneschi and L Vignozzi contributed equally to this work)

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    Effects of OCA treatment on the morphological and functional features of VAT in the experimental rabbits. (A, B and C) Representative images of the hematoxylin and eosin-stained VAT sections showing different adipocyte sizes among the experimental groups (magnification 20×, scale bar=50 μm). Adipocyte size was significantly increased in the HFD rabbits when compared with that in the CON and OCA-treated HFD rabbits. (D) Histomorphometric analysis of adipocyte diameter (μm) in the different experimental groups (n=3 for each group). (E, F and G) Immunohistochemical staining of hypoxyprobe adducts in VAT sections. Hypoxyprobe adducts were revealed in hypoxic cells (PO2 <10 mmHg) of VAT transverse sections by a MAB (magnification 10×, scale bar=50 μm). An intense hypoxyprobe positivity was detected in VAT isolated from the HFD rabbits (F), while only scanty positive labeling was present in VAT isolated from the CON (E) and OCA-treated HFD (G) rabbits. (H) Computer-assisted quantitative image analysis of three independent experiments (n=3 for each group). (I) Protein expression of perilipin in VAT extracts isolated from the experimental rabbits. Representative immunoblots with anti-perilipin and anti-actin primary antibodies and the corresponding graphical representation of optical density (OD) analysis of perilipin band intensity normalized over actin are shown (n=5 for each group). (J) Analysis of GLUT4 membrane translocation in VAT. The lower panel shows representative immunoblots with anti-GLUT4 primary antibody on the membrane (m) and cytosolic (c) fractions of VAT isolated from the CON, HFD, and OCA-treated HFD rabbits. The bar graph shows the optical density analysis of membrane:cytosolic GLUT4 ratio (n=5 for each group). Data are expressed as the percentage of CON values. *P<0.05, **P<0.01, and ***P<0.0001 vs CON; °P<0.05 and °°°P<0.0001 vs HFD. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-13-0109.

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    Amelioration of HFD-induced liver steatosis and inflammation by OCA. (A, B and C) Lipid accumulation was revealed in liver sections of the experimental rabbits by Oil Red O staining (magnification 10×, scale bar=50 μm). An abundant hepatic lipid deposition was found in the HFD rabbits (B) when compared with the CON rabbits (A). OCA dosing was able to markedly counteract lipid accumulation, mainly limited to the perilobular region occupied by the portal system (C). The quantitative computer-assisted analysis of Oil Red O staining is shown in (D). (E) Relative mRNA expression of steatosis marker (PPARγ) was evaluated using quantitative RT-PCR in liver samples of the CON (n=38), HFD (n=36), and HFD+OCA (n=18) rabbits. Data were calculated according to the comparative Ct method using the 18S rRNA subunit as the reference gene for normalization. Results are expressed as percentage over the CON. (F, G and H) Immunohistochemistry for TNFα in liver sections of the experimental rabbits (magnification 20×, scale bar=50 μm). Livers isolated from the HFD rabbits (G) exhibited an intense intrahepatocyte immunopositivity for anti-TNFα antibody, when compared with those isolated from the CON rabbits (F), which was significantly blunted by OCA dosing (H). The quantitative computer-assisted analysis of anti-TNFα staining is shown in (I). (J) Relative mRNA expression of inflammation marker (TNFα) was evaluated using quantitative RT-PCR in the liver samples of the CON (n=38), HFD (n=36), and HFD+OCA (n=18) rabbits. Data were calculated according to the comparative Ct method using 18S rRNA subunit as the reference gene for normalization. Results are expressed as percentage over the CON. *P<0.01, **P<0.001, and *** P<0.0001 vs CON; °P<0.05, °°P<0.01, and °°°P<0.0001 vs HFD. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-13-0109.

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    Amelioration of both spontaneous and DIM-induced adipogenic differentiation in rPADs by in vivo OCA dosing. (A) Lipid content (white arrows) in untreated rPADs isolated from each experimental group, as evaluated by Oil Red O staining. (B) Quantitative assessment of lipid content in untreated rPADs isolated from each experimental group, as evaluated by the AdipoRed assay. Results are expressed as relative fluorescence unit (RFU)/μg of protein (n=6 for each group). (C) Analysis of the lipid droplet content in untreated, AdipoRed-stained, rPADs isolated from all the rabbit groups. AdipoRed-positive cells were counted using the ImageJ Software and are expressed as the percentage of total cells. (D) Lipid content in DIM-induced rPADs isolated from each experimental group, as evaluated by Oil Red O staining. (E) Quantitative assessment of lipid content in DIM-induced rPADs isolated from each experimental group, as evaluated by the AdipoRed assay. Results are expressed as RFU/μg of protein (n=6 for each group). (F) Analysis of the lipid droplet content in DIM-exposed, AdipoRed-stained, rPADs isolated from all the rabbit groups. AdipoRed-positive cells were counted using the ImageJ Software and are expressed as the percentage of total cells. ^P<0.01 and ^^P<0.001 vs all the other groups; *P<0.01 vs CON; and °P<0.01 vs HFD. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-13-0109

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    Positive effect of OCA on lipid droplet fusion. rPADs isolated from the CON (A), HFD (B), and OCA-treated HFD (C) rabbits were imaged by confocal microscopy (scale bar=10 μm). Images were quantitatively analyzed using the Volocity 5 Software (Perkin-Elmer, Foster City, CA, USA) to measure the number (D) and volume (μm3; E) of lipid droplets within single cells. At least eight cells were analyzed for each group. (F and G) Relationship between the lipid droplet volume (expressed as μm3, ordinate) and the SNAP23 or SYNT5 mRNA expression (abscissa) in both untreated and DIM-induced rPADs as derived from univariate Spearman's regression analysis. *P<0.05, **P<0.01, and ***P<0.0001 vs CON; °°°P<0.0001 vs HFD.

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    Insulin sensitivity of DIM-exposed rPADs. Dose–response curves of radiolabeled 3H-glucose uptake in DIM-treated rPADs after exposure to increasing concentrations of insulin are shown. Results are expressed as percentage over 0 nM insulin (five different experiments, each performed in duplicate and using a different cell preparation per group). The relative EC50s and Emax values are reported in the text. *P<0.01 and **P<0.001 vs CON; °P<0.01 vs HFD+OCA.

  • AdoriniLPruzanskiMShapiroD2012Farnesoid X receptor targeting to treat nonalcoholic steatohepatitis. Drug Discovery Today17988997.(Review) (doi:10.1016/j.drudis.2012.05.012)

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