The prevalence of obesity has increased strikingly during the past three decades, particularly among minorities and socio-economically disadvantaged populations around the world( Reference Flegal, Carroll and Ogden 1 – Reference Whaley-Connell, Pulakat and Demarco 3 ). The main factor that leads to this condition is overnutrition, especially when characterised by the excessive intake of carbohydrates and fat( Reference Carvalheira, Calegari and Zecchin 4 – Reference Hwi, Zhang and Dae 9 ), which can trigger the metabolic syndrome (MS) defined as a constellation of metabolic abnormalities for CVD and diabetes. A consensus agreement by the International Diabetes Federation and the American Heart Association/National Heart, Lung and Blood Institute identifies the criteria of the MS as abdominal obesity, reduced HDL, elevated TAG, glucose intolerance and hypertension; a diagnosis requires any three of these five criteria( Reference Alberti, Eckel and Grundy 10 ).
The primary cause of the MS appears to be increased adiposity associated with insulin resistance( Reference Gonçalves, Glade and Meguid 11 , Reference Mayans 12 ). Furthermore, there is a strong relationship between obesity and inflammation( Reference Dupuy, Jaussent and Lacroux 13 ), since hyperadiposity produces adipokines, such as leptin, adiponectin and resistin, as well as proinflammatory cytokines such as IL-6, TNF-α and plasminogen activator inhibitor type 1, which are all involved in proinflammatory and prothrombotic responses( Reference Yudkin 14 ).
In obesity, inflammation that is triggered in adipose tissue may occur due to the excess of SFA in the diet. These fatty acids can be recognised by Toll-like receptor-4 (TLR-4), which is expressed by adipocytes and macrophages, leading to activation of NF-κB( Reference Hwang 15 – Reference Zeyda and Stulnig 18 ), stimulating the production of chemokines and proinflammatory cytokines( Reference Cai, Yuan and Frantz 19 ), as well as attracting immune cells from the circulation into the adipose tissue( Reference Kim and Sears 16 ). TLR-4 is a cell surface receptor that generates innate immune responses to pathogens by inducing signalling cascades of kinase and transcription factor activation, leading to the generation of proinflammatory cytokines, chemokines, eicosanoids and reactive oxygen species, all of which are effectors of innate immunity.
Thus, we can propose that excessive sugar and fat intake are factors that lead to inflammation in adipose tissue. Several studies have shown that inflammation is involved in the development of components of the MS associated with obesity, especially insulin resistance( Reference Marette 20 – Reference Festa, D'Agostino and Howard 22 ). However, few experimental studies that have emphasised the role of diet, obesity and inflammation evaluated the participation of the TLR-4 pathway as a function of time( Reference Maric, Woodside and Luheshi 23 – Reference Park, Lee and Chung 28 ). Therefore, additional studies are needed to characterise the inflammatory response in adipose tissue and insulin resistance as a function of time. Thus, the aim of the present study was to evaluate the manifestation of the MS and inflammation adipose tissue as a function of time in rats submitted to a high-sugar/fat diet (HSF).
Materials and methods
Animals and experimental protocol
The experimental protocol was approved by the local Ethical Committee for Animal Research of the University of Sao Paulo State University (permit number PE-47/2011). Male Wistar rats (10 weeks old, ±350 g) from the Animal Center of Botucatu Medical School, Sao Paulo State University (UNESP, Botucatu, SP, Brazil), were assigned to either a commercial chow diet (control diet; C; 12 % energy from fat) or an HSF (49·7 % energy from fat) with sucrose in the drinking water (300 g/l) for a period of 6, 12 or 24 weeks (C6, HSF6, C12, HSF12, C24, HSF24). The diet-induced obesity model was adapted from our previous study( Reference Nascimento, Sugizaki and Leopoldo 29 ) and it has been published previously( Reference Luvizotto, Nascimento and Imaizumi 30 ), which was used to mimic obesity from Western occidental dietary habits.
Rats were housed in individual cages in the animal facility at the Internal Medicine Experimental Laboratory, Botucatu Medical School, UNESP, under controlled ambient temperature (22–26°C) and lighting (12 h light–12 h dark) conditions. Dietary and water consumption was measured daily, and body weight was assessed weekly. Energy intake was calculated according to the formula: energy intake (kJ/d) = food consumption (g) × dietary energy (kJ/g). For the animals that received sucrose in drinking water (30 %), the energy intake was calculated according to the formula: volume consumed (ml) × 0·3 (equivalent to 30 % sucrose) × 16·7 (kJ per g of carbohydrate) + energy values offered by feeding (food consumption (g) × dietary energy (kJ/g)).
The animals were killed by decapitation after anaesthesia with sodium pentobarbital Q4 (50 mg/kg, intraperitoneal injection) and all efforts were made to minimise suffering. Blood from fasted animals was collected in tubes containing EDTA and centrifuged at 3500 rpm and the plasma was collected for analysis. Epididymal adipose tissue was selected for analysis because of its similar inflammatory patterns to visceral fat( Reference Gea-Sorlí, Bonjoch and Closa 31 ).
Adiposity index
The adiposity index was used as an indicator of obesity because it enables the precise evaluation of body fat percentage. Epididymal, retroperitoneal and visceral fat deposits were dissected from the rats. The sum of the fat deposits, normalised by body weight, was calculated to obtain the adiposity index: ((epididymal + retroperitoneal + visceral)/body weight) × 100( Reference Boustany, Bharadwaj and Daugherty 5 , Reference Ferron, Jacobsen and Sant'Ana 32 ).
Plasma analysis
Biochemical
After 12 h of overnight fasting, plasma analysis were carried out. An enzymic colorimetric kit was used to measure glucose (Bioclin®; Belo Horizonte), TAG (Bioclin®; Belo Horizonte) and NEFA (WAKO®; HR Series NEFA-HR( Reference Sowers 2 )). Spectrophotometry was performed with the Chemistry Analyser BS 200 automatic spectrophotometer (Mindray Medical International Ltd).
Insulin resistance
Insulin resistance was determined using the index of homeostasis model assessment (HOMA-IR) using the following formula( Reference Matthews, Hosker and Rudenski 33 ): HOMA-IR = fasting insulin (μU/ml) × fasting glucose (mmol/l)/22·5.
Hormones and inflammatory cytokines
Plasma levels of insulin, leptin, adiponectin, TNF-α and IL-6 were measured by ELISA. Insulin, leptin and adiponectin ELISA kits were purchased from Millipore Corporation and TNF-α and IL-6 ELISA kits were purchased from R&D Systems. A microplate spectrophotometer reader (SpectraMax 190; Molecular Devices) was used according to the manufacturer's instructions.
Analysis of epididymal adipose tissue
Adipokine levels
Epididymal adipose tissue (400 mg) was triturated with 2 ml of PBS (pH 7·4) and then centrifuged at 3000 rpm and 4°C for 10 min. Using the supernatants, TNF-α and IL-6 were measured using commercial ELISA kits (R&D Systems) according to the manufacturer's instructions. The results were normalised to protein amounts of each sample, quantified by the Bradford method( Reference Bradford 34 ).
Western blotting
The protein concentration of the whole epididymal adipose tissue extract (including the cell membrane, cytoplasm and nucleus) was determined by the Bradford method( Reference Bradford 34 ). Samples (25 µg of protein) were heated in Laemmli buffer at 100°C for 5 min, then loaded onto a 10 % SDS–polyacrylamide gel. Transfer to a nitrocellulose membrane was carried out at 4°C in the presence of methanol. Incubation with the primary antibodies (purchased from Santa Cruz Biotechnology) was performed overnight at 4°C in Tris-buffered saline solution containing Tween 20 (TBS-T) and 3 % non-fat dried milk. Antibody dilutions were: 1:200 for mouse anti-TLR-4 sc293072, 1:200 for mouse anti-β-actin sc47778, 1:100 for rabbit anti-phosphorylated NF-κB (ser536) sc33020, and 1:200 for mouse anti-total NF-κB sc8008. After incubation overnight at 4°C in TBS-T containing 1 % non-fat dried milk with the Abcam secondary antibodies (dilution 1:10 000) anti-rabbit ab97069 and anti-mouse ab98808, protein was revealed using the chemiluminescence method according to the manufacturer's instructions (ECL SuperSignal® West Pico Chemiluminescent Substrate; Thermo Scientific). Band intensities were evaluated using Scion Image Software (Scion Corporation).
Histological analysis
Adipose tissue was fixed in 4 % formaldehyde and embedded in paraffin. Two consecutive sections from each sample were cut (4 µm) and stained with haematoxylin/eosin. The entire slide was scanned using a 3DHISTECH Panoramic MIDI System attached to a Hitachi HV-F22 colour camera and ten fields/slide were analysed under 40× magnification in a blinded manner. The inflammatory reactions are reported as the number of inflammatory cells per high-power field. Using the same slides, the mean area of adipocytes was calculated using a method previously described by Osman et al. in 2013( Reference Osman, Selway and Kępczyńska 35 ).
Statistical analysis
Results are expressed as means and standard deviations. Comparisons among groups were performed using two-way ANOVA for independent groups and were completed using Tukey's post hoc test. SigmaPlot 11.0 software (Systat Software Inc.) was used for statistical analyses. Differences were considered significant at P < 0·05. The statistical power for the main outcome variables was above 80 %.
Results
Body weight and body fat
There was no difference in energy intake between the C and HSF groups at any time (Table 1). The HSF caused changes in the body composition of the animals. At the end of 24 weeks, the HSF group had a greater average weight than the rats in the C24, HSF6 and HSF12 groups. The adiposity index was higher in all HSF groups compared with their respective controls, and in HSF12 and HSF24 compared with HSF6 (Table 1).
Table 1. Nutritional profile of the control diet (C) group and high-sugar/fat diet (HSF) group†
(Mean values and standard deviations, n 8)
C6, control diet for 6 weeks; HSF6, high-sugar/fat diet for 6 weeks; C12, control diet for 12 weeks; HSF12, high-sugar/fat diet for 12 weeks; C24, control diet for 24 weeks; HSF24, high-sugar/fat diet for 24 weeks; IBW, initial body weight; FBW, final body weight.
A,B Mean values within a row with unlike uppercase letters were significantly different among the C groups (6 v. 12 v. 24 weeks) (P < 0·05).
a,b,c Mean values within a row with unlike lowercase letters were significantly different among the HSF groups (6 v. 12 v. 24 weeks) (P < 0·05).
* Mean value was significantly different from that for the C group at the same time point (P < 0·05).
† Comparisons among groups were performed using two-way ANOVA for independent groups and were completed using Tukey's post hoc test.
Plasma biochemical and hormonal measurements
An increase in TAG, glucose, insulin and leptin levels and a decreased level of adiponectin was detected in all animals in the HSF group compared with the C rats. However, when comparing animals subjected to the same diet for different periods of time, only leptin was increased in HSF12 and HSF24 rats compared with the HSF6 group. Animals that received HSF showed insulin resistance only at 24 weeks compared with control animals characterised by increased HOMA-IR (Table 2). There was no difference in NEFA levels among the groups.
Table 2. Plasma biochemical and hormonal measurements†
(Mean values and standard deviations, n 8)
C6, control diet for 6 weeks; HSF6, high-sugar/fat diet for 6 weeks; C12, control diet for 12 weeks; HSF12, high-sugar/fat diet for 12 weeks; C24, control diet for 24 weeks; HSF24, high-sugar/fat diet for 24 weeks; HOMA-IR, homeostasis model assessment.
A,B Mean values within a row with unlike uppercase letters were significantly different among the C groups (6 v. 12 v. 24 weeks) (P < 0·05).
a,b Mean values within a row with unlike lowercase letters were significantly different among the HSF groups (6 v. 12 v. 24 weeks) (P < 0·05).
* Mean value was significantly different from that for the C group at the same time point (P < 0·05).
† Comparisons among groups were performed using two-way ANOVA for independent groups and were completed using Tukey's post hoc test.
Adipose tissue and serum adipokine measurements
No differences were found in the plasma levels of TNF-α and IL-6 when comparing different diets or different durations (data not shown). In adipose tissue, animals in the HSF24 group exhibited elevated levels of these cytokines compared with the C24 group, as well as the HSF6 and HSF12 groups (Fig. 1). However, HSF12 had lower levels of TNF-α and IL-6 compared with C12.
Fig. 1. Cytokine levels (pg/g protein) in epididymal adipose tissue in control diet (C) and high-sugar/fat diet (HSF) groups over 6, 12 and 24 weeks (n 8 animals/group). (a) IL-6 level; (b) TNF-α level. Values are means, with standard deviations represented by vertical bars. * Mean values were significantly different (P < 0·05).
Adipose tissue area
Table 3 shows the mean area of adipocytes in control animals and those fed the HSF in the three periods of the experiment. Note that there was an increase in the area of the adipocytes in animals fed the HSF only at 24 weeks compared with the C24, HSF6 and HSF12 groups. Fig. 2 shows images taken for the assessment of inflammatory cell infiltration. A greater number of cells was observed in the HSF24 group.
Fig. 2. Inflammatory cells in adipose tissue. (a) Control group; (b) group fed high-sugar/fat diet for 6 weeks (HSF6); (c) group fed high-sugar/fat diet for 12 weeks (HSF12); (d) group fed high-sugar/fat diet for 24 weeks (HSF24) (n 8 animals/group). 40× Magnification.
Table 3. Mean area of adipocytes in epididymal adipose tissue of control diet (C) and high-sugar/fat diet (HSF) groups†
(Mean values and standard deviations, n 8)
C6, control diet for 6 weeks; HSF6, high-sugar/fat diet for 6 weeks; C12, control diet for 12 weeks; HSF12, high-sugar/fat diet for 12 weeks; C24, control diet for 24 weeks; HSF24, high-sugar/fat diet for 24 weeks.
A,B Mean values within a column with unlike uppercase letters were significantly different among the C groups (6 v. 12 v. 24 weeks) (P < 0·05).
a,b Mean values within a column with unlike lowercase letters were significantly different among the HSF groups (6 v. 12 v. 24 weeks) (P < 0·05).
* Mean value was significantly different from that for the C group at the same time point (P < 0·05).
† Comparisons among groups were performed using two-way ANOVA for independent groups and were completed using Tukey's post hoc test.
Western blotting
Fig. 3 shows the protein expression of TLR-4 and NF-κB in epididymal adipose tissue. At the end of 24 weeks, the animals in the HSF24 group showed higher TLR-4 expression than those in the C24 and HSF6 groups. Similar to TLR-4, the expression of NF-κB increased in epididymal adipose tissue after 24 weeks in the HSF group when compared with animals in the C24 group.
Fig. 3. Relative expression of protein in epididymal adipose tissue in control diet (C) and high-sugar/fat diet (HSF) groups over 6, 12 and 24 weeks (n 8 animals/group). (a) Toll-like receptor-4 (TLR-4); (b) NF-κB. Values are means, with standard deviations represented by vertical bars. * Mean values were significantly different (P < 0·05).
Discussion
In the present study using Wistar male rats fed an HSF, we evaluated the temporal relationship between the manifestation of metabolic parameters and the effects of inflammation in adipose tissue via TLR-4. According to the WHO, obesity is defined as an excessive accumulation of body fat( 36 ). In 2014, Strissel et al.( Reference Strissel, Denis and Nikolajczyk 37 ) associated obesity with chronic low-grade inflammation, called metainflammation( Reference Ouchi, Parker and Lugus 38 ), which differs from the classic inflammatory response against injury or pathogens( Reference Hotamisligil 39 ). Chronic consumption of an HSF is associated with metabolic changes, since it triggers an increase in body weight and obesity( Reference Hwi, Zhang and Dae 9 ). These factors are associated with high blood pressure, as well as biochemical and hormonal changes, such as increased blood glucose, TAG, NEFA, leptin and insulin, as well as reduced adiponectin( Reference Ye 40 , Reference Soumaya 41 ). However, it is still unclear how the duration of this diet is related to metabolic changes and whether these changes occur before or after inflammation.
HOMA-IR has been used to assess overall insulin sensitivity in human subjects and rats with different degrees of insulin sensitivity( Reference Cacho, Sevillano and de Castro 42 ). Therefore, HOMA-IR is a good predictor of whole-body insulin sensitivity. In our study, the modification of parameters including glucose, TAG, leptin and insulin, as well as low adiponectin, started as early as 6 weeks on the diet. There was also a greater adiposity index observed in these animals. All these conditions persisted up to 24 weeks, even without higher energy intake. This underscores the notion that diet components are an important factor in the manifestation of the MS. The HSF is a combination of palatable foods with high energy density, reflecting the Western dietary pattern. These data corroborate the HSF as a trigger of metabolic and hormonal changes, abdominal circumference, and expansion of fat mass( Reference Sampey, Vanhoose and Winfield 43 , Reference Panchal, Poudyal and Iyer 44 ). More specifically, a higher proportion of SFA (myristic (C14), palmitic (C16) and stearic (C18)) in relation to unsaturated fats (mono- and polyunsaturated) is associated with high adiposity and central fat deposit( Reference Paniagua, Escandell-Morales and Gil-Contreras 45 ). High levels of carbohydrates are also able to mobilise fat from the periphery to central deposits and reduce the activity of adiponectin in peripheral tissues( Reference Paniagua 46 ). When there is an expansion of fat mass, there is also adipocyte hypertrophy, which is responsible for the production of adipokines, including TNF-α and IL-6( Reference Monteiro and Azevedo 47 ). In our work, at 6 and 12 weeks, although the animals presented an increase in the adiposity index, hypertrophy was not present and was only observed at 24 weeks, accompanied by inflammation and increased TNF-α and IL-6 levels. Therefore, the inflammatory state was observed only together with adipocyte hypertrophy, suggesting that adipose tissue is made up of mature and immature adipocytes (low fat content). Under excessive energy supply conditions, immature adipocytes would be responsible for the accumulation of fat, giving the adipose tissue a uniform appearance that still does not characterise hypertrophy.
Two distinct macrophage populations can be identified in adipose tissue: M1 and M2( Reference Chatzigeorgiou and Chavakis 48 ). The M1 profile produces proinflammatory cytokines that affect cell proliferation and promote insulin resistance, while the M2 population is associated with an anti-inflammatory phenotype that protects against metabolic disorders( Reference Kraakman, Murphy and Jandeleit-Dahm 49 , Reference Fjeldborg, Pedersen and Møller 50 ). Lean individuals express a balance in the M1/M2 profile in adipose tissue, while obese individuals initially show an increase in the M2 profile, a defence mechanism to combat possible inflammation( Reference Fjeldborg, Pedersen and Møller 50 ). Our data show that, at 12 weeks, the animals showed a reduction in TNF-α and IL-6, providing evidence for this compensatory response in adipose tissue. This mechanism can occur to reduce inflammation and metabolic deterioration of the tissue. However, at 24 weeks, due to the hypertrophy of adipocytes and increased body fat, a shift may have occurred in the profile of these macrophages towards M1, causing an increase in proinflammatory cytokines. Corroborating this hypothesis, a murine study by Shaul et al.( Reference Shaul, Bennett and Strissel 51 ) also showed an enhanced M2 phenotype in adipose tissue in obese mice after 12 weeks on a high-fat diet compared with mice fed the same diet for 8 weeks.
Increased adiposity is associated with the activation and migration of inflammatory cells into the adipose tissue, as well as proinflammatory cytokine secretion and development of low-grade chronic inflammation( Reference Nishimura, Manabe and Nagasaki 52 ). Besides the hypertrophy of adipose tissue and macrophage profile in tissue, another factor that can enhance this inflammatory condition is the activation of TLR-4 receptors( Reference Cai, Yuan and Frantz 19 ). Our results show an increase in expression of adipocytes at 24 weeks. The literature shows that this activation can occur by an increased release of fatty acids by adipose tissue, by SFA intake( Reference Shaul, Bennett and Strissel 51 ) or by the change in intestinal flora and lipopolysaccharide stimulus( Reference Erridge and Samani 53 ). In the present study, we can attribute the increase in TLR-4 to the dietary fat stimulus, since elevated NEFA in circulation were not found. TLR-4 activates the transcription factor NF-κB, leading to increased production of proinflammatory cytokines( Reference Shi, Kokoeva and Inouye 54 ), reinforcing the importance of this pathway.
In obesity, the degree of inflammation correlates with the extent of insulin resistance, a mechanism involving TNF-α, which interferes with the phosphorylation of the insulin receptor, impairing its glucose uptake function( Reference Schenk, Saberi and Olefsky 55 ). Our work shows that insulin resistance is present, together with the inflammatory response, in adipose tissue after 24 weeks, along with adipocyte hypertrophy. Thus, these data allow us to hypothesise that insulin resistance is influenced by inflammation of the adipose tissue, which in turn is associated with adipocyte hypertrophy and TLR-4 activation. We can conclude that the MS occurs independently of inflammation in adipose tissue and that inflammation is associated with adipocyte hypertrophy, which varies according to the duration of exposure to an HSF.
Final considerations
Although the present study was carried out in rats, the mechanisms related to the development of the MS and inflammation may be similar to those involved in clinical obesity, since after the onset of inflammation in adipose tissue, dependent on adipocyte hypertrophy, the organism may develop new co-morbidities over time, and potentiate the pre-existing ones. The results of the present study are clinically important because they provide information that may promote interventions to prevent adipocyte hypertrophy and inflammation, being a preventive measure in the development of co-morbidities arising from this process.
Acknowledgements
We thank Mario B. Bruno, José Carlos Georgette and Renata Capela for their technical support.
We thank Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP (2011/14132-0, 2011/14593-8, 2015/10626-0) for providing financial support.
The author contributions were as follows: A. F. N. and C. R. C. designed the research; F. V. F., C. B.-R., I. O. M., M. C. D., R. A. M. L. and A. L. A. F. conducted the research; A. F. N. and C. R. C. analysed the data; F. V. F., A. F. N. and C. R. C. wrote the paper.
The authors declare no conflicts of interest.
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