1. Introduction
Vascular dysfunction, including compromised vasodilation and vasoconstriction, is an important complication of chronic obesity [
1]. Metabolic disorders associated with obesity, such as dyslipidemia, low-grade systemic inflammation, and increased oxidative stress, can lead to the development of atherosclerosis, hypertension, and some other cardiovascular disorders [
2]. Therefore, there is a need to find ways to prevent and treat the early development of obesity with its complications. A good experimental model of obesity and vascular dysfunction is homozygous recessive Zucker rats (
fa/
fa), which have a mutation in a gene-encoding leptin receptor, resulting in a lack of sensitivity to circulating leptin in the blood [
1,
3]. In Zucker rats, leptin is unable to inhibit neuropeptide Y secretion in the hypothalamus that potentiates appetite, and thus a meaningful increase in dietary intake and body weight is observed together with the occurrence of the aforementioned metabolic disorders [
3].
An increased contribution of plant-based food is one of the ways to prevent obesity and associated metabolic disorders, which is partly due to the relatively low caloric value of such food [
4]. However, food from plants is also a good source of bioactive compounds that can directly bring benefits to the cardiovascular system, a good example of which are polyunsaturated fatty acids (PUFAs) with α-linolenic acid and linoleic acid as their main representatives in nature [
5]. Besides their nutritional importance, these PUFAs, especially from the n-3 family, can improve lipid metabolism and the inhibition of lipid synthesis in the body [
6,
7]. PUFAs are also believed to prevent lipid accumulation in the arteries and decrease the development of hypertension. However, the consumption of PUFAs can also bring some negative consequences to the body, which especially applies to the fatty acids of the n-6 family that can be metabolized to pro-inflammatory eicosanoids [
7].
An interesting and relatively less known source of fatty acids is the seeds of industrial hemp (
Cannabis sativa L.), as they contain a large amount of PUFAs, including linoleic and α-linolenic acids (approx. 53% and 18% of total fatty acids) and some small amount of other fatty acids that are uncommon in vegetable oils, such as γ-linolenic acid [
8,
9]. The oil from hemp seeds is characterized as having an optimal ratio of n-3 to n-6 PUFAs (1:3), so important for the adequate functioning of the cardiovascular system; however, hemp oil contains trace amounts of trans fatty acids, which are generally thought to raise the risk of atherosclerosis by inhibiting the synthesis of other PUFAs in arterial cells [
7,
9]. Nevertheless, Al-Khalifa et al. [
10] demonstrated that hearts from rats fed with hemp seeds exhibited significantly better postischemic recovery of maximal contractile function and relaxation during reperfusion compared to the control group. The authors concluded that this was due to PUFAs from hemp seeds. However, hemp seeds are also rich in peptides that have been recognized as a potential antihypertensive agent [
11] and lignan amides with potential anti-inflammatory and cardiovascular activities [
12]. Moreover, in the latest study conducted in one of our laboratories, relatively strong hypolipidemic effects of dietary hemp in genetically obese rats were observed, and the oil fraction was only partially responsible for these effects [
9].
The aforementioned findings suggest that the consumption of hemp seeds is beneficial for the cardiovascular system compromised by obesity. However, the extent to which PUFAs derived from hemp seeds can be responsible for these beneficial effects has not yet been studied nor the mechanisms underlying their vascular protection. Thus, we aimed to compare the effects of hemp seeds (12% diet) with the corresponding amount of lipid fractions from hemp seed oil (4% diet) on the cardio-vascular system in genetically obese Zucker rats. We hypothesized that supplementation with either the seeds or the oil can improve metabolic dysfunction of genetic origin and that the seeds are more effective due to a wider range of potentially bioactive compounds.
2. Materials and Methods
2.1. Drugs and Chemicals
Acetylcholine chloride, sodium nitroprusside, and noradrenaline hydrochloride were obtained from Sigma-Aldrich (St. Louise, MO, USA); potassium chloride from Chempur (Piekary Śląskie, Poland); pinacidil, NS1619, and U-46619 from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions (10 mM) of these drugs were prepared in distilled water, except for noradrenaline, which was dissolved in NaCl (0.9%) + ascorbic acid (0.01% w/v) solution; pinacidil, NS-1619 were dissolved in DMSO, and U-46619 in ethanol.
These solutions were stored at −20 °C, and appropriate dilutions were made in Krebs-Henseleit solution (KH in mmol/L: NaCl 115; CaCl2 2.5; KCl 4.6; KH2PO4 1.2; MgSO4 1.2; NaHCO3 25; glucose 11.1) on the day of the experiment. The maximal solvent concentration in the medium was less than 0.01% (vol/vol). At these concentrations, solvents did not alter the reactivity of the studied arteries.
Hemp seeds were purchased from Ekogram (Zielonki, Poland), and unrefined, cold-pressed hemp seed oil was obtained from Ol’Vita (Panków, Poland).
2.2. Chemical Composition of Hemp Seeds and Hemp Seed Oil
Following methods were applied: gravimetric method at a high temperature 105 °C/∼580 °C for dry matter (DM) content and ash; enzymatic–gravimetric method for total dietary fiber; Kjeldahl method for crude protein; the Soxhlet extraction for crude fat; and gas chromatography with flame ionization detection (Hewlett Packard 5890, Wilmington, DE, USA) for fatty acids methyl esters, as previously described [
5].
2.3. Experimental Protocol
All efforts were made to minimize animal suffering. Male Zucker rats (Charles River, Sulzfeld, Germany) at 8 weeks of age were randomly allocated to 4 groups (n = 6/group) and fed for a further 4 weeks with experimental diets in form of pellets. The lean controls (LC) and obese controls (OC) were fed with a standard rat chow, whereas the other 2 obese groups were fed a modified diet in which either hemp seeds (12% diet; HS) or hemp seed oil (4% diet; HO) were included. All these diets were prepared in such a manner so that they had the same amount of carbohydrate (52%), protein (18%), fat (8.3%), and fiber (5%). Diets fed to the O+HS group and O+HO group also had a similar fatty acid profile.
The rats were individually housed in plastic cages under a controlled environment (a 12 h light–dark cycle, a temperature of 21 ± 1 °C, relative humidity of 50–70%, and 20 air changes per hour) [
13].
2.4. Experimental Procedures
The mixture of ketamine + xylazine (100 + 10 mg/kg BW) was used for intraperitoneal anesthesia [
14]. The whole blood was kept in tubes containing heparin + EDTA as an anticoagulant and centrifuged at 3000×
g for 10 min to separate the blood plasma, which was stored at –80 °C until further analysis. The thoracic arteries were carefully isolated and kept in a Krebs–Henseleit buffer at +4 °C.
2.5. Blood and Heart Tissue Analysis
The traditional blood plasma lipids (the total cholesterol—TC, high-density lipoprotein cholesterol—HDL, triglycerides—TG; mmol/L), gamma-glutamyl transferase (GGT, U/L), uric acid (µmol/L), urea (mmol/L), creatinine level (µmol/L), albumin (µmol/L), and total protein (g/L) were measured with a biochemical autoanalyzer (Horiba, Kyoto, Japan) [
14].
The malondialdehyde-thiobarbituric acid (MDA-TBA) adduct was quantified at 532/553 nm (Ex/Em) with a Fluorometric Assay Kit (ab118970), and the values were expressed as µmol/L of the blood plasma and ng/g of heart tissue.
The antioxidant capacity of water- and lipid-soluble compounds of the blood plasma (ACW and ACL, respectively; mg/L) were determined by a photo-chemiluminescence detection method using Photochem (Analytik Jena AG, Germany). This method is based on the generation of free radicals that are partially eliminated through a chemical reaction with antioxidants present in the plasma sample, and the remaining radicals are quantified by luminescence generation. The lipid fraction was separated using methanol, n-hexane, and centrifugation. Calibration curves are based on Ascorbate and Trolox as standards for ACW and ACL, respectively.
2.6. Glucose Tolerance Test
Rats were given a 50% glucose solution by oral gavage (2 g/kg of body weight) after overnight starvation, 4 days before the final termination. Blood samples were collected from the tail tip, and glucose was measured with a glucometer (Accu-Chek Active, Roche Diagnostics, Germany) at 0, 15, 30, 60, 90, 120, and 180 min.
2.7. Blood Pressure Measurements
Mean arterial pressure, MAP (mmHg) and heart rate, HR (bpm) were monitored on the day before the blood collection with the noninvasive tail-cuff method (LE5001, Panlab, Harvard Apparatus, Barcelona, Spain).
2.8. Thromboxane-A2 Quantification
This was completed following Majewski et al. [
2]. Briefly, after a stabilization period in KHS at +37 °C for 30 min (pH 7.4), aortic rings from each group of rats were followed by 2 wash periods of 10 min using 200 μL of KHS. Once fresh KHS was replaced, arteries were exposed to noradrenaline (0.1 μM, 2 min) and then to the cumulative acetylcholine concentrations (0.1 nM–10 μM) at 1 min intervals. The medium was collected and stored at −80 °C until further analysis. Production of thromboxane-A
2 was monitored by measuring the stable metabolite thromboxane-B
2. This was completed using the appropriate enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA). Results are expressed as pg/mg of tissue.
2.9. Vascular Reactivity Studies
This was previously described in detail by Majewski et al. [
14,
15]. Briefly, isolated aortic rings of 4 mm length were aerated in a stagnant 5 mL Graz Tissue Bath System (Harvard Apparatus, March-Hugstetten, Germany) for 60 min, under 1 g pre-load tension, and precontracted with noradrenaline (0.1 μM). Then, the cumulative concentrations of either acetylcholine (0.1 nM–10 μM), sodium nitroprusside (0.001 nM–10 μM), pinacidil (10 nM–10 μM), or NS-1619 (1 nM–10 μM) were added into the bath chambers to study the vasodilator response. In another set of experiments, vasoconstriction was studied with cumulative concentrations of noradrenaline (0.1 nM–10 μM) and U-46619 (0.1 nM–1 μM).
2.10. Data Analysis and Statistics
A nontraditional lipid profile was calculated based on TC, HDL, and TG as log
10,
, TC
minus HDL, VLDL (calculated as:
), LDL (calculated as TC
minus HDL
minus VLDL),
,
and
.
NonHDL was calculated as TC
minus HDL [
14,
16,
17]. MAP was calculated as DP + 1/3(SP − DP), where DP is the diastolic blood pressure and SP is the systolic blood pressure.
The contraction induced by high KCl (75 mM) was expressed in mg of developed tension; meanwhile, contraction with noradrenaline and U-46619 was expressed as % of KCl-induced response. Vascular relaxation was expressed as a percentage of the contractile response to noradrenaline NA (0.1 μM). This concentration of NA was chosen based on the preliminary studies with cumulative doses of NA added into the incubation chambers. The cumulative concentration–response curves were analyzed by a nonlinear regression model, which determined the area under the curve (AUC), maximal response (Emax, %), and the potency (pEC50). The group comparison was performed by either a parametric (ANOVA) or non-parametric test (Kruskal–Wallis test), with n = 6. The Gaussian distribution of residuals and homoscedasticity of variance were tested. The Grubbs’ test was performed to detect outliers. The post hoc tests were run only when F achieved the necessary level of statistical significance (p ≤ 0.05). The group comparison was performed by Mann–Whitney’s test. Results are expressed as means ± SD (and means ± SEM for vascular studies). This research was randomized and stayed blinded for laboratory analyses. The level of significance was when p ≤ 0.05.
4. Discussion
Previously, we had reported that ground seeds from dietary hemp (
Cannabis sativa L.) more effectively attenuate metabolic disorders compared with the oil fraction from hemp seeds [
9]. Now, we have further investigated the influence of dietary supplementation with hemp seeds (12% of diet) vs. corresponding concentration of hemp seed oil (4% of diet) on vascular dysfunction, blood pressure, and heart rate, the blood plasma lipid profile, oral glucose tolerance, antioxidant capacity, and renal functioning in obese Zucker rats, a model of obesity.
As was stated before, experimental supplementation neither modified the body weight gain nor the food intake of supplemented obese Zucker rats [
9].
The influence of the oil from hemp seeds on lipid metabolism is well documented, contrary to the effect of the ground seeds, which is poorly studied as of yet. Therefore, we have undertaken further research. Both ground seeds and the oil were able to affect the lipid metabolism (decrease in the plasma HDL cholesterol), although the effectiveness of the seeds was much more indicated (decrease in the plasma total cholesterol). The plasma triglycerides concentration was not significantly decreased by the seeds, but it was by the oil, which is in accordance with our previous results [
9]. We have further calculated a nontraditional lipid profile, which is an even better marker of atherogenicity [
14,
16,
18]. Surprisingly, the effect of hemp oil was much more pronounced compared to the seeds, as indicated by a decrease in the AIP: log
10, the cholesterol ratio:
, VLDL, and by an increase in
and
. Neither hemp seeds nor hemp oil improved the impaired glucose tolerance that was induced in the obese group of rats, yet these favorable effects of hemp seeds and oil were not as defined on blood glucose as those on the lipids.
Hemp seeds are a good source of fatty acids (33.2%), see
Table 1. In our study, the fatty acid profile of the seeds and the oil from hemp was found to be similar. The main PUFAs determined were linoleic acid, α-linolenic acid, and γ-linolenic acid, with an abundance of ∼52%, 18%, and 4%. Moreover, hemp seeds are also a good source of protein (26.3%) and dietary fiber (27.5%), which may explain the observed differences in the blood plasma lipids. Highly digestible proteins and dietary fiber can trigger a rise in protein synthesis of smooth muscles [
19] and increase the gut microbial glycolytic activity: β-glucosidase as well as α- and β-galactosidase [
9].
In addition, this study is the first to describe the reactivity of isolated thoracic arteries in hemp-supplemented obese Zucker rats. This specific rat model is characterized by a number of metabolic disorders, including vascular dysfunction and increased oxidative stress. Supplementation with hemp seeds and seed oil beneficially potentiated (already decreased,
Figure 8B) vasoconstriction in response to noradrenaline. However, this neither changed the membrane depolarization induced by high KCl nor the response to the thromboxane-A
2 analog, U-46619. It is worth mentioning that metabolic dysfunction observed in obese Zucker rats decreased depolarization and enhanced U-46619-induced contraction, as was presented in
Figure 8A,C. Moreover, supplementation with hemp did not decrease the thromboxane-A
2 level in blood vessels under basal and acetylcholine-stimulated conditions in obese Zucker rats.
Next, we studied impaired vascular relaxation observed in obese Zucker rats. The attenuated relaxant response to acetylcholine (
Figure 9A) was improved by the seeds but not by the oil. Surprisingly, the vasodilator response to exogenous nitric oxide (which is attenuated in obese Zucker rats (
Figure 9B) was not modified with dietary hemp. This indicates that the sensitivity of the smooth muscles of rat thoracic aorta to nitric oxide was not modified during supplementation and that it was the endothelial functioning that was improved by the seeds but not by the oil. In rat thoracic arteries, K
ATP and BK
Ca channels are also engaged in vascular tone regulation to compensate for the attenuated vascular relaxation [
2]. The relaxant response to the K
ATP channel opener was down-regulated in obese rats, which points to a decreased sensitivity (
Figure 9C). Experimental supplementation with hemp improved the impaired functioning of these channels and shifted that response to the left. However, the sensitivity was not fully restored to the level observed in the lean controls. Next, we examined the impaired vasodilator response with a BK
Ca channel opener (
Figure 9D). In our study, supplementation with hemp increased both the sensitivity and the maximal response, which was more pronounced in rats fed with seeds (increased sensitivity). Our results point to an improvement in the functioning of K
ATP and BK
Ca channels in response to the dietary hemp, with a more beneficial effect from the seeds than the oil.
Despite these beneficial effects on the vascular system, neither HO nor HS had any beneficial impact on impaired mean arterial pressure and heart rate of obese Zucker rats.
We noticed an increased plasma antioxidant capacity of lipid- and water-soluble compounds (
Figure 4A,B) as well as MDA in the blood plasma and the heart (
Figure 4I,J) in obese Zucker rats compared to the lean controls, perhaps as a response to the increased oxidative stress, which up-regulated the mechanism(s) responsible for the antioxidant defense and potentiated lipid peroxidation. Both dietary seeds and the oil decreased the lipid peroxidation in the blood plasma and in the heart. However, the effectiveness of the seeds was more indicated, which was reflected by a decrease in the plasma antioxidant capacity of water-soluble compounds. This was not observed for hemp oil and the plasma antioxidant capacity of lipid-soluble compounds.
The decreased plasma antioxidant capacity of water-soluble compounds by the seeds is associated with the decreased plasma uric acid (
Figure 4C), which is strongly hydrophilic, but not with the plasma levels of albumin (
Figure 4F) nor bilirubin (data not shown). All these components, together with vitamin C, are considered the main blood plasma antioxidants in humans [
20,
21]. Paradoxically, plasma uric acid positively correlates and predicts the development of obesity, hypertension, and cardiovascular disease [
21]. Thus, the decreased plasma uric acid by the seeds in the present study can be considered beneficial for the body, especially when looking at its role in the development of gout. Support of this supposition can be found in the study by Opyd et al. [
9], who showed that dietary supplementation with hemp seeds improved the antioxidant status of the liver in Zucker rats by increasing glutathione levels and decreasing a marker of lipid peroxidation. This means that a considerable decrease in the plasma antioxidant capacity and uric acid level does not automatically exclude benefits coming from hemp seed supplementation to the organ’s antioxidant defense system.
Moreover, gamma-glutamyl transferase and blood plasma creatinine were neither modified by obesity itself (
Figure 4E,H) nor by hemp supplementation, as opposed to blood plasma total protein level, which was decreased by HO. It is worth mentioning that obesity increased blood plasma albumins, total protein, and urea (
Figure 4D,F,G,).