The Effect of Hemp (Cannabis sativa L.) Seeds and Hemp Seed Oil on Vascular Dysfunction in Obese Male Zucker Rats

Seeds of industrial hemp (Cannabis sativa L.) contain a large amount of protein (26.3%), dietary fiber (27.5%), and fatty acids (33.2%), including linoleic, α-linolenic, and some amount of γ-linolenic acid. In our study, obese male Zucker rats (n = 6) at 8 weeks of age were supplemented for a further 4 weeks with either ground hemp seeds (12% diet) or lipid fractions in the form of hemp seed oil (4% diet). Hemp oil decreased blood plasma HDL-cholesterol (x0.76, p ≤ 0.0001), triglycerides (x0.55, p = 0.01), and calculated atherogenic parameters. Meanwhile, hemp seeds decreased HDL-cholesterol (x0.71, p ≤ 0.0001) and total cholesterol (x0.81, p = 0.006) but not the atherogenic index. The plasma antioxidant capacity of water-soluble compounds was decreased by the seeds (x0.30, p = 0.0015), which in turn was associated with a decrease in plasma uric acid (x0.18, p = 0.03). Dietary hemp seeds also decreased plasma urea (x0.80, p = 0.02), while the oil decreased the plasma total protein (x0.90, p = 0.05). Hemp seeds and the oil decreased lipid peroxidation in the blood plasma and in the heart (reflected as malondialdehyde content), improved contraction to noradrenaline, and up-regulated the sensitivity of potassium channels dependent on ATP and Ca2+. Meanwhile, acetylcholine-induced vasodilation was improved by hemp seeds exclusively. Dietary supplementation with ground hemp seeds was much more beneficial than the oil, which suggests that the lipid fractions are only partially responsible for this effect.


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 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.

Data Analysis and Statistics
A nontraditional lipid profile was calculated based on TC, HDL, and TG as log 10 [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 (E max , %), and the potency (pEC 50 ). 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.

Results
The composition of hemp seeds and hemp seed oil was determined in order to prepare the experimental diets. Crude fat was calculated as 100:33.2, so the concentration of hemp seeds was increased three-fold compared with the oil; see Table 1.

Blood Analysis
Supplementation with HO decreased the total protein (x0.90, p = 0.05), but not the

Blood Pressure Measurements
The mean arterial pressure (MAP, x1.30, p = 0.0244, Figure 6A) and heart rate (HR, x0.94, p = 0.0010, Figure 6B Blood plasma fasting glucose only tended to be different among the dietary groups p ≥ 0.1054, with the highest level observed in HS; see Table S1.

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

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 ( TG HDL ), the cholesterol ratio: TC HDL , VLDL, and by an increase in nonHDL HDL and LDL HDL . 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 αand 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 acetylcholinestimulated 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 downregulated 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,).

Conclusions
Dietary supplementation with ground hemp seeds was far more beneficial than with oil, which suggests that the lipid fractions, mainly including PUFAs, are only partially responsible for this effect. In both cases, dietary hemp supplementation was unable to attenuate the development of obesity with its complications, despite the cholesterollowering effect, some improvement in the vascular functioning, and changes in blood plasma antioxidant status.