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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Objective

To investigate microvascular alterations in an experimental model of metabolic syndrome induced by a high-fat diet (HFD) associated with salt supplementation (0.5% NaCl).

Design and Methods

Wistar Kyoto rats were fed standard chow (control group, CONT) or HFD for 20 weeks. The functional capillary density (FCD) was assessed using intravital fluorescence videomicroscopy.

Results

The HFD group presented a higher systolic blood pressure, plasma glucose and insulin levels, total and LDL-cholesterol levels, triglycerides, and visceral and epididymal fat when compared with the CONT group. When compared with the CONT group, the HFD group showed a lower FCD in the skeletal muscle (P < 0.05) but not in the skin (P > 0.05). The HFD group also had a lower capillary-to-fiber ratio in the skeletal muscle (P < 0.01). The capillary volume density-to-fiber volume density ratio in the left ventricle of the HFD was also reduced (P < 0.01). Finally, rats fed with HFD showed ventricular hypertrophy and increased cardiomyocyte diameter (P < 0.01).

Conclusions

The long-term administration of a HFD associated with salt supplementation to rats generates an experimental model of metabolic syndrome characterized by central body fat deposition, insulin resistance, glucose intolerance, hypertriglyceridemia, hypercholesterolemia, arterial hypertension, cardiac remodeling, and rarefaction of the microcirculation in the heart and skeletal muscle.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

The global obesity epidemic is rapidly evolving as a major global health issue because it is frequently associated with several cardiometabolic diseases that have high mortality and morbidity, such as arterial hypertension, diabetes, coronary artery disease, and stroke [1]. The lack of adequate physical activity, coupled with an increase in the consumption of fat-laden, high-calorie diets, contributes to the growing prevalence of obesity [2].

Metabolic syndrome (MS) is a cluster of several risk factors for cardiovascular disease and diabetes and is associated with insulin resistance, glucose intolerance, central body fat distribution, dyslipidemia, and hypertension [3]. The metabolic alterations that characterize MS are multifactorial and complex, including both environmental and genetic influences [3].

In the last decade, studies have demonstrated that functional and structural alterations in the microcirculation, such as impaired capillary recruitment and capillary rarefaction, respectively, may represent a pathophysiological link between several components of MS, including obesity, insulin resistance, and hypertension [4, 5]. Microvascular dysfunction leads not only to increased peripheral vascular resistance and blood pressure but may also decrease insulin-mediated glucose uptake in the skeletal muscles [4]. Therefore, the elucidation of such alterations is of paramount importance for managing patients with MS.

Due to the importance of investigating the pathophysiologic basis of MS, several animal models have been used. The obese Zucker rat, which has a deficiency in the leptin receptor gene [6], is commonly used and is accepted as a genetic model. In this model, the elevated systemic vascular resistance appears to result from structural alterations in the skeletal muscle microcirculation [7], which elevates the blood pressure [8]. Moreover, in this genetic model, abnormalities in the microvascular networks can compromise glucose tolerance by reducing peripheral glucose utilization.

High-fat diets are also generally accepted as a method to generate a valid rodent model for MS [9], even if diets with very different fatty acid compositions described in the literature result in dissimilar metabolic effects. In general, the rodent models of MS induced by high-fat diets are characterized by central obesity, insulin resistance, and changes in the plasma lipid profiles [10, 11]; therefore, these diets reproduce the main features of human MS. Moreover, because MS in humans is largely caused by lifestyle factors including excessive dietary intake and physical inactivity, the animal model that most closely reflects this situation is the high-fat diet model.

The association between the ingestion of large quantities of salt with the development of hypertension is supported by extensive epidemiological evidence in humans [12]. Moreover, in patients who present with MS, the blood pressure response to sodium intake is greater than that in subjects without this syndrome [13]. A similar association of salt intake and an increase in blood pressure has been reported in experimental studies with animals fed high levels of salt. This association is not only observed in genetically selected Dahl salt-sensitive rats [14] but also in Wistar rats [15]. Additionally, high salt intake results in microvascular rarefaction in the skeletal muscles of normal rats [16].

However, no studies have directly characterized the microcirculatory profile in models of MS induced by a high-fat diet (HFD). Therefore, the main purpose of the present study was to investigate whether long-term HFD with a salt supplementation could modify the functional and/or structural capillary density in the skeletal muscles and hearts of rats. We also studied the microcirculation in the ear skin as a control microvascular bed because it does not play an important role in the regulation of systemic vascular resistance. Additionally, structural alterations of the myocardium, including left ventricle and cardiomyocyte hypertrophy, and collagen deposition were also assessed.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Diet-induced metabolic syndrome in rats

All experiments were conducted in accordance with internationally accepted principles for the care and use of laboratory animals and were approved by the Oswaldo Cruz Foundation Animal Welfare Committee (protocol # P 0034-08).

The experiments were performed in male Wistar Kyoto rats (WKY, Oswaldo Cruz Foundation Animal Facilities, Brazil) housed under controlled light (12:12 h light-dark cycle) and temperature (22 ± 1 °C) conditions.

At four weeks of age, the rats were randomly divided into two experimental groups and were fed either a standard chow (Nuvilab - CR1, Nuvital Nutrients Ltd, Colombo, Brazil) (CONT; n=10) or a high-fat diet (HFD; n=10) for 20 weeks. The composition of the commercial standard chow used in the present study is in agreement with the guidelines of the American Institute of Nutrition revised in 1993 for rats in this period of life [17]. The CONT diet contained 23% proteins, 71% carbohydrates, 6% lipids and 1.3% NaCl, and the HFD contained 14% proteins, 56% carbohydrates, 30% lipids and salt supplementation (standard chow + corn starch + condensed milk + animal fat + 0.5% NaCl; Table 1). The main fat source of the high fat diet was saturated fat (lard). The rats' body weight and food intake were measured daily. At the end of the diet period, total blood was collected and the visceral and epididymal adipose tissue deposits were dissected and weighed. Plasma was separated by centrifugation at 3000 rpm for 15 min at 4°C, and the plasma aliquots were stored at −80°C until measurement.

Hemodynamic measurements

The systolic arterial pressure (SAP) and heart rate (HR) were measured using a computerized tail-cuff plethysmography system (Visitech blood pressure analysis system, model BP-2000, Apex) in conscious animals in the morning (8 am to 12 pm). At least 1 week before recording the arterial pressure, the rats were acclimatized for 3 consecutive days using the pre-warmed tail-cuff device.

Intravital fluorescence videomicroscopy

In the morning of the terminal experiments, the animals were anesthetized with pentobarbital (75 mg/kg, i.p.); the anesthesia was complemented with an i.v. injection of 5 mg/kg of pentobarbital immediately before the administration of the neuromuscular-blocking agent. The rats were intubated with a polyethylene tube via a tracheotomy, immobilized with pancuronium (1 mg/kg i.v.), and artificially ventilated with room air using a small animal ventilator (Ugo Basile, Varese, Italy). The right jugular vein was catheterized to permit the injection of the anesthetic agents and the fluorescent dye. The rats' central temperature was monitored with a rectal probe, and the body temperature was maintained at 38 ± 0.5°C using a homeothermic blanket system (Harvard Apparatus, Boston).

The skin of the ear was scraped, and the animals were placed prone on a Plexiglas pad. The gracilis muscle was exposed through an incision in the right thigh and was covered with an oxygen-impermeable plastic wrap. The animals were then placed under an upright fixed-stage intravital microscope (Olympus BX51/WI, USA) coupled to a CCD digital video camera system (Optronics, Goleta). An Olympus objective with a 10× magnification was used in the experiments resulting in a total magnification of 100× at the video monitor. After the intravenous injection of 0.15 ml of 5% fluorescein-isothiocyanate (FITC)-labeled dextran (molecular weight 150,000), microscopic images of the muscle and skin were successively obtained in real time by the counting of capillaries using the Saisam software (Microvision, Evry, France). The functional capillary density, defined as the total number of spontaneously perfused capillaries per square millimeter of surface area (1 mm2), was determined in random microscopic fields over a 4-min period. Using intravital fluorescence videomicroscopy, it is possible to identify the network of capillaries in tissues such as skeletal muscles and the skin. In the skin, capillaries are arranged as interconnected networks, and capillary loops are perpendicularly oriented to the skin surface. In skeletal muscles, the capillaries are arranged along the longitudinal axis of the muscle fibers; thus, the capillaries run parallel to each other.

After the experiment, the animals were deeply anesthetized, and the left ventricle and gracilis muscle were immediately dissected and placed in 4% neutrally buffered paraformaldehyde for morphological analysis.

Metabolic measurements

Oral glucose tolerance tests (OGTTs) were performed in animals that had fasted for 6 h. Blood glucose levels were measured 0, 30, 60, and 120 min after the administration of a glucose overload (2.0 g/kg), and the areas under the glucose curves (AUCs) were calculated. An automatic glucose monitor was used to the measure blood glucose levels in the venous blood (OneTouch Ultra 2®, LifeScan Inc. Johnson & Johnson, Milpitas). The plasma insulin levels were measured using a radioimmunoassay (ImmuChem™ Coated Tube 125I RIA Kit, MP Biomedicals, Orangeburg). Insulin resistance was evaluated using the Homeostasis Model of Assessment-Insulin Resistance (HOMA-IR), calculated using the following formula:

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The total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) levels were directly measured using enzymatic methods (Colesterol Liquiform®, Labtest, Brazil). The low-density lipoprotein cholesterol (LDL-C) concentration was estimated indirectly using the Friedewald equation:

  • display math

Plasma levels of catecholamines

Plasma adrenaline and noradrenaline levels were determined using a competitive catecholamine enzyme immunoassay (2-Cat A-N research ELISA; Labor Diagnostika Nord) according to the manufacturer's protocol. All measurements were taken in duplicate.

Histochemical analysis of skeletal muscle

The tissue samples were dehydrated in a graded series of ethanol (70%, 95%, and 100%) and embedded in paraffin. The paraffin blocks were cut into 5-μm sections and stained with a FITC-conjugated Griffonia simplicifolia I lectin at a 1:150 dilution in a dark humidified chamber at room temperature for 45 min to label capillary endothelial cells. The structural capillary density (number of capillaries per mm2) and the structural fiber density (number of muscle fibers per mm2) were identified and recorded with a fluorescence microscope (Olympus BX51 and FluoView SV 300 scanning unit, Olympus) and analyzed using the Saisam software. The structural capillary density of the skeletal muscles was evaluated using at least seven microscopic fields from randomly selected tissue sections. The capillary-to-fiber ratio was calculated by dividing the capillary density by the fiber density and was considered as an anatomic index of angiogenesis.

Histochemical analysis of the left ventricle

The tissue samples were dehydrated in a graded series of ethanol (70%, 95%, and 100%) and embedded in paraffin. The left ventricular structural capillary density was determined using the orientator method as previously described [18]. Briefly, the orientator method describes an approach to generate isotropic, uniform, and random sections of biological specimens, which allows for a quantitative study of three-dimensional anisotropic structures on two-dimensional sections. The myocardium is an anisotropic structure, but isotropic sections are necessary for a stereological study. The technique was performed by cutting the organ using the “orthrip” method [18], which turns the samples into uniformly isotropic sections by dividing the fragment three times consecutively; the first section is random, the second section is orthogonal to the first, and the third section is orthogonal to the second. The paraffin blocks were cut into 5-μm sections and stained with a FITC-conjugated Griffonia simplicifolia I lectin at a 1:150 dilution in a dark humidified chamber at room temperature for 45 min. For each animal, at least seven microscopic fields were randomly examined from the three sections of tissue. The volume density of the capillaries (Vv[cap]) was calculated as follows: Vv[cap] = Pp/PT (%), where Pp is the number of points hitting the capillaries and PT is the total number of test points (PT = 64 in the present case). The fiber volume density (Vv[fib]) was similarly calculated. The ratio of the capillary volume density to the fiber volume density (Vv[cap] /Vv[fib]) was calculated to negate any influences of cardiac hypertrophy on myocardial capillary density.

Assessment of left ventricular weight

The atria were separated from the ventricles and the left ventricle (including the interventricular septum) was separated from the right ventricle and weighed according to the liquid volume displacement method of Scherle [19]. The left ventricle weight/body weight ratio (LVW/BW) was then calculated.

Assessment of cardiac fibrosis and cardiomyocytes diameter

Heart sections were prepared and embedded in paraffin wax. Five-micrometer-thick sections were cut, mounted onto glass slides, and stained with Sirius red or hematoxilin-eosin. Sirius red-stained sections were captured at ×100 magnification (Olympus BX50 and Olympus UC30 scanning unit; Olympus) and collagen content was assessed using Image Pro-Plus® software (version 6.3). Hematoxilin-eosin-stained sections were captured at ×400 magnification and the diameter of 100 cardiomyocytes from each sample was calculated with Saisam 5.1.3 software.

Statistical analysis

The results are expressed as the mean ± SEM for each group, and comparisons between the groups were made using unpaired t-tests. Differences with p values of less than 0.05 were considered statistically significant. All calculations were made using a commercially available, computer-based statistical package (GraphPad InStat 5.0, GraphPad Software, La Jolla).

Drugs

The following drugs were used: sodium pentobarbital, pancuronium bromide, FITC-labeled dextran, FITC-conjugated Griffonia simplicifolia I lectin, and glucose, all of which were purchased from Sigma Chemical, St. Louis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Body weight and visceral fat

The administration of the HFD did not induce any significant change in body weight (CONT 402.9 ± 14.8 and HFD 385.8 ± 6.5 g; P > 0.05) or food intake (CONT 3.7 ± 0.3 and HFD 4.1 ± 0.2 g/100g of body weight/day; P > 0.05) when compared with the control group (Table 1). On the other hand, the visceral fat (CONT 4.4 ± 0.5 and HFD 9.0 ± 0.9 g; P < 0.0001) and the epididymal fat (CONT 4.4 ± 0.3 and HFD 5.9 ± 0.6 g; P < 0.05) contents were higher in the rats with MS when compared with the group that received the normal diet. Food energy intake in the HFD group was significantly increased (82.5 ± 4.5 kcal/day; P < 0.001), when compared to the control group (60.2 ± 1.2 kcal/day) (Table 2).

Table 1. Body and fat weight, food intake, plasma catecholamines, and cardiovascular parameters in the control (CONT) and the high-fat diet (HFD) groups
 CONTHFD
  1. SAP: systolic arterial pressure; HR: heart rate.

  2. Values represent the mean ± SEM, n = 10 for each group.

  3. a

    P < 0.05 and

  4. b

    P < 0.001 versus the CONT group.

Body weight (g)402.9 ± 14.8385.8 ± 6.5
Food intake (g/100g body weight/day)3.7 ± 0.34.1 ± 0.2
Food energy intake (kcal/day)60.2 ± 1.282.5 ± 4.5b
Visceral fat weight (g)4.4 ± 0.59.0 ± 0.9b
Epididymal fat weight (g)4.4 ± 0.35.9 ± 0.6a
Noradrenaline (pmol/mL)3.5 ± 0.68.2 ± 0.4b
Adrenaline (pmol/mL)11.8 ± 0.511.4 ± 0.3
SAP (mmHg)146 ± 7.2168 ± 2.7a
HR (bpm)382 ± 14.4435 ± 8.0a
Table 2. Description of the diets used in the control (CONT) and high-fat diet (HFD) groups
g/100g of dietCONTHFD
Protein
Commercial chow2313.2
Condensed milk-1.4
Carbohydrates
Commercial chow7116
Corn starch-30
Condensed milk-10
Fat
Commercial chow62.5
Condensed milk-1.6
Animal fat (lard)-25

Arterial blood pressure, heart rate, and plasma catecholamines

The HFD induced an increase in systolic arterial pressure (CONT 146 ± 7.2 and HFD 168 ± 2.7 mmHg; P < 0.05) and HR (CONT 382 ± 14.4 and HFD 435 ± 8.0 beats/min; P < 0.05) when compared with the control group (Table 2). Plasma levels of noradrenaline were significantly increased in the HFD group (CONT 3.5 ± 0.6 and HFD 8.2 ± 0.4 pmol/mL; P < 0.001), while plasma levels of adrenaline did not change significantly (CONT 11.8 ± 0.5 and HFD 11.4 ± 0.3 pmol/mL; P < 0.001).

Metabolic parameters

The fasting plasma glucose (CONT 108.4 ± 3.2 and HFD 122.1 ± 3.8 mg/dL; P < 0.05) and the plasma insulin (CONT 27.74 ± 4.3 and HFD 41.20 ± 2.9 μIU/mL; P < 0.05) levels were significantly elevated in the HFD group compared to the CONT group (Figure 1). The area under the curve of the glucose levels during the oral glucose tolerance test was also significantly elevated in the HFD group (CONT 14.924 ± 752 and HFD 18.171 ± 488 mg/dL/min; P < 0.0001). The HFD group also presented insulin resistance, compared to the CONT group, as characterized by the HOMA-IR (CONT 6.26 ± 1.0 and HFD 10.0 ± 1.2; P < 0.05) (Figure 1).

image

Figure 1. Fasting plasma levels of glucose and insulin, area under the curve (AUC) of the plasma glucose level and the HOMA-IR in the control (CONT) and the high-fat diet (HFD) groups. HOMA-IR: homeostasis model assessment for insulin resistance. Values represent the mean ± SEM, n = 10 for each group. * P < 0.05 and ** P < 0.01 versus the CONT group.

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The total cholesterol levels were also significantly elevated in the HFD group (CONT 47.86 ± 2.3 and HFD 62.91 ± 4.1 mg/dL; P < 0.05), including the LDL-cholesterol (CONT 11.60 ± 2.0 and HFD 18.59 ± 2.0 mg/dL; P < 0.05) and the triglycerides (CONT 30.03 ± 6.3 and HFD 50.76 ± 6.7 mg/dL; P < 0.05). However, the HDL levels were not significantly affected by the HFD (CONT 29.22 ± 1.5 and HFD 34.31 ± 3.3 mg/dL; P > 0.05) (Figure 2).

image

Figure 2. Plasma levels of total cholesterol, HDL cholesterol, LDL cholesterol and triglycerides in the control (CONT) and the high-fat diet (HFD) groups. HDL: high-density lipoprotein; LDL: low-density lipoprotein. Values represent the mean ± SEM, n =10 for each group. * P < 0.05 versus the CONT group.

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Functional capillary density

The HFD group showed a significantly lower functional capillary density in the skeletal muscle (CONT 250 ± 12 and HFD 157 ± 12 capillaries/mm2; P < 0.001) but not in the skin (CONT 256 ± 10 and HFD 234 ± 15 capillaries/mm2; P > 0.05) when compared with the control group (Figure 3).

image

Figure 3. Capillary perfusion in the skeletal muscle (upper panel) and the skin (lower panel) in the control (CONT) and the high-fat diet (HFD) groups. Values represent the mean ± SEM, n =10 for each group. ***P < 0.001 versus the CONT group.

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Structural capillary density

When compared with the control group, the HFD group showed a significantly lower capillary-to-fiber ratio in the skeletal muscle (CONT 1.86 ± 0.07 and HFD 1.53 ± 0.06 capillaries/muscle fiber; P < 0.01, Figures 4 and 6). The capillary volume density-to-fiber volume density ratio (Vv[cap]/Vv[fib]) in the left ventricle of the HFD group was also significantly reduced when compared with the control group (CONT 0.24 ± 0.024 and HFD 0.16 ± 0.006; P < 0.01, Figures 4 and 6).

image

Figure 4. The capillary-to-fiber ratio in the skeletal muscle (upper panel) and the capillary volume density-to-fiber volume density ratio in the left ventricle (lower panel) in the control (CONT) and the high-fat diet (HFD) groups. Values represent the mean ± SEM, n = 10 for each group. **P < 0.01 versus the CONT group.

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Ratio of left ventricle weight/body weight (LVW/BW), cardiomyocyte diameter and collagen deposition in the left ventricle

The ratio of LVW/BW was significantly increased in the HFD group (CONT 1.7 ± 0.1 and HFD 2.0 ± 0.1 mg/g; P < 0.05, Figures 5 and 6), indicating the development of ventricular hypertrophy in the group fed with HFD. Cardiomyocyte diameter in the left ventricle was also significantly increased in the HFD group (299.7 ± 11.8 μm; P < 0.01) compared with the CONT group (257.8 ± 7.7 μm) (Figures 5 and 6). Finally, collagen deposition in the left ventricle was not significantly increased in the HFD group (32.9 ± 3.8 pixels ×103; P > 0.05) compared with the WKY group (27.0 ± 2.1 pixels ×103) (Figures 5 and 6).

image

Figure 5. Left ventricle weight/body weight ratio (upper panel), left ventricle cardiomyocytes diameter (middle panel), and left ventricle collagen deposition (bottom panel) in the control (CONT) and the high-fat diet (HFD) groups. LVW/BW: Left ventricle weight/body weight ratio. Values represent the mean ± SEM, n = 10 for each group. *P < 0.05 and **P < 0.01 versus the CONT group.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

The main findings of this study are as follows: (i) a long-term high-fat diet with salt supplementation in rats induces metabolic and hemodynamic alterations representative of the MS, which establishes an experimental model of the disease; (ii) the diet also results in marked central body fat distribution, which is a hallmark of MS in humans; and (iii) functional and structural rarefaction of the microvessels is also present in this model and confirms the importance of the microcirculation in the pathophysiology of the MS.

The present study showed that a long-term HFD can expand central adiposity in Wistar rats, as shown by the marked increase in the visceral and epididymal fat storage. Visceral adiposity plays a key role in the development of MS in humans, and visceral adiposity is associated with all the MS criteria independently of insulin sensitivity and abdominal subcutaneous fat area [20]. Thus, our diet-induced experimental model of MS reproduces the main features of the disease. Conversely, the total body weight and food intake of the animals were not different between the HFD and control groups at the end of the experimental period. Actually, total body weight or weight gain are not always correlated with central fat deposition. For instance, Bonomo et al. showed that a small increase in body weight (around 10%) is accompanied by a marked increase (2.3 times) in central adipose tissue deposition in a murine experimental model of maternal hypoprolactinemia at the end of lactation [21]. In another study, wistar rats that received a HFD for 7 weeks had similar total body weight and weight gain at the end of the treatment, compared to rats that receive a low-fat diet [22].

We also demonstrated that the increased visceral fat deposition was accompanied by hypertriglyceridemia and significant elevations in plasma cholesterol levels (total and LDL cholesterol), which are dependent on the fatty acid consumption of the diets [23]. Additionally, increased salt intake increases plasma fatty acids levels, which contributes to alterations in the lipid profile of the animals [24]. The increased efflux of fatty acids and the high circulating levels of triglycerides probably also participates in the induction of hyperglycemia, impaired glucose tolerance and insulin resistance in these animals [24], which are all important features of MS. In this context, a high-salt diet enhances insulin signaling and induces insulin resistance in Dahl salt-sensitive rats [25].

Additional and important features of the experimental model used in the present study are the cardiovascular alterations. In fact, elevations in the arterial pressure and heart rate are consistent findings in patients with MS [26] and constitute robust markers of high cardiovascular risk and the overactivity of the sympathetic nervous system. Interestingly, in the present study, we showed that plasma levels of noradrenaline were markedly elevated in the HFD group, thus confirming the involvement of increased sympathetic activity in the development of the MS. In fact, several components of MS are associated with indirect and direct markers of adrenergic overdrive [27]. Moreover, our rat model of a diet-induced increase in visceral adiposity associated with moderate hypertension suggests a role of metabolic alterations, including dyslipidemia and dysglycemia, in the development of hypertension. Even though we did not use a high-salt diet in the present study, the chronic increase in salt intake in our experimental model also probably contributed to the elevations in arterial pressure. The vasopressor effects of a high-salt diet are, at least in part, independent of direct vasopressor effects and vascular hypertrophy, which are a consequence of chronically elevated arterial pressure [28]. An important contributor to hypertension in salt-sensitive animal models and humans is endothelial dysfunction and, in particular, altered vascular reactivity due to increased oxidative stress and the consequent reduction of nitric oxide bioavailability [29]. Moreover, the endothelial dysfunction characteristic of MS is involved in the pathophysiology of arterial hypertension and plays an important role in microvascular damage due to the elevated plasma glucose levels [29].

The present study showed that the number of spontaneously perfused capillaries in the skeletal muscle of rats that were fed a HFD was significantly reduced when compared with the control animals. On the other hand, the capillary density of the skin, a microvascular bed that is not directly involved in the regulation of peripheral vascular resistance, was not altered by the HFD. This reduction in the functional capillary density is also present in spontaneously hypertensive rats and can be reversed by chronic anti-hypertensive treatment [30, 31]. The functional alterations in the microcirculation, such as capillary rarefaction and impaired capillary recruitment, result in reduced tissue perfusion and can contribute to the development of hypertension and insulin resistance [32, 33]. In a genetic model of obesity in obese Zucker rats, muscle insulin resistance is accompanied by impaired hemodynamic responses to insulin, including reduced capillary recruitment [34]. In fact, insulin recruits skeletal muscle capillaries via a nitric oxide-dependent mechanism, and the increase in capillary recruitment may contribute to the subsequent glucose uptake [35].

Finally, the present study showed that the HFD induces a structural microvascular rarefaction in the skeletal muscle and myocardium of the animals, which is in agreement with our previous studies that showed this same alteration in spontaneously hypertensive rats [30, 31]. In contrast to functional rarefaction, which represents the active closure of small arterioles and capillaries, the structural degeneration of microvessels results from the apoptosis of endothelial cells, which has been demonstrated in different experimental models of hypertension [36, 37]. Anatomic microvascular rarefaction may have important physiological consequences because it contributes to the maintained elevation of the total peripheral resistance and adversely affects oxygen, glucose, and insulin delivery to the tissues, which further contributes to the development of insulin resistance and glucose intolerance, independently of the elevations in the arterial pressure [38].

In our study, the animals fed with a HFD also developed left ventricular hypertrophy (LVH), confirming previous studies in rodents [22]. This cardiac remodeling was further confirmed by a significant increase in cardiomyocyte diameter. On the other hand, the collagen content in the left ventricle was not modified by the HFD. LVH has often been observed in most models of diet-induced obesity or MS, including mice, rats, rabbits, and dogs [39]. Increased sympathetic tone, which was also identified in our experimental model, contributes to the development of concentric LVH through hemodynamic factors, that is, elevated blood pressure and increased cardiac contractility, in addition to the direct cardiac hypertrophic effects catecholamines [40].

Limitations to this study must be considered. First, we did not address the types of muscle fibers involved in the microvascular alterations observed in our experimental model of high fat diet-induced metabolic syndrome, which are known to differentially affect glucose and insulin availability. Second, even if functional capillary density (FCD) is a microcirculatory parameter classically considered to reflect tissue perfusion, FCD is not directly matched to microvascular blood flow. Thus, in future studies microcirculatory blood flow, as well as the complex pathophysiological mechanisms involved in cardiometabolic alterations in our experimental model should be further evaluated.

In summary, we characterized the functional and structural microcirculatory alterations, as well as left ventricular remodeling, associated with metabolic and hemodynamic components in a rat model of high-fat diet-induced metabolic syndrome. Taken together, these findings could be relevant for human pathology and indicate that a moderate increase in salt intake associated with a high-fat diet may lead not only to metabolic and systemic cardiovascular alterations but also to microvascular rarefaction and an acceleration of end-organ damage6.

image

Figure 6. Representative photomicrographs of transverse sections of the skeletal muscle (A) and stereological (B) and transverse (C and D) sections of the left ventricle of control (CONT) and high-fat diet (HFD) rats. Images in (A and B) are representative slides used to determine structural capillary density with capillaries stained with FITC-conjugated G. simplicifolia lectin. Magnification, ×200; scale bar, 100 μm. (C) shows photomicrographs of hematoxilin-eosin-stained sections captured at ×400 magnification. The diameter of cardiomyocytes was calculated with Saisam 5.1.3 software; scale bar, 100 μm. (D) shows photomicrographs of Sirius red-stained sections. Collagen deposition was evaluated using Image Pro Plus software. Magnification, ×100; scale bar, 100 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References