Acute and sustained inflammation and metabolic dysfunction induced by high refined carbohydrate-containing diet in mice

Authors

  • Marina C. Oliveira,

    1. Department of Basic Nursing, Nursing School, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
    2. Imunopharmacology, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Zélia Menezes-Garcia,

    1. Imunopharmacology, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
    2. Department of Microbiology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Milene C. C. Henriques,

    1. Department of Basic Nursing, Nursing School, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
    2. Imunopharmacology, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Frederico M. Soriani,

    1. Imunopharmacology, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Vanessa Pinho,

    1. Imunopharmacology, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
    2. Department of Morphology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Ana M. C. Faria,

    1. Imunopharmacology, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Andrezza F. Santiago,

    1. Imunopharmacology, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Denise C. Cara,

    1. Department of Morphology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Danielle G. Souza,

    1. Imunopharmacology, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
    2. Department of Microbiology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Mauro M. Teixeira,

    1. Imunopharmacology, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Adaliene V. M. Ferreira

    Corresponding author
    1. Imunopharmacology, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
    • Department of Basic Nursing, Nursing School, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
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  • Funding agencies: Financial assistance from Capes, FAPEMIG and CNPq is gratefully acknowledged.

  • Relevant conflicts of interest/financial disclosures: The authors declare no conflict of interest.

Correspondence: Adaliene V. M. Ferreira (adaliene@gmail.com)

Abstract

Objective

The effects of high-refined carbohydrate-containing diet (HC) on inflammatory parameters and metabolic disarrangement of adipose tissue are poorly understood. The aim of this study was to evaluate the timing and progression of metabolic and inflammatory dysfunction induced by HC diet in mice.

Design and Methods

BALB/c mice were fed chow or HC diet for 1 and 3 days, 1, 2, 4, 6, 8, 10, and 12 weeks.

Results

Animals given HC diet exhibited acute and sustained increase in visceral adiposity, glucose intolerance, low insulin sensitivity, hyperlipemia, acute increase in mRNA expression of ACC, LPL, PPARγ, SREBP-1, and ChREBP and altered circulating levels of adiponectin, resistin, and leptin. There was leucocyte rolling and adhesion on adipose tissue microvessels already at 3 days and until 8 weeks of HC diet. Adipose tissue of mice had increased number of macrophages (M1 and M2), lymphocytes (CD8+ and CD4+Foxp3+), and neutrophils (GR1+) already at 3 days after initiation of HC diet. Overall, concentration of cytokines and chemokines, TNF-α, IL-6, IL-10, TGF-β1, CCL2, and CXCL1, in adipose tissue was elevated throughout the experimental period. Levels of IL-10 and TGF-β1 tended to reach baseline levels at 12 weeks of HC diet.

Conclusions

We describe a novel murine model of fat pad expansion induced by HC diet that is characterized by early onset and sustained adipose tissue inflammation and metabolic disarrangement. The acute inflammatory response in adipose tissue occurs very early and is sustained, suggesting that adipose tissue inflammation is a homeostatic mechanism to regulate nutrient overload and adipose expansion.

Introduction

Obesity is increasing at alarming rates throughout the world and is associated with many health complications, including type 2 diabetes, cardiovascular disease, insulin resistance, and metabolic syndrome [1]. The exact pathogenesis of obesity and metabolic disorders remains unclear, but it appears to be a complex combination of many factors that contribute to the development of these syndromes [2]. Recently, numerous studies have shown that dietary factors are important in modulating the development of increased adiposity and metabolic dysfunction [3, 4]. Previous reports have shown the correlation between refined carbohydrates and fat diet consumption and the increased prevalence of obesity and type 2 diabetes in the occident [5, 6]. However, the mechanisms by which specific nutrient influence adiposity development and metabolic dysfunction still need to be better clarified.

Previous reports have shown that animals fed for a long time with high fat diet develop increase in adiposity associated with chronic low grade systemic inflammation [7]. Several reports support the idea that the adipose tissue could be the primary source of proinflammatory factors [7, 8] and an important link between inflammation and metabolic dysfunction [9]. The expansion of visceral fat tissue produces a wide range of pro- and anti-inflammatory mediators that have been linked to the development of insulin resistance and glucose intolerance [9, 10]. At the cellular level, macrophages, lymphocytes, and adipocytes are known to interact and regulate the inflammatory cascade and metabolism [8, 11].

Increased consumption of diet containing fat or refined carbohydrate contributes to obesity and related diseases [12, 13]. Therefore, approaches using different experimental diets should be useful to understand the metabolic and inflammatory impairment induced by increased fat deposition. Although many studies have shown the effect of obesity induced by high fat diet, little is known about the timing and progression of metabolic and inflammatory dysfunction induced by adipose tissue expansion in animals fed high-refined carbohydrate-containing (HC) diets. The present work describes a new mouse model of fat pad expansion induced by HC diet and demonstrates that this diet promotes rapid and sustained increase in adiposity and metabolic disorders associated with inflammation in adipose tissue and systemically.

Methods and Procedures

Animal, diet, and tissue collection

Male BALB/c mice at 5-7 weeks of age were obtained from the animal care center of Universidade Federal de Minas Gerais (CEBIO-UFMG) and kept in an environmentally controlled room under a 14/10 h light-dark cycle. Animals had free access to tap water and food and were maintained according to ethical guidelines of our institution. The experimental protocol was approved by the Animal Ethics Committee of the University (protocol 060/2010). To analyze the time-course of metabolic and inflammatory changes induced by HC diet, animals were fed standard laboratory chow (LABINA) or experimental diet for various periods as indicated in the Results section. The HC diet was composed of 45% condensed milk, 10% refined sugar, and 45% chow diet. The macronutrient composition of the chow diet (4.0 kcal/g) was 65.8% carbohydrate, 3.1% fat, and 31.1% protein; the HC diet (4.4 kcal/g) was 74.2% carbohydrate, 5.8% fat, and 20% protein. It is important to note that HC diet contains at least 30% refined sugars, mostly sucrose.

Mice were collectively housed and weighed once a week. Food intake was measured twice a week for 12 weeks. At the end of the dietary treatment, animals were anesthetized with ketamine (130 mg/kg) and xylazine (0.3 mg/kg) and killed. Samples of blood, epididymal, retroperitoneal, and mesenteric white adipose tissues were collected.

PCR assay

Total RNA was isolated from epididymal adipose tissue by the Trizol method. Real-time PCR was performed on an ABI PRISM 7500 sequence-detection system (Applied Biosystems, Warrington, UK) with SYBR Green PCR Master Mix (Applied Biosystems) after utilizing M-MLV reverse transcriptase (Promega, Madison, WI) for a reverse transcription reaction of 2 μg of RNA. The relative level of gene expression was determined by the 2−ΔΔCt method using mice fed chow diet and was normalized to the ribosomal 18S expression. The following primer pairs, designated as forward (For) and reverse (Rev) for the investigated RNA sequences, were utilized: (i) acetyl-CoA carboxylase (ACC) For, 5′-TCCGCACTGACTGTAACCACAT-3′; ACC Rev, 5′-TGCTCCGCACAGATTCTTCA-3′; (ii) lipoprotein lipase (LPL) For, 5′-AGTCTGGCCTCGAACTAAACTATGTAT-3′; LPL Rev, 5′-TCCCAGGACACAGGAAGCTAA-3′; (iii) peroxisome proliferator-activated receptor gamma (PPARγ) For, 5′-ACAGACAAGATTTGAAAGAAGCGGTGA-3′; PPARγ Rev, 5′-TCCGAAGTTGGTGGGCCAGA-3′; (iv) sterol regulatory element-binding protein-1 (SREBP-1) For, 5′-AAGCAAATCACTGAAGGA CCTGG-3′; SREBP-1 Rev, 5′-AAAGACAAGGGGCTACTCTGGGAG-3′; (v) carbohydrate responsive element-binding protein (ChREBP) For, 5′-GCATCCTCATCCGACCTTTA-3′; ChREBP Rev, 5′-GATGCTTGTGGAAGTGCTGA-3′.

Oral glucose tolerance and insulin sensitivity tests

For oral glucose tolerance test, D-glucose (2 mg/g body weight) was given orally to mice that were fasted overnight and performed after the first week after initiation of diets. Glucose levels were monitored from tail blood samples at 0, 15, 30, 60, and 90 min after glucose overload using an Accu-Check glucometer (Roche Diagnostics, Indianapolis, IN). Insulin sensitivity test was performed after i.p. injection of insulin in overnight-fed mice (0.75 units/kg body weight; Sigma, St. Louis, MO). Tail blood samples were taken at 0, 15, 30, 60, and 120 min after insulin injection for measurement of blood glucose levels.

Adipocyte isolation and “in vitro” lipolysis measurements

Adipocytes were isolated from epididymal fat pads, as described by Rodbell [14]. Briefly, digestion with collagenase (1 mg/ml) was carried out at 37°C with constant shaking (140 cycles/min) for 40 min. Cells were filtered through nylon mesh and washed three times with buffer plus 1% bovine fatty acid free-serum albumin. Lipolysis was measured by following the rate of glycerol release, as previously described [15]. After washing, adipocytes were incubated at 37°C for 20 h for basal and isoproterenol (ISO, 1.0 μmol/L) stimulated lipolysis in the absence or presence of insulin (25 ng/ml). At the end of the incubation period, an aliquot of the infranatant was removed for enzymatic determination of glycerol released into the incubation medium (KATAL, Belo Horizonte, MG, Brazil).

Determination of serum parameters

Fasting glucose, cholesterol, and triglycerides levels were assayed using enzymatic kits (KATAL Belo Horizonte, MG, Brazil). The fasting serum levels of insulin, adiponectin, resistin, and leptin were determined by ELISA (all R&D systems Europe Ltd., Abington, UK). The insulin resistance index assessed by homeostasis model was calculated as follows: HOMA-IR = fasting glucose level (mmol/L) × fasting insulin level (μU/mL) ÷ 22.5.

Histology

Samples from epididymal white adipose tissue were fixed at room temperature in phosphate-buffered formaldehyde solution for 48 h and then incubated in 70% ethanol. Samples were then dehydrated and embedded in paraffin and 7 μm sections of the tissue were stained with hematoxylin-eosin. Images of six fields from each animal were captured using a digital camera coupled to a microscope (200×). The area of 50 cells was measured in each animal using Image Pro-Plus software (Media Cybernetics, USA) and was used ImageJ (National Institutes of Health, Bethesda, Maryland, USA) to calculate mean adipocyte area (μm2).

Total and differential blood cell counts

Blood was collected from the tail vein of mice and total white blood cells counted using a Neubauer chamber. Peripheral blood smears were stained with May-Grünwald-Giemsa and differential white blood cell count determined under oil immersion (1000×) using standard morphologic criteria.

Intravital microscopy in mouse epididymal adipose tissue

Intravital microscopy was performed as previously described [16]. Briefly, mice were anesthetized by i.p. injection of 130 mg/kg ketamine and 0.3 mg/kg xylazine. The epididymal adipose tissue was exposed, and the number of leukocytes rolling and adherent to the vascular wall was evaluated. Leukocytes were fluorescently labeled by i.v. administration of rhodamine 6G (1.5 mg/kg body weight; Sigma, St. Louis, MO) and observed on a fluorescence microscope (Nikon H550L, 20× objective lens). Rolling leukocytes were defined as cells passing through a transverse imaginary line to the vessel for 60 s and moving at a velocity less than that of erythrocytes. Leukocytes were considered adherent to the venular endothelium if they remained stationary for a period of 30 s or longer in a vessel fragment of approximately 100 μm. The two parameters were measured in two or three different vessels and averaged for each animal.

Analysis of adipose tissue-derived stromal vascular cells (AT-SVC) by flow cytometry

AT-SVC were obtained from epididymal adipose tissue of mice fed chow and HC diet during 3 days. The cells were isolated by the method of Rodbell as described above [14]. AT-SVC were washed with DMEM supplemented with 10% FCS, counted, labeled with conjugated antibodies for F4/80, CD11c, CD11b, GR1, CD8, CD4, Foxp3 (BD Pharmingen), and their respective isotype controls. Cells were analyzed with a FACSCalibur, and data were analyzed by FlowJo (TreeStar).

ELISA assay

IL-6, TNF-α, IL-10, TGF-β1, CCL2, and CXCL1 assays were performed using DuoSet ELISA kits and according to the instructions provided by the manufacturer (R&D System, Inc., Minneapolis, USA).

Statistical analysis

Results are expressed as means ± SEM and analyzed using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA). All data were analyzed for normality of distribution using Kolmogorov-Smirnov test and were found to be normal. Comparison between two groups was performed using Student's t test and multiple comparisons performed using one-way ANOVA with Student-Newman-Keuls post-hoc analysis. Statistical significance was set at P < 0.05.

Results

Kinetics of adiposity in animals fed HC diet

On average, body weight gain (Figure 1A), and weakly food intake (Figure 1B) were similar in animals given chow or HC diet at all times evaluated. Furthermore, no differences were found for daily energy intake of animals given chow (18.1 ± 1.8 kcal) and HC diet (19.0 ± 2.0 kcal), P > 0.05. Despite the unchanged food intake and body weight gain, animals given HC diet exhibited considerable increase in visceral adiposity, as evaluated by adiposity index (Figure 1C). Changes in adiposity were already noticeable at day 1 and peaked at 2 weeks after the initiation of the HC diet. Thereafter, the adiposity index was persistently elevated and tended to drop toward the end of the 12 weeks observation period (Figure 1C). The same trend was also observed to the individual weight of epididymal, retroperitoneal, and mesenteric adipose tissue mass. Morphometric analysis of adipocyte area showed a similar pattern of increase (Figure 1D and E), i.e., rapid increase of adipocyte size to peak at the second week after initiation of the HC diet. We examine whether increased adiposity observed in mice fed HC diet is associated with change in the expression of key enzymes and transcription factors involved in lipogenesis and fatty acids storage. High expression of ACC and LPL (Figure 2A and B) were noticeable at 1 day after the initiation of the HC diet and drop toward control levels throughout the observation period. These enzymes are under coordinate transcriptional regulation by PPARγ, SREBP-1, and ChREBP. In accordance, the mRNA levels of these transcription factors increased after one day of HC diet and fall thereafter to baseline levels throughout the observation period (Figure 2C-E).

Figure 1.

Time-course evaluation of body weight gain, food intake, visceral adiposity index, and adipose tissue morphometry of experimental animals. (A) Body weight gain and (B) cumulative food intake of chow and HC-fed mice throughout the experimental period, (C) adiposity index (sum of epididymal, retroperitoneal, and mesenteric adipose tissue mass divided by body weight × 100 expressed as adiposity percent), and (D) adipocyte area of mice given HC diet as compared to chow diet. (E) Representative photomicrographs of hematoxylin–eosin stained epididymal adipose tissue (200×) of mice given chow and HC diet for 1 day, 3 days, 2 weeks, and 12 weeks. Bars indicate 50 μm. The data represents means ± SEM of 6-8 animals per group. *P < 0.05 vs. chow diet.

Figure 2.

The mRNA expression in epididymal adipose tissue of ACC (A), LPL (B), PPARγ (C), SREBP-1 (D), and ChREBP (E) of mice fed chow and HC diet for 1 and 3 days, 2, 8, and 12 weeks. The data represents means ± SEM of 3-4 animals per group. *P < 0.05 vs. chow diet.

Changes in lipid and glucose metabolism and adipokine profile in animals fed HC diet

There were significant changes of total cholesterol, triglycerides, and glucose in serum of animals given HC diet as compared to animals given chow diet (Table 1). Changes in levels of triglycerides and glucose started at day 3 after initiation of diet and were persistent throughout the observation period. Levels of cholesterol were already increased at day 1, persisted at high levels till week 6 and tended to elevate further thereafter.

Table 1. Serum lipids, glucose, adiponectin, resistin, and leptin levels of chow and HC diet-fed mice over time
 CHC diet (days)HC diet (weeks)
13124681012
  1. The data represents means ± SEM of 6 animals per group.
  2. aP < 0.05 vs. chow diet.
Total cholesterol (mg/dL)84.3 ± 2.0103.9 ± 3.2a103.9 ± 4.7a104.2 ± 3.8a108.2 ± 5.7a111.1 ± 6.0a101.9 ± 8.3a121.3 ± 7.3a120.5 ± 13.2a131.5 ± 8.5a
Triglycerides (mg/dL)46.5 ± 2.246.7 ± 6.371.1 ± 6.6a67.0 ± 6.1a66.5 ± 6.6a65.0 ± 9.7a67.7 ± 10.0a67.6 ± 8.4a68.1 ± 6.7a65.4 ± 6.3a
Glucose (mg/dL)144.4 ± 8.2157.9 ± 15.0186.2 ± 14.0a217.8 ± 17.5a216.0 ± 22.7a227.7 ± 8.9a211.4 ± 21.3a185.7 ± 7.1a241.1 ± 14.6a206.6 ± 17.9a
Adiponectin (mcg/mL)3.2 ± 0.13.4 ± 0.33.0 ± 0.13.2 ± 0.12.8 ± 0.31.7 ± 0.2a1.0 ± 0.1a1.0 ± 0.1a0.7 ± 0.04a1.0 ± 0.1a
Resistin (ng/mL)21.0 ± 1.727.2 ± 1.2a30.3 ± 2.2a14.3 ± 1.217.4 ± 0.415.8 ± 2.015.5 ± 0.615.1 ± 0.916.0 ± 2.614.2 ± 0.5
Leptin (ng/mL)3.0 ± 0.34.5 ± 0.4a5.1 ± 0.4a4.0 ± 0.4a4.6 ± 0.5a4.6 ± 0.2a3.9 ± 0.3a4.2 ± 0.4a6.0 ± 0.5a4.4 ± 0.5a

The altered glucose and lipid metabolism was associated with changes in serum levels of adiponectin, resistin, and leptin (Table 1). Serum levels of adiponectin in animals given HC diet were similar to normal chow till the second week after start of the diet. However, from then on, levels of adiponectin fell markedly and reach very low levels from week 6 of HC diet initiation, 32% of control. Serum levels of resistin increased rapidly (days 1 and 3) in mice fed HC diet but normalized to background levels thereafter. Changes in levels of leptin started at day 1 after initiation of HC diet and were persistent increased throughout the observation period.

Glucose tolerance tests were performed at all time points after the first week of the initiation of HC diet. Data for weeks 1, 2, 8, and 12 are presented in Figure 3A-D. Other time points followed a similar pattern and tolerance curves are not shown. Animals given HC diet had significant intolerance to glucose challenge. Indeed, there was an increase of 27, 23, 33, and 24% in the area under the curve in animals given HC when compared to chow diet at weeks 1, 2, 8, and 12, respectively.

Figure 3.

Evaluation of insulin resistance at different time points of increased adiposity induced by HC diet. Oral glucose tolerance test (OGTT) at 1 (A), 2 (B), 8 (C), and 12 (D) weeks and insulin sensitivity test at 8 (E) and 12 (F) weeks of HC diet were measured. (G) Fasting insulin levels and (H) insulin resistance index (HOMA-IR) of mice fed during 3 days and 12 weeks. To evaluate the antilipolytic effect of insulin, the adipocytes were incubated at basal and isoproterenol (ISO)-stimulated lipolysis in the absence (ISO) or presence of insulin (INS). Glycerol release from (I) 3 days and (J) 12 weeks of chow and HC diet feeding. The data represents means ± SEM of 6-8 animals per group. *P < 0.05 vs. chow diet. #P < 0.05 vs. basal chow diet. P < 0.05 vs. ISO chow diet.

Insulin sensitivity tests were performed at weeks 8 and 12 after initiation of HC diet. As it can be seen in Figure 3E and F, animals fed HC diet were less sensitive to insulin than animals given chow diet and there was an increase of 40% and 43% in the area under the curve in animals given HC at weeks 8 and 12 when compared to chow diet, respectively.

The fasting serum level of insulin was similar in animals given HC diet for 3 days or 12 weeks as compared to animals fed chow diet (Figure 3G). However, the HOMA-IR index was higher in mice fed HC diet for 12 weeks (Figure 3H).

Since increased adiposity may change lipolytic events in adipocytes, the influence of HC diet on lipolysis and anti-lipolytic actions of insulin was determined in adipocyte cultures. Adipocytes from mice fed chow or HC diet for 3 days or 12 weeks were analyzed at the basal state and after stimulation with isoproterenol in the presence or absence of insulin. In adipocytes from animals given chow diet, stimulation with isoproterenol enhanced the release glycerol, and this was inhibited by insulin. In contrast, the inhibitory effects of insulin were lost in adipocytes from animals given HC diet for 3 days or 12 weeks (Figure 3I and J). In addition, basal lipolysis was increased in adipocytes from animals fed HC diet for 12 weeks as compared to animals fed chow diet.

Changes in adipose tissue and systemic inflammation in animals fed HC diet

Number of leukocytes in peripheral blood of mice fed chow and HC diet is shown in Table 2. HC diet induced an increase in total number of blood leukocytes that was already detectable at day 1 and was elevated throughout the observation period. It is interesting to note that leukocytes in blood tended to reach normal levels around week 4 after start of HC diet and to increase very significantly thereafter. Overall, increase in total number of leukocytes was secondary to increase in number of both mononuclears and neutrophils cells (Table 2).

Table 2. White blood total and differential cell count (×105/mL) in chow and HC diet-fed mice over time
 CHC diet (days)HC diet (weeks)
13124681012
  1. The data represents means ± SEM of 6–8 animals per group.
  2. aP < 0.05 vs. chow diet.
Leukocytes64.2 ± 4.699.4 ± 3.1a88.8 ± 8.1a146.8 ± 11.0a115.7 ± 16.4a76.8 ± 12.0278.5 ± 18.6a264.0 ± 13.6a174.3 ± 17.9a148.0 ± 11.3a
Mononuclear55.8 ± 4.289.3 ± 3.5a83.7 ± 7.9a136.2 ± 10.1a98.9 ± 15.8a70.6 ± 10.7196.2 ± 11.1a227.0 ± 12.4a148.7 ± 14.7a113.8 ± 8.0a
Neutrophil7.1 ± 0.910.5 ± 1.34.1 ± 0.910.6 ± 2.718.5 ± 3.2a5.7 ± 1.265.5 ± 10.5a37.0 ± 4.4a29.6 ± 3.5a35.6 ± 4.3a

To visualize leukocyte-endothelial cell interactions in the microcirculation of adipose tissue in living mice, these events were followed in vivo using intravital microscopy (Figure 4). There was increased rolling and adhesion of leukocytes to the microcirculation, which was observed from day 3 after start of diet. Akin to what was observed in the number of blood leukocytes, adhesive events tended to fall around weeks 2 and 4 and increase significantly thereafter (Figure 4A and B). It was not technically possible to perform intravital microscopy in animals given HC diet for 10 or 12 weeks as it was difficult to find vessels with usual blood flow. This is similar to findings from a previous study showing slow blood flow in animals with high tissue fat content [17].

Figure 4.

Visualization of leukocyte-endothelium interaction during different times of adiposity induction by HC diet. Intravital microscopy was used to assess the (A) rolling and (B) adhesion of leukocytes in epididymal adipose tissue vessels in vivo. The data represents means ± SEM of six animals per group. *P < 0.05 vs. chow diet. Flow cytometry analysis of derived stromal vascular cells from epididymal adipose tissue at 3 days in chow and HC diet feeding mice for the expression of (C) macrophages (F4/80), M1 macrophages (F4/80+CD11b+CD11c+), and M2 macrophages (F4/80+CD11b+CD11c-), (D) neutrophil (GR1+), and (E) T (CD8+) and regulatory T cells (CD4+Foxp3+). The data represents means ± SEM of 7-8 animals per group. *P < 0.05 vs. chow diet.

To gain better insight into the inflammatory cell population of adipose tissue of animals fed HC diet at short time we performed the flow cytometry analyses in collagenase-digested stromal vascular fraction 3 days after diet. The number of leukocytes expressing F4/80 (Mϕ), F4/80+CD11b+CD11c+ (M1) and F4/80+CD11b+CD11c− (M2) was increased in animals fed HC diet in relation to animals fed chow diet (Figure 4C). Number of neutrophils (GR1+), cytotoxic T (CD8+), and regulatory (CD4+ Foxp3+) lymphocytes population also increased in animals fed HC diet (Figure 4D and E).

To evaluate whether HC diet could influence the production of cytokines, levels of TNF-α, IL-6, IL-10, TGF-β1, CCL2, and CXCL1 in adipose tissue were measured by ELISA (Figure 5). Concentrations of TNF-α and IL-6 increased significantly in adipose tissue of mice fed HC diet throughout the experimental period, with the exception of the second week after start of treatment (Figure 5A and B). The latter observation is consistent with the decreased rolling and adhesion of leucocytes on adipose microvessels at week 2 (Figure 4A and B). The concentrations of IL-10 and TGF-β1 in adipose tissue increased continuously till the second week of the start of HC diet and tended to fall thereafter to baseline levels at week 12 (Figure 5C and D). Concentrations of the macrophage-active chemokine CCL2 increased from day 1 after the start of HC diet and peaked at week 1. Thereafter levels fell very significantly but were still above baseline levels in animals given HC diet at all time points (Figure 5E). The adipose CXCL1 levels, a cytokine with neutrophil chemoattractant activity, were transiently increased in animals fed HC diet throughout the experimental period. The higher levels of CXCL-1 were at 8-12 week of HC diet feeding (Figure 5F).

Figure 5.

Kinetics of cytokines (A) TNF-α, (B) IL-6, (C) IL-10, (D) TGF-β1, and chemokines (E) CCL2 and (F) CXCL1 production in epididymal adipose tissue in chow and HC diet-fed mice over time. The data represents means ± SEM of 6-8 animals per group. *P < 0.05 vs. chow diet.

Discussion

Recent studies have shown that expansion of adipose tissue leads to chronic low-grade inflammation that contributes to the development of metabolic disorders, including dyslipidemia, insulin resistance, and type 2 diabetes [1]. The present study has the following major findings: (i) a high-refined carbohydrate-containing diet induced rapid and sustained fat tissue expansion without weight gain; (ii) the rapid fat accumulation is related to the high mRNA levels of key enzymes and transcription factors involved in lipogenesis and fatty acid storage; (iii) adiposity was associated with marked metabolic dysfunction, as demonstrated by hyperglycemia, hyperlipidemia, glucose intolerance, insulin resistance, and altered circulating levels of adipokines; (iv) adiposity was also associated with rapid and sustained adipose tissue inflammation with increased concentration of cytokines and leukocyte interactions with the microvasculature.

The very acute effects of high-refined carbohydrate-containing diet on adiposity was surprising since animals fed for just 1 or 3 days presented strikingly increased fat mass, as seen in visceral fat pads, metabolic alterations in glucose, and lipid metabolism and in immune cell response. Long-term feeding with high-refined carbohydrate-containing diet (for 12 weeks) maintained high adiposity and metabolic and inflammatory disarrangement in animals. Most experimental models of obesity use high-fat diet feeding for long period of time [11, 18] while studies using either high-carbohydrate or high fat diet for a short-time analysis are scarce [19].

The increased adiposity induced by chronic high carbohydrate-containing diet feeding consisting of refined sugar is thought to be due to the high glycemic index of this type of diet, which in turn increases postprandial insulin levels and favors insulin-stimulated glucose oxidation and incorporation into total lipids, resulting in increased fat pads [20]. In addition to the hyperinsulinemia-induced lipogenesis in animals fed high-refined carbohydrate diet, increased enzymes and transcription factors involved in lipid metabolism may be contributing to short-term HC diet in fat accumulation. Previous studies have shown that dietary nutrients are able to regulate metabolic fluxes and homeostasis through gene expression control [21], and this modulation could happen with short-term diet feeding. Indeed, in this study, mice fed HC diet presented a rapid increase in the mRNA expression of key enzymes (ACC and LPL) and transcription factors (PPARγ, ChREBP, and SREBP-1) involved in lipogenesis and fat storage. Lin et al. [22] found that 1 and 2 days of feeding high-fat diet caused an increase in transcriptional profile in genes involved in hepatic lipogenesis. Dietary composition may also influence differential gene expression that could contribute to gene modulation induced by different diet components. Our data have shown that the adipose tissue is readily responsive to dietary carbohydrates, a similar pattern observed to the effect of HC diet on liver response [23]. In respect to the long-term HC diet feeding, as presented by Shankar et al. [24], a wide range of other genes involved in glycolysis, pentose phosphate pathways, fatty acid and triglycerides biosynthesis, and lipolysis could be related to maintenance of adipose tissue fat mass.

The adipose tissue consists not only of adipocytes but also of stromal and vascular cells, including fibroblasts, vascular endothelial cells, and inflammatory cells [25]. The stromal vascular fraction is known to be essential for adipose tissue inflammation, and it is well known that inflammatory cell population change during the development of obesity [25, 26]. A previous report [27] has shown that leukocyte-endothelial cell-platelet interactions are apparently selectively enhanced in obese visceral adipose tissue in ob/ob and in 4 weeks high fat diet-induced obese mice. We performed in vivo intravital microscopy to verify leukocyte rolling and adhesion induced by high-refined carbohydrate-containing diet in the epididymal microvasculature. There was an increase in rolling and adhesion of leukocytes already 3 days after the start of HC diet. There were even higher numbers of leukocyte-endothelial cell interactions in animals given HC diet for chronic periods (8 weeks). A detailed analysis of leukocytes present at 3 days after initiation of HC diet shows that there was an increase in macrophage (M1 and M2), neutrophil, and lymphocytes in adipose tissue. An increase in number of macrophages and lymphocytes in adipose tissue has been previously demonstrated in animals fed high-fat diet at long-term [8, 11] and, more recently, in animals fed HF diet for shorter periods [19].

The macrophage content of adipose tissue is correlated with adipocyte size and macrophages are the primary source of TNF-α and other proinflammatory molecules in adipose tissue [28]. It has been suggested that the polarization of adipose tissue macrophage into a proinflammatory phenotype, often referred to as M1-polarized macrophage that gradually replace M2-polarized cells, is an important link between adipose tissue inflammation and glucose intolerance [29]. We observed that, after short periods of HC diet, there was an increase in both M1 and M2 macrophage populations. Concurrently, increased numbers of T reg lymphocytes were also present indicating a counter-regulatory mechanism to suppress the acute increase in inflammation induced by HC diet. Indeed, the high concentrations of IL-10 and TGF-β1 in adipose tissue-derived stromal vascular cells of mice given HC diet during 3 days could be related to a high content of T reg lymphocytes in this tissue. Feuerer et al. [30] demonstrated that cytokines differentially synthesize by fat-resident regulatory and conventional T cells directly affected the synthesis of inflammatory mediators. In fact, T reg cells produce large amounts of IL-10 and also seem to respond to it since a number of genes downstream of the IL-10 receptor are upregulated in fat T reg cells. The anti-inflammatory effect of TGF-β1 also induces a regulatory signaling pathway that increases the Foxp3 expression and, consequently, promotes the generation and expansion of T reg cells [31]. Therefore, changes in immune cell phenotype in adipose tissue seem to curb overactive immune responses induced by acute nutrient overload.

In a previous report [32], it was shown that circulating neutrophils are activated in obese people, suggesting that infiltrating neutrophils into adipose tissue may actively enhance the release of many substances capable of causing cellular damage and dysfunction. A transient acute neutrophil infiltrate (3-7 days) has been shown in animals fed high-fat diet [33]. The rapid influx of macrophages and neutrophils into adipose tissue could contribute to, at least in part, the early development of metabolic dysfunction observed in animals fed HC diet. In addition to this, it seems possible that the acute inflammation induced by nutrient overload would trigger the classic inflammatory response induced by infection or injury; albeit at a lower magnitude.

The altered production of pro- and anti-inflammatory molecules by adipose tissue has been implicated in metabolic disarrangements induced by fat pad expansion that includes the development of insulin resistance and consequently glucose intolerance [34]. Our study shows that the early increase of local and systemic inflammatory processes induced by HC diet is associated with increase in serum lipid levels, glucose intolerance, and insulin resistance. Our work, in agreement with others, supports the notion that an imbalance of pro- and anti-inflammatory cytokine secreted by adipose tissue favors the development of metabolic dysfunction [35]. We found that animals fed HC diet for short periods showed moderate increase in adipose tissue cytokines TNF-α and IL-6, and the chemokine CCL2. At this early time, there was concomitant significant increase in anti-inflammatory cytokines IL-10 and TGF-β1. The increased levels of IL-10 in animals fed HC diet could, in turn, contribute to attenuate the proinflammatory cytokine profile observed in animals fed HC diet for 8-10 weeks. Since IL-10 and TGF-β1 levels dropped in adipose tissue, the proinflammatory cytokines IL-6 and TNF-α increased. It could imply a role for IL-10 and TGF-β as homeostatic regulators of adipose inflammation and consequently insulin sensitivity in this animal model of fat pad expansion.

Another mechanism involved in insulin resistance induced by obesity is the ability of chronic inflammation to affect the capability of adipocytes to store triglycerides. In this case, many cytokines such as TNF-α and IL-6 are able to increase adipocyte lipolysis, which in turn increases free fatty acid circulation and contributes to the overall insulin resistance state [34, 36]. After performing adipocyte lipolysis tests at 3 days and 12 weeks of HC diet we found that, in short-term as well as long-term HC diet, insulin was incapable of decreasing lipolysis induced by the β-agonist, isoproterenol. In addition, the high basal lipolysis seen in the chronic stage could contribute to a paracrine loop involving free fatty acid and inflammatory cytokines that in turn may feed a vicious circle aggravating inflammation and adipocyte dysfunction.

Several clinical and experimental observations support an association between adiponectin and resistin levels and obesity linked metabolic dysfunction [35]. Studies performed in obese people and animal models have shown that low adiponectin levels are associated with reduced insulin sensitivity and a chronic inflammation [37, 38]. Our data showed that long-term HC diet feeding is able to decrease serum levels of adiponectin, which could contribute to adipocyte dysfunction and induction of chronic metabolic disorders observed in our study. Unlike adiponectin, resistin is described to be elevated in obese people [39]. Previous report for animals fed high fat diet have shown that high resistin levels are associated with proinflammatory properties and impaired insulin sensitivity [40]. In the present work, HC diet caused a rapid and transient augmentation in resistin levels that could contribute to acute metabolic dysfunction. In our model, chronic metabolic dysfunction was not accompanied by changes in resistin levels.

Our study supports the idea that a HC diet consisting of refined sugar induces rapid fat tissue expansion which is associated with leukocyte influx into adipose tissue vessels. Infiltration of macrophages, neutrophils and lymphocytes in fat tissue could then contribute to increase cytokines level, such as IL-6, TNF-α, IL-10, TGF-β1, CCL2, and CXCL1. Increased cytokines levels in adipose tissue along with high serum resistin levels could contribute to insulin resistance and metabolic dysfunction in short-term HC diet fed mice. Long-term HC diet intake may establish systemic and local chronic inflammation that could be maintained by lower adiponectin and anti-inflammatory cytokines levels, such as IL-10 and TGF-β1, in serum and adipose tissue, respectively. The altered immune response appears to contribute to the metabolic disarrangement observed in obesity induced by HC diet. Overall, this study presents a new mouse model of fat pad expansion induced by HC diet that has rapid and sustained adipose tissue inflammation and metabolic disorders. The acute inflammatory response in adipose tissue occurs very early and is sustained, suggesting that adipose tissue inflammation is a homeostatic mechanism to regulate nutrient overload and adipose expansion. Defining the molecular mechanisms by which nutrient overload is sensed and causes leukocyte recruitment into adipose tissue should provide novel avenues to understand obesity and its systemic consequences.

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