High fat high sucrose diet-induced dyslipidemia in guinea pigs

Abstract

Easy access to high-calorie and fat-dense fast food has resulted in unhealthy dietary and lifestyle changes worldwide, which affects both developed and developing economies. This predisposes populations to a considerable number of metabolic and inflammatory conditions, such as diabetes, nonalcoholic fatty liver disease (NAFLD), and cardiovascular disease (CVD). Guinea pigs have been proposed as a model to study high-fat diet-induced metabolic disease due to their similar antioxidant metabolism and lipid profile to humans, and their susceptibility to atherosclerosis and endothelial disease. This study aims to evaluate cardiovascular and metabolic disorders induced by high-fat high-sucrose diet (HFHSD) in guinea pigs. Two to three-week-old male guinea pigs were fed a normal diet (ND) or HFHSD for 12 wk. Guinea pigs fed a HFHSD developed glucose intolerance, dyslipidemia, and liver, cardiac, and kidney damage. However, hypertension, dysautonomia, endothelial disease, and obesity were absent in these HFHSD guinea pigs. Taken together, these results show that guinea pigs fed a HFHSD are a nonobese model of metabolic disorders, resulting in important cardiac damage. Moreover, our findings suggest that NAFLD may be an important risk factor for diet-induced CVD.

NEW & NOTEWORTHY In this study, we show a new animal model for diet-induced disease metabolic disorders without obesity in guinea pigs. Moreover, results suggest a strong relation between liver disease and increased cardiovascular risks.

INTRODUCTION

Easy access to high-calorie and fat-dense fast food, particularly in so-called “food-deserts,” has resulted in unhealthy dietary and lifestyle changes worldwide, which affects both developed and developing economies (1). The consumption of a high-fat diet shifts the energy balance and leads to ectopic lipid accumulation, especially in the liver, resulting in inflammation, oxidative stress, and fibrosis, which leads to organ damage and loss of function (2). This predisposes these populations to a considerable number of metabolic and inflammatory conditions, such as diabetes, nonalcoholic fatty liver disease (NAFLD), and cardiovascular disease (CVD), (2) and makes them more susceptible to COVID-19-induced complications (3). Although obesity is a well-characterized risk factor for the development of NAFLD and recent research has focused on understanding the pathways driving the pathological processes associated with obesity-induced NAFLD, poor diet appears to be a major factor in NAFLD progression (i.e., liver cell death, inflammation, or fibrosis) independent of obesity or metabolic syndrome.

More importantly, mice fed a high-fat high-sugar diet develop steatohepatitis with fibrosis [nonalcoholic steatohepatitis (NASH)] (4); however, the fibrosis in this model is mild, and the disease is not complicated by severe hepatic inflammation and/or lipid dysfunction. Furthermore, it is known that no murine model can replicate the full spectrum of human NASH with regard to both the metabolic and histological characteristics, but only mimic specific features, therefore these animal models contribute to the elucidation of NASH pathogenesis, but none of them fully replicate human NASH pathogenesis (5). The association between diet and atherosclerosis has been under investigation since the 20th century, but the exact relationship between dietary constituents and atherosclerotic progression has not been completely elucidated (6). Many species have been challenged with different fat/cholesterol/carbohydrate proportions in their diet, and although mice and rats challenged with high-fat and high-sucrose diets can develop fatty livers (similar to humans), they are resistant to developing atherosclerosis and endothelial disease, thus necessitating knockout models for studying atherosclerosis in mice (7, 8).

There are many studies in the literature which show that diet-induced metabolic disorders can lead to lipid accumulation in kidneys in mice and rats (9, 10). In spontaneously hypertensive rats, a high-fat diet worsens renal damage (11). However, little is known about kidney function in high-fat diet guinea pig models. One study showed that hypercholesterolemia plays an important role in the pathogenesis of glomerulosclerosis in a high-fat diet guinea pig model (12). Although metabolic disorders such as obesity and type II diabetes are important risk factors for cardiovascular complications in patients with liver dysfunction, significantly less attention is given to the association of liver dysfunction and CVD in lean patients (13). A recent study showed that there is an association between liver disease and a higher incidence of cardiovascular events even in patients with body mass index (BMI) < 25 kg/m² (14), which may be mediated by altered lipid and lipoprotein metabolism (13). Regardless, the exact relationship between CVD and liver dysfunction remains unclear, especially because most rodent models of diet-induced metabolic disorder are also associated with increased fat mass. Clearly, there is a need to create animal models that recapitulate the physiology, histology, outcomes, and transcriptomic changes seen in humans in response to a high-fat diet.

Guinea pigs have been proposed as a model to study high-fat diet-induced metabolic disease due to their similar lipid profile to humans, and their susceptibility to atherosclerosis and endothelial disease (15). Moreover there exist several differences in the response to high-fat diets between rats and guinea pigs (16). The differential response to high-fat diet between guinea pigs and other rodent models may be due to guinea pigs’ evolutionary loss of functional hepatic l-gulonolactone oxidase (LGO) (17), which inhibits them from producing ascorbic acid (AA). The loss of LGO activity also occurred (independently) in the haplorrhini suborder of primates (18), but interestingly, guinea pigs and haplorrhini (including humans), evolved similar ways to distribute antioxidant capabilities within the circulation and tissue parenchyma to counteract the loss of AA (19) which enhances the utility of guinea pig models in studying human disease states. However, there are few studies with these animals, and the effects of different diets and the time to diet-induced injury are not well established in guinea pigs. Therefore, this study aimed to evaluate cardiovascular and metabolic disorders induced by a 12-wk exposure to high-fat high-sucrose diet in guinea pigs.

METHODS

Animal Preparation

Animal handling and care followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the University of California San Diego Institutional Animal Care and Use Committee approved the experimental protocol. Guinea pigs weighing between 200 and 220 g were used. Guinea pigs were fed regular chow (ND; Envigo TD.2040), or a commercially available high-fat and high-sucrose diet (HFHSD; Envigo TD.110484; 35% sucrose, 15% cocoa butter, 0.25% cholesterol) for 12 wk before experimental protocols.

Glucose Tolerance Test

Glucose tolerance testing (GTT) was performed after 12 wk of diet. Animals were fasted for 6 h before GTT. Glucose (2.0 g/kg body weight) was then given orally. Blood glucose concentration was determined from fresh saphenous vein samples collected 0, 15, 30, 60, 90, and 120 min after glucose administration (AccuChek Advantage RocheDiagnostics). The area under the curve (AUC) was calculated using GraphPad Prism 6 (GraphPad Software Inc, San Diego, CA).

Dual Energy X-Ray Absorptiometry

Measurement of body composition. Body composition was assessed in all animals using DEXA scan (PIXImus2; Madison, WI). Each animal was anesthetized for the duration of the procedure (5–7 min) with 1.5%–2% isoflurane in 30% oxygen gas using a nose cone. Each animal was placed on the scanner bed in the prone position, with the limbs stretched away from the body. Based on the attenuation of two energy levels, the system provides quantitative data on the fat tissue content, the lean tissue content, and the total tissue mass within a region of interest. To evaluate visceral fat, three different regions of interest were defined from the whole body scan.

Cardiac Output and Cerebral Blood Flow

Magnetic resonance imaging (MRI) was employed to study cardiac output cerebral and cardiac blood flow. Briefly, guinea pigs lightly anesthetized with isoflurane (0.75%/volume for immobilization) were subjected to MRI studies in a 4.7-Tesla, 40-cm bore Bruker system (Billerica, MA), equipped with a 12-cm diameter shielded gradient insert and a home-built radio frequency coil. Perfusion images were acquired using a continuous imaging technique (spin echo). Measurements were performed under normoxia (21% O₂) and after 10 min of exposure to 10% O₂.

Artery and Vein Catheterization

Animals were placed on a heating pad to maintain the core body temperature at 37°C for any procedures or experimental protocols that were performed under anesthesia. Guinea pigs were anesthetized with isoflurane (Drägerwerk AG, Lübeck, Germany) in compressed room air (flow rate 1.5 LPM) slowly, by increasing the isoflurane 0.4% every 3 min until a surgical depth of anesthesia was achieved, typically 3%. This ensured that the animals did not stop breathing due to airway irritation by isoflurane and prevented variations in heart rate (HR). Animals were instrumented with catheters in the right carotid artery and left jugular vein and catheters were exteriorized dorsally. Animals were allowed to recover for 24 h before any experimental procedures were performed.

Hematological Parameters

Hematocrit was measured from centrifuged arterial blood samples taken in heparinized capillary tubes. Arterial blood was collected in heparinized glass capillaries (50 μL) and immediately analyzed for pO₂, pCO₂, pH, electrolytes, lactate, and Hb content (ABL90; Radiometer America, Brea, CA).

Blood Pressure, Heart Rate, Heart Rate Variability, Blood Pressure Variability, and Spectral Analysis

Twenty-four hours after surgery, the arterial cannula was connected to a pressure transducer and recording system (MP150, Biopac, Santa Barbara, CA), and blood pressure signals were recorded for 10 min. Heart rate variability was calculated in time and frequency domains using the Cardioseries v2.4 software. BP and HR were recorded in the awake guinea pigs during 10 min for the analysis of pulse interval (PI) and systolic blood pressure (SBP) in the time domain, obtaining the total variance of PI (VAR PI) and the variance of SBP (VAR SBP). For the frequency domain, the interpolated waves of these same baseline periods were divided into segments of 512 beats, with an overlap of 50%, and were processed by Fast Fourier Transform. One spectrum was obtained for each segment and the oscillatory components were quantified in two different frequencies: low frequency (LF) from 0.10 to 0.75 Hz and high frequency (HF) from 0.75 to 3.00 Hz. The results are represented by absolute values (ms² and mmHg²), the percentage of total spectrum (%) and normalized units (nu) (percentage of LF and HF bands only). The very low oscillations (<0.20 Hz) were considered nonstationary (20).

Vasoreactivity Measurements

After recording mean arterial blood pressure (MAP), baroreflex sensitivity was evaluated via sequential injections of phenylephrine (Phe, 1 and 2 µg, Sigma Aldrich) and sodium nitroprusside (Np 1 and 2 µg, Sigma Aldrich), with 3 min between doses for blood pressure to return to baseline. The index of baroreflex sensitivity was calculated as the ratio of the change in HR to the change in MAP (ΔHR/ΔMAP), representing the indices of bradycardia and tachycardia for phenylephrine and sodium nitroprusside, respectively. To evaluate the loss of endothelial sensitivity, animals were dosed with acetylcholine (2 and 4 µg, with a 10 min interval between doses). Results are expressed as change in MAP. Degree of nitric oxide synthase (NOS) activity was evaluated via administration of the NOS inhibitor l-NG-nitro arginine methyl ester (l-NAME, 12 mg/kg, Sigma Aldrich), and the results are expressed as ΔMAP, where a smaller ΔMAP indicates a loss of NOS activity.

Harvesting Tissues

Two hours after l-NAME injection, guinea pigs were anesthetized and 10 mL of blood was collected from the indwelt arterial catheter and centrifuged to separate the plasma. Guinea pigs were euthanized with Fatal Plus (sodium pentobarbital, 300 mg/kg), and urine, kidneys, liver, spleen, heart, and lungs were harvested. Markers of inflammation, function, and organ injury were evaluated. These analyses were performed by the UC San Diego Histology Core via ELISA and flow cytometric analysis of tissue homogenates and plasma. The kits and methods used for these analyses are described in Supplemental Table S1 (all Supplemental material is available at https://doi.org/10.6084/m9.figshare.13542944).

Statistical Analysis

All values are expressed as means ± SE. Data between groups were analyzed using a Student’s t test. Data between groups and different O₂ levels were analyzed using two-way analysis of variance (ANOVA), with Tukey's post hoc when necessary. In each figure, n is indicative of the number of animals per group. All statistics were calculated using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA). Changes were considered significant if P < 0.05.

RESULTS

Body Composition and Glucose Tolerance

No differences were observed between the initial or final body weight between groups. HFHSD diet decreased total fat mass (ND: 28.0 ± 0.7% vs. HFHSD: 21.5 ± 0.8%) and nonvisceral fat compared with ND (ND: 24.0 ± 0.7% vs. HFHSD: 16.3 ± 0.6%). On the other hand, HFHSD increased visceral fat (ND: 4.0 ± 0.2% vs. HFHSD: 5.2 ± 0.3%), and the visceral/nonvisceral fat ratio compared with ND (ND: 0.17 ± 0.01% vs. HFHSD: 0.33 ± 0.01%), suggesting that HFHSD changes lipid storage sites in guinea pigs (Table 1). GTT was performed to evaluate the degree of glucose intolerance after 12 wk of HFHSD. The AUC for guinea pigs fed a HFHSD was 16% higher than that of ND-fed guinea pigs, indicating glucose intolerance (Table 1).

Table 1.

Body composition and glucose tolerance test

	ND	HFHSD
Initial body weight, g	202.0 ± 7.7 (n = 11)	217.9 ± 5.4 (n = 11)
Final body weight, g	633.1 ± 8.3 (n = 11)	619.9 ± 10.8 (n = 11)
Total fat tissue, %	28.0 ± 0.7 (n = 6)	21.5 ± 0.8† (n = 8)
Visceral fat tissue, %	4.0 ± 0.2 (n = 6)	5.2 ± 0.3† (n = 8)
Nonvisceral fat tissue, %	24.0 ± 0.7 (n = 6)	16.3 ± 0.6† (n = 8)
Visceral/nonvisceral fat tissue ratio, %	0.17 ± 0.01 (n = 6)	0.32 ± 0.01† (n = 6)
GTT (AUC), mg/dL/120 min	26,795 ± 917 (n = 11)	31,223 ± 2,166† (n = 7)

Data are presented as means ± SE. AUC, area under the curve; GTT, glucose tolerance test; HFHSD, high-fat high-sucrose diet; ND, normal diet. †P < 0.05 compared to ND.

Hematological Parameters

HFHSD did not significantly impact tHb, pH, pO₂, pCO₂, or lactate. However, HFHSD significantly decreased serum sodium (ND: 145.5 ± 0.4 mmol/L vs. HFSD: 139.5 ± 1.1 mmol/L), and chloride (ND: 112.3 ± 0.8 mmol/L vs. HFHSD: 106.2 ± 1.4 mmol/L), but did not impact potassium or calcium (Supplemental Table S2; see https://figshare.com/s/ac4905a562d1aaa30fb9), indicating changes in kidney tubule reabsorption.

Lipid Profile

HFHSD increased total cholesterol (150%), LDL (217%), HDL (38%), and ox-LDL (67%) compared with ND controls, confirming that HFHSD induces dyslipidemia in guinea pigs (Fig. 1).

Figure 1.

Lipid profile in guinea pigs after 12 wk of normal diet (ND) or high-fat high-sucrose diet (HFHSD). A: total cholesterol. B: low density lipoprotein (LDL). C: high density lipoprotein (HDL). D: oxidized LDL (oxLDL). †P < 0.05 compared with ND. ND (n = 9); HFHSD (n = 7).

Cardiovascular Measurements

HFHSD did not increase the MAP of guinea pigs, however it did increase SBP variability, as indicated by SBP SD (ND: 4.4 ± 0.2 mmHg vs. HFHSD: 5.7 ± 0.5 mmHg) and variance (ND: 20.0 ± 2.2 mmHg² vs. HFHSD: 35.6 ± 5.8 mmHg²) and an increase in the LF (nonstatistical, 32%) suggesting increased vascular sympathetic modulation (Fig. 2). Surprisingly, HFHSD did not induce changes in indices of HRV or spectral analysis (Supplemental Table S3), whereas most other rodent models experience changes in HRV and heart rate spectra following a high-fat diet. Moreover, we observed no differences in vascular reactivity, baroreflex, or l-NAME-induced hypertension between groups, indicating that 12 wk of HFHSD does not induce endothelial dysfunction in guinea pigs (Supplemental Table S4). HFHSD increased heart interleukin-6 (IL-6) (20%), tumor necrosis factor-α (TNF-α) (21%), monocyte chemoattractant protein-1 (MCP-1) (27%), troponin (21%), atrial natriuretic peptide (ANP) (20%), and C-reactive protein (CRP) (16%) relative to ND. All together these markers give an overview of heart function and damage and suggest that HFHSD induced heart damage (Fig. 3).

Figure 2.

Mean arterial pressure (MAP) and blood pressure variability in guinea pigs after 12 wk of normal diet (ND) or high-fat high-sucrose diet (HFHSD). A: MAP. B: systolic blood pressure standard deviation (SD). C: systolic blood pressure variance. D: low frequency band of systolic blood pressure (LF SBP). †P < 0.05 compared with ND. ND (n = 11); HFHSD (n = 13).

Figure 3.

Cardiac inflammation and injury after 12 wk of normal diet (ND) or high-fat high-sucrose diet (HFHSD). A: cardiac interleukin- 6 (IL-6). B: cardiac tumor necrosis factor-alpha (TNF-α). C: cardiac monocyte chemoattractant protein-1 (MCP-1). D: cardiac troponin. E: C-reactive protein. F: atrial natriuretic peptide (ANP). †P < 0.05 compared with ND. ND (n = 9); HFHSD (n = 7).

Organ Injury and Systemic Inflammation

HFHSD increased aspartate aminotransferase AST (ND: 25.2 ± 1.0 U/L vs. HFD: 38.1 ± 2.4 U/L) and alanine aminotransferase ALT (ND: 27.3 ± 1.3 U/L vs. HFHSD: 43.0 ± 1.2 U/L), but did not affect liver chemokine (C-X-C motif) ligand-1 (CXCL-1) (Fig. 4). Moreover, HFHSD decreased serum creatinine (ND: 1.4 ± 0.1 mg/dL vs. HFHSD: 1.1 ± 0.1 mg/dL) and increased blood urea nitrogen (BUN) (20%), but did not alter urine-neutrophil gelatinase-associated lipocalin (U-NGAL) (Fig. 4). These are classical markers of liver and kidney injury and they reveal HFHSD-induced injury for both organs. There were no differences in the systemic inflammatory markers IL-6, IL-10, or CXCL-1 between groups. Epinephrine was 26% higher for the HFHSD group compared with the ND group, but norepinephrine was no different between groups. Serum, spleen, and liver ferritin were unaffected by diet, but total bilirubin was increased with HFHSD (ND: 5.1 ± 0.3 mg/dL vs. HFHSD: 6.1 ± 0.2 mg/dL) (Table 2).

Figure 4.

Liver and kidney injury after 12 wk of normal diet (ND) or high-fat high-sucrose diet (HFHSD). A: aspartate aminotransferase (AST). B: alanine aminotransferase (ALT). C: liver chemokine ligand-1 (CXCL-1). D: serum creatinine. E: blood urea nitrogen (BUN). F: urine-neutrophil gelatinase-associated lipocalin (U-NGAL). †P < 0.05 compared with ND. ND (n = 9); HFHSD (n = 7).

Table 2.

Systemic inflammation, catecholamines, and iron metabolism

	ND	HFHSD
IL-6, pg/mL	188.6 ± 7.8	184.2 ± 11.4
IL-10, pg/mL	186.7 ± 6.5	202.6 ± 8.7
CXCL-1, pg/mL	119.0 ± 4.4	124.7 ± 1.4
Spleen CXCL1, pg/mg	210.6 ± 9.2	246.4 ± 12.0
Norepinephrine, pg/mL	697.0 ± 16.8	746.1 ± 28.0
Epinephrine, pg/mL	220.0 ± 19.9	277.8 ± 16.0†
Serum ferritin, µg/L	241.0 ± 15.1	226.2 ± 10.7
Liver ferritin, µg/g	206.8 ± 28.2	220.5 ± 12.1
Spleen ferritin, µg/g	305.3 ± 27.0	280.6 ± 14.2
Total bilirubin, mg/dL	5.1 ± 0.3	6.1 ± 0.2†

Data are presented as means ± SE. CXCL-1, chemokine ligand-1; HFHSD, high-fat high-sucrose diet; IL, interleukin; ND, normal diet. †P < 0.05 compared with ND. ND (n = 9); HFHSD (n = 7).

Brain and Artery Flow during Normoxia and Hypoxia

The HFHSD group had lower HR and cardiac output (CO) during normoxia compared with the ND group, but there were no significant differences in SV between groups. The slight differences in HR between these, and those presented above may be a result of the groups differential response to isoflurane. When subjected to hypoxia, SV and CO increased for the ND group, but no changes were observed in the HFHSD group (Fig. 5), exacerbating the difference in CO between groups during hypoxia. During normoxia, there were no differences in cerebellum, pons, or thalamus flow between groups, but cortical and hippocampal flow was lower for the HFHSD group compared with the ND group (Fig. 6). Animals fed a HFHSD showed lower aortic and LCCA flow (Fig. 6F), but similar ICA flow (Fig. 6G) compared with those fed a ND. During hypoxia both groups experienced increased cortical (Fig. 6A) and hippocampal flow (Fig. 6D) compared with normoxia, but these flows remained lower for the HFHSD group than the ND group. Only the ND group experienced increased flow in the pons (Fig. 6C) and thalamus (Fig. 6E) compared with normoxia. Cerebellum flow was similar for both groups, and neither group experienced significant changes in flow due to hypoxia (Fig. 6B). Hypoxia did not induce changes in LCCA or ICA flow in either group, but aortic flow significantly increased in ND animals in response to hypoxia (Fig. 6).

Figure 5.

MRI-measured cardiac flow in anesthetized guinea pigs during normoxia and hypoxia after 12 wk of normal diet (ND) or high-fat high-sucrose diet (HFHSD). A: heart rate (HR). B: stroke volume (SV). C: cardiac output (CO). †P < 0.05 and ††P < 0.01 compared with ND; ‡P < 0.05 compared with normoxia. n = 4/group.

Figure 6.

MRI-measured brain and artery flow in anesthetized guinea pigs during normoxia and hypoxia after 12-wk exposure to normal diet (ND) or high-fat high-sucrose diet (HFHSD). A: cortex flow. B: cerebellum flow. C: pons flow. D: hippocampus flow. E: thalamus flow. F: left common carotid artery (LCCA) flow. G: internal carotid artery (ICA) flow. H: aorta flow. †P < 0.05, ††P < 0.01, and ††††P < 0.0001 compared with ND; ‡P < 0.05 compared with normoxia. n = 4/group.

DISCUSSION

The main finding of this study is that guinea pigs fed a HFHSD for 12 wk developed dyslipidemia, glucose intolerance, and damage in vital organs, without an increase in body weight or total fat mass. Other studies where guinea pigs were fed a high-fat diet showed similar results, with no body weight increase, and without increase in fat mass (12, 21, 22). These results are surprising since a HFD and HFHSD in other species typically results in obesity. Although guinea pigs did not develop obesity, they developed hepatic, cardiac, and cardiovascular injury that is atypical for other rodent models.

There were no differences in MAP between groups; however, guinea pigs fed a HFHSD showed increased blood pressure variability and vascular sympathetic modulation compared with ND guinea pigs. There were not significant differences in ΔMAP due to systemic acetylcholine administration between groups, indicating that the differences in blood pressure variability may not stem from vascular injury. Moreover, there were no differences in any of the HR variability parameters, which is surprising because other species with diet-induced metabolic disease develop autonomic dysfunction (23, 24). This lack of autonomic dysfunction could be related to diet composition or time under diet, and reinforces that there are numerous differences between species that must be considered when designing diet and metabolic studies. Moreover, the evolutionary loss of functional hepatic l-gulonolactone oxidase which inhibits them from producing ascorbic acid could be the explanation for the differences found between species when challenged with HFHSD. Moreover, no endothelial dysfunction was found. This could be due to time under diet, and/or diet composition. However, in this study, no morphological parameters of the vessels were evaluated; it is possible that morphological changes occurred due to HFHSD, without provoking functional changes.

In addition, troponin, CRP, ANP, cardiac IL-6, TNF-α, and MCP-1 were assessed to indirectly evaluate cardiac function and injury (25, 26). All these parameters were increased in animals fed a HFHSD suggesting that guinea pigs fed a HFHSD develop cardiac injury. Since our data suggest a lack of vascular injury, the observed changes in BP variability may be due to cardiac injury. Indeed, others have suggested that BP variability may have prognostic value during heart failure, and can accurately predict survival independent of ejection fraction or oxygen consumption (27).

To evaluate cerebral and cardiac blood flow, we subjected the guinea pigs to MRI during normoxia and hypoxia. Under the mild anesthesia required for immobilization during MRI, guinea pigs fed a HFHSD had significantly lower HR than those fed a ND. In addition, guinea pigs fed a HFHSD had no compensatory response to hypoxia whereas SV increased in response to hypoxia for animals fed a ND, ultimately resulting in significantly decreased CO for HFHSD animals compared with ND controls, and no compensatory increase in CO to offset impaired oxygen uptake during hypoxia in HFHSD animals. These compensatory changes in CO (or lack thereof, in the case of HFHSD animals), translate directly to changes in aortic and ICA flow; the flow in these vessels must change in response to hypoxia in order to ensure adequate oxygen supply to highly sensitive tissues, but this response is stunted in HFHSD animals. On the other hand, some of the LCCA blood supply is normally directed to the skin, which can be easily redirected to the more tenuous tissues supplied by the LCCA, such as the skull and meninges. Interestingly, diet-induced obesity speeds recovery from hypoxia in mice due to obesity-associated upregulation of IL-1RA, (28) moreover a high-carbohydrate diet improved resistance to hypoxic stress in zebrafish by increasing glycogen deposition, (29) which could deemphasize the need for increased blood flow during hypoxia.

Adipose tissue is the major site for cholesterol storage, and during the pathogenesis of obesity, up to half of the cholesterol may reside within this tissue (30). Adipose tissue is an endocrine tissue, and when its mass is increased during obesity, it produces and releases several hormones and cytokines, which cause inflammation and increase cardiovascular risk (31). In our study, we observed a slight decrease in adipose tissue due to exposure to a HFHSD, which may explain the relatively minimal changes in inflammatory cytokines observed, but we observed increased visceral fat content, which can significantly contribute to organ injury and dysfunction. More importantly, animals fed a HFHSD doubled total cholesterol and increased plasma LDL content almost four times. Hypercholesterolemia, like that observed herein, has be shown to impair adipocyte differentiation and maturation, which may have affected adipose tissue development observed in HFHSD guinea pigs, especially since the diet was started at a young age. This decreases the amount of cholesterol that can be sequestered by the adipocytes and decreases the adipose tissues’ storage capacity (30). When the adipose tissue is saturated or is dysfunctional, ectopic lipid deposits can form, mostly in liver, kidney, and muscle tissue, and these lipid deposits lead to inflammation, fibrotic injury, and loss of function.

Elevated AUC from the GTT revealed that guinea pigs fed a HFHSD developed glucose intolerance. This is expected and regularly found in all rodent species subjected to HFHSD (10). Glucose intolerance is the first indicator of type 2 diabetes, and is usually found before changes in serum glucose and insulin (32). However, the etiology of glucose intolerance remains unclear. Lifestyle and diet, as well as genetic predisposition, are all likely associated with glucose intolerance (32). It is important to note that patients with glucose intolerance and prediabetes are usually asymptomatic, making it difficult to diagnose (33). Moreover, it is well established that patients with glucose intolerance have a higher risk of developing type 2 diabetes and its comorbidities, such as hypertension and kidney disease (33). Although we did not observe typical signs of type 2 diabetes (e.g., elevated basal glucose), nor hypertension, it is possible that these morbidities could develop in guinea pigs if HFHSD treatment was extended beyond the 12 wk in this study, as we did observe markers indicative of the beginning stages of kidney disease.

Guinea pigs fed a HFHSD had higher cholesterol than those fed a ND. Cholesterol has been implicated in kidney injury via lipid accumulation, resulting in glomerulosclerosis and monocyte infiltration (12). More recent studies have shown similar results in mice (9) and rats (10, 11), but perhaps more interesting is that the presence of lipid accumulation and injury occurs before a loss of kidney function in mice (9). We observed kidney injury, as indicated by elevated BUN in the HFHSD group. The HFHSD group also possessed lower serum creatinine than ND controls, which could indicate that these animals are in the beginning stages of kidney disease, as increased glomerular filtration rate precedes a loss of function. Serum sodium and chloride decreased slightly for guinea pigs fed a HFHSD, however the values remained within physiological limits.

HFHSD animals had higher levels of AST and ALT compared with ND animals, two classical markers of liver damage, suggesting some degree of liver disease. De Ogburn et al. (34) showed that excessive dietary cholesterol ingestion (induced by 0.25% cholesterol, the same as the present study) by guinea pigs results in hepatic steatosis, independent of the carbohydrate content. Many other studies conducted in guinea pigs with high-fat diets have shown that guinea pigs develop liver disease (21, 22, 35). This diet-induced hepatic injury may be a result of the impaired oxidative status of guinea pigs, as hepatic injury typically occurs from a positive feedback loop of oxidative stress coupled with compromised microcirculatory function. In other animal models, vitamin C (ascorbic acid) could break this feedback loop and understate the degree of hepatic injury that would be observed in humans, as vitamin C helps ensure adequate endothelial nitric oxide synthase (eNOS) cofactor BH4 and thus prevents eNOS uncoupling (36). Indeed, in our study, liver CXCL1 concentration did not increase in animals fed a HFHSD, suggesting that the liver injury we observed may be independent of inflammation-induced injury. To confirm this, other markers of inflammation should be measured in future studies. In addition, we observed liver injury with no increases in body weight or total fat mass. Although nonalcoholic fatty liver disease (NAFLD) is commonly associated with obesity, it is also present in the lean population. As such, guinea pigs fed a HFHSD may represent a unique animal model to study comorbidities of NAFLD (21).

It is well known that NAFLD and CVD have many risk factors in common, such as obesity, insulin resistance, and type 2 diabetes. However, recent studies have suggested that liver disease itself could be an important risk factor for CVD (13, 14). The results in the present study also suggest that liver dysfunction could be an important risk factor to cardiac damage, since we observed cooccurring liver dysfunction and cardiac injury in this lean model. Dyslipidemia is frequently found in patients with NAFLD, with increased LDL. The literature shows that the liver plays an important role in regulating the lipoprotein’s metabolism, production and/or clearance. Moreover, the liver is the major site of triglycerides and cholesterol metabolism, making it clear that there is cross talk between hepatic metabolic dysfunction found in the NAFLD and altered lipoprotein metabolism and composition (13).

Conclusions

Taken together, these results show that guinea pigs fed a HFHSD are a nonobese model of metabolic disorders, culminating in important cardiac damage. Other target organs such as the liver and kidney also developed injury in guinea pigs subjected to HFHSD diet. Moreover, our findings suggest that NAFLD can be an important risk factor for diet-induced CVD.

GRANTS

This work was supported by the National Institutes of Health Grants R01HL126945 and R01HL138116, and the Department of Defense under Grant W81XWH‐18‐1‐0059.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.R.M., A.F.P., and P.C. conceived and designed research; C.R.M. and A.M.E. performed experiments; C.R.M., F.D.S., and P.C. analyzed data; C.R.M., A.T.W., F.D.S., and P.C. interpreted results of experiments; C.R.M., A.T.W., and F.D.S. prepared figures; C.R.M. and A.T.W. drafted manuscript; C.R.M., A.T.W., F.D.S., A.F.P., and P.C. edited and revised manuscript; C.R.M., A.T.W., A.M.E., F.D.S., A.F.P., and P.C. approved final version of manuscript.