Involvement of peroxisome proliferator-activated receptors in cardiac and vascular remodeling in a novel minipig model of insulin resistance and atherosclerosis induced by consumption of a high-fat/cholesterol diet
© Yongming et al.; licensee BioMed Central. 2015
Received: 20 October 2014
Accepted: 27 December 2014
Published: 16 January 2015
A long-term high-fat/cholesterol (HFC) diet leads to insulin resistance (IR), which is associated with inflammation, atherosclerosis (AS), cardiac sympathovagal imbalance, and cardiac dysfunction. Peroxisome proliferator-activated receptors (PPARs) and nuclear factor ĸB (NF-κB) are involved in the development of IR-AS. Thus, we elucidated the pathological molecular mechanism of IR-AS by feeding an HFC diet to Tibetan minipigs to induce IR and AS.
Male Tibetan minipigs were fed either a normal diet or an HFC diet for 24 weeks. Thereafter, the minipigs were tested for physiological and biochemical blood indices, blood pressure, cardiac function, glucose tolerance, heart rate variability (HRV), and PPAR-associated gene and protein expression levels.
HFC-fed minipigs exhibited IR through increased body weight, fasting blood glucose levels, plasma cholesterol and its composition, and insulin and free fatty acid (FFA) levels; decreased insulin sensitivity; impaired glucose tolerance; and hypertension. Increased C-reactive protein (CRP) levels, cardiac dysfunction, depressed HRV, and the up-regulation of PPAR expression in the abdominal aorta concomitant with down-regulation in the heart tissue were observed in HFC-fed minipigs. Furthermore, the levels of NF-κBp65, IL-1β, TNF-α, MCP-1, VCAM-1, ICAM-1, MMP-9, and CRP proteins were also significantly increased.
These data suggest that HFC-fed Tibetan minipigs develop IR and AS and that PPARs are involved in cardiovascular remodeling and impaired function.
KeywordsTibetan minipig Insulin resistance Atherosclerosis Myocardial ischemia PPARs NF-ĸB High-fat/cholesterol diet Heart rate variability
Hyperlipidemia is a common predicament in modern society owing to changes in lifestyle and food consumption in humans. Diet plays an important role in the management of lipoprotein and lipid concentrations in blood. Long-term, high-fat consumption leads to insulin resistance (IR), because saturated fatty acids interfere with the action of insulin .
IR is a precursor and the primary characteristic of type 2 diabetes. Based on an epidemiological study, the incidence of diabetes is rapidly increasing with the population aging, and approximately 9.7% of adults have diabetes in China . Moreover, the mortality of heart disease is 4-fold higher in diabetes patients . In fact, myocardial ischemia (MI), caused by atherosclerosis (AS) of coronary arteries, more frequently occurs without prior symptoms in these patients . Therefore, the pre-diabetic pathological state of IR is concerning.
IR has been implicated in AS promotion and impaired endothelial-dependent diastolic function . Data from patients with IR or elevated fasting blood glucose, increased heart rate, reduced heart rate variability (HRV) [6,7], and cardiac sympathovagal imbalance  and data from animal experimental and clinical studies all confirm that IR is associated with impairment of selective signaling pathways and that IR leads to peripheral vascular and myocardial structural and functional changes [9-11]. Therefore, it is necessary to establish an ideal animal model to have a better understanding of the pathological process involved in IR, hyperlipidemia, and AS, which will be helpful to further develop new therapeutic agents.
Pigs are a potentially useful animal model because, unlike mice and rabbits, their anatomy, physiology, feeding and sleeping habits, neo-intimal formation, and thrombosis are very similar to those of humans. Pigs can recapitulate the formation of diabetes- and inflammation-induced AS lesions [12,13], which are currently, mostly induced by toxicity to drugs (e.g.,streptozotocin and alloxan), causing pancreatic β-cell ablation, and AS lesions are also induced by feeding with an atherogenic diet [14,15], rather than natural development subsequent to metabolic syndrome or diabetes. In a recent study, Göttingen minipig fed a high-fat diet for 12 weeks develops obesity, IR, and lipid disorders , and the obesity-prone Ossabaw minipig fed a high-fat/cholesterol (HFC) diet for 9 weeks develops IR, impaired glucose tolerance, dyslipidemia, hypertension, and early coronary intimal hyperplasia . However, these models do not exhibit chronic ischemia formation. Many minipig varieties in China are used in experimental studies, such as Bama minipig, WZS minipig, Guizhou minipig, and Tibetan minipig. In our previous studies using the Chinese minipig model of cardiovascular diseases, we found that HFC-fed Tibetan minipigs could also develop IR, impaired glucose tolerance, dyslipidemia, hypertension, AS, and cardiac dysfunction . These conditions are very similar to IR caused by related complications in clinical cases; thus, this disease model is a novel minipig model of IR-AS and warrants further study.
Peroxisome proliferator-activated receptors (PPARs) belong to a superfamily of the type II nuclear hormone receptors, are ligand-activated transcription factors, which have three subtypes (PPAR α, β/δ, and γ), and may participate in lipid metabolism regulation and AS development . PPARs also inhibit the activity of inflammation-related transcription factors (such as NF-ĸB), which inhibit inflammatory effects of a variety of inflammatory diseases. PPARs are gaining research interest with regard to metabolic and cardiovascular diseases, and PPAR expression in the target organ may play an important role in changing organ structure and function; moreover, PPAR subtypes function in cardiovascular diseases and may also influence lesion development. However, the specific active mechanism of PPARs in cardiovascular remodeling and dysfunction is unclear.
Therefore, we hypothesized that IR-AS with spontaneous chronic ischemia in HFC-fed Tibetan minipig maybe associated with inflammation, IR, autonomic dysfunction, and activation of PPARs and the NF-ĸB signaling pathway. In this work, we successfully recapitulated the IR-AS Tibetan minipig model induced by an HFC diet. We examined blood biochemical indices, blood pressure, cardiac function, glucose tolerance, HRV, and expression levels of genes and proteins associated with PPARs and the NF-ĸB signaling pathway. We aimed to elucidate whether HFC-fed Tibetan minipig developed IR-AS and to understand the cause of cardiac impairment and pathological molecular mechanisms. Consequently, our results provide an experimental basis for the relationship between IR and cardiovascular diseases and drug treatment.
Blood biochemical index measurements
At 24 weeks, blood samples were drawn from minipigs in a supine position by performing anterior vena cava puncture within 30 s after a fasting period of 12 h . Blood samples were then separated to analyze total cholesterol(TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and fasting blood glucose (FBG), which were measured by an automatic biochemical analyzer (7020, HITACHI, Japan) using kits corresponding to each component (Shanghai Shenneng-DiaSys Diagnostic Technology Co., Ltd., China) for each test. The serum free fatty acid (FFA), insulin, and C-reactive protein (CRP) levels were measured using homologous specific ELISA kits (Shanghai Xinran Biotech Co, Ltd., China) according to the manufacturer’s instructions. The homeostasis model assessment IR (HOMA-IR) method was used to evaluate insulin resistance. HOMA-IR was calculated using the following formula: FBG × insulin/22.5 .
Intravenous glucose tolerance test (IVGTT)
The IVGTT was performed in each minipig after 8 and 24 weeks of assigned diet consumption. Minipigs were acclimated to restraint in a specialized fixed sling for 3 days before IVGTT. They were anesthetized with isoflurane (maintained at 2–3% by mask with supplemental O2), and then right jugular veins were catheterized percutaneously . Minipigs were allowed to recover for 3 h before IVGTT to avoid any effects of isoflurane on glucose levels . Blood samples were obtained at the baseline time point (0 min), then glucose (0.5 g/kg) was injected intravenously over 2 min, and further samples were obtained at 15, 30, 60, 90, and 120 min after injection for measurement of blood glucose and insulin concentrations. From the IVGTT, the insulin sensitivity index (S2) for minipigs was calculated as previously described .
Blood pressure and cardiac function measurement
Minipigs were acclimated to a low-stress sling for blood pressure measurements. Non-invasive blood pressure was monitored using a non-invasive physiological signal telemetry system (EMKA Technologies S.A.S., France) by using a tail cuff method at the beginning of the experiment and 12 weeks into the experiment. Mean blood pressure (MBP), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were analyzed by ECGAUTO software (EMKA Technologies). After 24 weeks, all minipigs were anesthetized with 10 mg/kg of intramuscular xylazine hydrochloride (Huamu Animal Health Products Co., Ltd., Jilin, China) and intubated, and anesthesia was maintained by inhalation of 0.5–2% isoflurane combined with oxygen and room air . During anesthesis, minipigs were placed on heating pads maintained at 37°C, and the core temperature was measured via a rectal probe (Life Window 6000; Digicare Animal Health, Boynton Beach, FL, USA). All minipigs were equipped with two polyethylene blood vessel catheters. One catheter was inserted into the left ventricle via the right common carotid artery to record cardiac function parameters, and another catheter was inserted into the left femoral artery to record blood pressure parameters. After stabilizing for 20 min (i.e., after intramuscular injection of xylazine hydrochloride) at 80 min, when the effects of xylazine hydrochloride on blood pressure are negligible , the signals were recorded continuously by using a non-invasive physiological signal telemetry system (EMKA Technologies). MBP, SBP, DBP, left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and the maximum rate of left ventricular pressure rise and fall (±dp/dtmax) were analyzed by ECGAUTO software (EMKA Technologies).
The physiological telemetry test was performed for each minipig every 8 weeks. All minipigs were acclimatized to the experimental jackets for 3 days before each test. Minipigs were shaved, and surface ECG electrodes were attached. Then, specially designed jackets were placed on each minipig to protect the ECG electrodes and lead wires, and minipigs were returned to their cages. After stabilization for 2 h, vital signs of each animal (Lead I and II ECG) were monitored for 24 h and recorded using a Non-Invasive Physiological Signal Telemetry System (EMKA Technologies). ECG signals were recorded at a sampling rate of 500 Hz by EMKA IOX data acquisition software. ECG-AUTO software (EMKA Technologies) was used for ECG, activity, and HRV analyses. Data from the 24 h sampling period were divided into 30-minsegments of RR data. R waves were detected, and the RR interval (RRI) tachogram was calculated as raw HRV in sequence order. Similarly, from the same recordings, HRV was analyzed for time and frequency domains. The time-domain parameters obtained were as follows: mean of normal RRIs, standard deviation of all normal RRIs (SDNN), and square root of the mean square successive differences between successive normal intervals (rMSSD). From this tachogram, data sets of 1024 points were re-sampled at 286 ms. A Hamming window was applied to each data set to minimize spectral leakage. A fast Fourier transform was used to obtain the power spectrum of the fluctuation. We identified frequency bands of very low frequency (VLF; 0.003–0.01 Hz), low frequency (LF; 0.01–0.07 Hz), and high frequency (HF; 0.07–1.0 Hz) according to a previous study ; the sympathetic and vagal modulation ratio balance was defined by the LF/HF ratio.
The percentage of carcass fat was calculated according to the following equation: carcass fat weight/body weight × 100%. Body mass index was measured as in humans  and was calculated by the following equation: (body weight in kg)/(square of the minipig length from the end of the snout to the base of the tail).
Myocardium ischemia size (MIS) determination
MIS was assessed by nitrotetrazolium blue chloride (NBT; Sinopharm Chemical Reagent Co., Ltd., China) staining, as described previously . Briefly, at 24 weeks, the heart was excised, washed in chilled saline, weighed immediately, and then dissected to remove the atria and right ventricle. The left ventricle was frozen at − 20°C, cut in a semi-frozen state into 5 equally thick sections, and incubated in 0.1% NBT solution for 15 min at 37°C. The non-ischemia area stained blue, whereas the ischemia area was not stained. The area of ischemia was measured in each slice with Image J software (version 1.4, National Institutes of Health, USA) and was expressed as the percentage of the total left ventricular area.
Upon anesthesis at 24 weeks, the chest of each minipig was opened to achieve euthanasia. Samples of the aorta, arteries, heart, pancreas, liver, and adipose tissues were removed from each animal. The thoracic and abdominal portions of the aortas were longitudinally incised and fixed with 10% buffered formalin for 24 h, followed by staining with Oil Red O stain for lipid deposition analysis. The Oil Red O-stained area relative to the entire surface was calculated using Image J software. The heart, pancreas, abdominal aorta, and left branch of the coronary artery were fixed overnight with 10% phosphate-buffered formalin, dehydrated, embedded in paraffin, sectioned into 5-μm-thick slices, and stained with hematoxylin and eosin (H&E). The intima-media thickness (IMT) was measured at the point of greatest thickness in sections from the standardized sample sites by using Image J software.
RNA extractions and quantitative real-time PCR (qPCR)
Total RNA was extracted from the abdominal aorta and myocardium tissue using TAKARA RNAiso Plus Reagent according to the instructions. For each sample, first-strand cDNA was synthesized using the Prime Script RT Reagent Kit (TaKaRa, Dalian, China). The sequences of the primers were as follows: porcine GAPDH sense, 5′-AGGTCGGAGTGAACGGATTTG-3′, GAPDH anti-sense 5′-ACCATGTAGTGGAGGTCAATGAAG-3′; PPAR-α sense, 5′-GCTGCTCAGCACCAAGGT-3′, PPAR-α anti-sense, 5′-ACCTCCTTCATGCTCTCCTCTAT-3′; PPAR-β/δ sense 5′-CTACAAGTGCCAGTGTGAGGAG-3′, PPAR-β/δ anti-sense 5′-GGTTGGTGAAGAAGAGGTAGGC-3′; and PPAR-γ sense 5′-GACGCAGATTTTGTAGACG-3′, PPAR-γ anti-sense 5′-CCCTACTGGTTTCTGGATG-3′. The expected sizes of synthesized cDNA were 125 bp for GAPDH, 115 bp for PPAR-α, 95 bp for PPAR-β/δ, and 81 bp for PPAR-γ. qPCR was performed in an automated thermal cycler (Minioption™ Real-time PCR System; BIO-RAD) in a final volume of 25 μL, containing 2 μL of cDNA solution, 12.5 μL of SYBR® Premix Ex Taq (TaKaRa, Dalian, China), 1 μL of each primer (10 μmol/L), and 8.5 μL of dH2O. The cycling reaction was performed according to the manufacturer’s instructions via a standard two-step PCR. GAPDH was used as a housekeeping gene to normalize target gene expression, and the expression levels were determined by the comparative CT method.
Abdominal aorta and myocardial protein extraction and western blotting analysis
Abdominal aorta and myocardial proteins were extracted according to the method provided by the manufacturer of the extraction kit (Key Gen Bio TECH, Nanjing, China).The concentrations of total protein in the supernatant were determined by the BCA method, and then 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol were added to denature proteins at 100°C for 10 min. Fifty micrograms of protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidenedifluoride membranes. The membranes were blocked with 5% non-fat milk in 20 mmol/L Tris-buffered saline (pH 7.4) containing 500 mmol/L NaCl and 0.05% Tween 20 (TBST) and then incubated with primary antibody (1:150 dilution, PPAR-α, SC-1982; PPAR-γ, SC-1981; and GAPDH, SC-166545: Santa Cruz Biotechnology, USA; PPAR-β/δ, ab23673: Abcam, USA) overnight at 4°C. Thereafter, the membranes were washed three times with TBST and incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Membranes were again washed three times in TBST. Finally, signals were detected using the Odyssey Infrared Fluorescent Scanner (LI-COR, USA) and quantified by Image J software.
Fresh abdominal aortas were sliced into 7-μm-thick frozen sections, blocked with 3% hydrogen peroxide solution for 10 min to eliminate the activity of endogenous peroxidase, and then incubated overnight at 4°C in the following polyclonal antibody solutions: anti-NF-ĸB p65, anti-interleukin 1 beta (IL-1β), anti-tumor necrosis factor alpha (TNF-α), anti-monocyte chemoattractant protein-1 (MCP-1), anti-vascular cell adhesion molecule 1 (VCAM-1), anti-intercellular adhesion molecule 1 (ICAM-1), anti-matrix metalloproteinase 9 (MMP-9), and anti-CRP (Zhongshan GoldenBridge, Beijing, China). Subsequently, samples were washed with phosphate- buffered saline three times, and the sections were incubated with secondary antibodies for 1 h. After incubation, the sections were stained using 3, 3′-diaminobenzidine tetrahydrochloride (DAB) as a color reagent and counterstained with hematoxylin. The stained slides were viewed with a light microscope (Nikon Eclipse 80i), and digital images of six randomly selected, high-power (×400 magnification) fields were captured on NIS- ElementS.F.2.30 software (Nikon United Kingdom Ltd., Surrey, UK). Protein expression was quantified with Image Pro Plus 5.0 software. This software discriminates between positive- and negative-stained pixels. A visual check was also performed to ensure accurate discrimination of immunolabeled regions. Positive expression is represented as the percentage of the total area.
Data are expressed as means ± SEM. SPSS 17.0 software (SPSS, Chicago, IL, USA) was used to perform statistical analysis. Data were compared using unpaired student’s t tests. A two-tailed P value <0.05 was considered statistically significant.
General characteristics of Ctr and HFC-fed Tibetan minipigs
Phenotypic characteristics of Tibetan minipigs fed a control (Ctr) or high-fat/cholesterol atherogenic (HFC) diet
Starting body weight (kg)
16.73 ± 0.72
16.30 ± 0.54
End body weight (kg)
33.43 ± 1.29
49.40 ± 1.07b
End body mass index (kg/m2)
41.98 ± 1.35
57.05 ± 1.93b
Total cholesterol (mg/dL)
96.08 ± 7.96
406.60 ± 42.26b
33.77 ± 3.42
120.60 ± 5.34b
42.76 ± 5.55
186.30 ± 21.93b
23.61 ± 2.31
25.38 ± 3.69
Free fatty acids (nmol/L)
0.51 ± 0.01
0.61 ± 0.01b
Fasting blood glucose (mmol/L)
4.64 ± 0.10
5.47 ± 0.20b
Fasting insulin (mU/L)
18.05 ± 0.74
21.61 ± 0.49b
3.71 ± 0.09
5.24 ± 0.17b
Up-regulation of PPAR gene and protein expression in the abdominal aorta of HFC-fed Tibetan minipigs
Down-regulation of PPAR gene and protein expression in the heart of HFC-fed Tibetan minipigs
NF-κB pathway activation and up-regulation of inflammatory cytokines in the abdominal aorta of HFC-fed Tibetan minipigs
In the current study, we aimed to establish a Tibetan minipig model of IR-AS by feeding an HFC diet, which resulted in significant weight gain, dyslipidemia, glucose intolerance, hypertension, inflammation, and activated the PPARs and NF-ĸB signaling pathways. These abnormalities are known to develop in IR and AS; thus, the Tibetan minipig model may be helpful to further understanding of human IR and cardiovascular disease development.
Other investigators have induced IR in pig models by using high-energy diets enriched with cholesterol and fat, but IR reportedly develops at different times, e.g., 2 weeks  and 16 weeks [31,32], which may be related to composition of diet and pig strains. A recent report stated that the IVGTT-derived S2 is more sensitive to evaluate insulin sensitivity, which has been shown to correlate with clamp insulin sensitivity in pigs . Interestingly, we also found that 8 weeks of feeding an HFC diet were sufficient to induce glucose intolerance and IR in Tibetan minipigs.
Additionally, HFC-fed Tibetan minipigs had significantly increased plasma FFA concentration and blood pressure and depressed HRV with increased sympathetic activities. Wang et al.  also reported that a high-fat/ high-carbohydrate diet fed to pigs could induce artery IR, vascular dysfunction, and hypertension. Accumulating evidence links elevated plasma FFA with IR, hypertension [34,35], and activation of the sympathetic nervous system and aggravates MI . FFAs are regarded as potential biochemical markers of post-infarct myocardial remodeling . Further, IR is also associated with left ventricular remodeling  and often is accompanied by hemodynamic stress, such as systemic hypertension, which could cause left ventricular pressure overload and result in heart failure . Therefore, a long term HFC diet increases plasma FFA and aggravates IR, which leads to severe cardiac autonomic nervous dysfunction. Specifically, the interaction between the sympathetic nervous system and inflammation plays an important role in the development of vascular stiffening and AS . Moreover, increased FFA was associated with augmented myocardial oxygen consumption , limited myocardial blood supply, and cardiac dysfunction, all of which further promote chronic MI in HFC-fed minipigs.
The most interesting, and possibly still largely underestimated, role of FFAs in cell signaling is their ability to bind to nuclear receptors such as PPARs. PPAR-α, PPAR-β/δ, and PPAR-γ are the ligand-activated transcription factors that function as the master regulators of insulin sensitivity, glucose homeostasis, fatty acid and lipoprotein metabolism, inflammation, and AS [41-47]. Considering this function, any dysregulation of these metabolic pathways may result in obesity, diabetes, and cardiovascular diseases. Previous animal experiments have identified that PPAR-α or PPAR-β/δ deficiency could reduce myocardial fatty acid oxidation, increase glucose utilization and myocardial hypertrophy, and increase ischemic heart sensitivity [42,48]. Similar to PPAR-α and PPAR-β/δ deficiency, PPAR-γ-defective mice also exhibit severe myocardial hypertrophy [43,44]. Interestingly, PPAR-α and PPAR-β/δ expression in heart tissue of HFC-fed minipigs is significantly down-regulated (Figure 6). Thus, we speculate that PPAR-α and PPAR-β/δ may have dominant roles in regulating myocardial fatty acid metabolism, instead of PPAR-γ; further, PPAR-γ may use an indirect mechanism to achieve cardioprotective effects, probably by mobilizing fat metabolism and directing glucose and fatty acid transport to the heart.
Our findings describe a novel minipig model where by feeding Tibetan minipigs an HFC diet produced IR-AS, including dyslipidemia, IR, glucose intolerance, hypertension, and inflammatory and vascular AS lesions. PPARs are involved in cardiovascular remodeling and impaired function. We propose that this Tibetan minipig model may be helpful to further understanding of human IR and cardiovascular disease development.
The authors gratefully acknowledge financial support from the Zhejiang Provincial Natural Science Foundation of China (LY12C04002) and the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents.
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