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The Journal of Nutrition

Nutrition and Disease

Dietary Hemp Seeds More Effectively


Attenuate Disorders in Genetically Obese

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Rats than Their Lipid Fraction
Paulina M Opyd, Adam Jurgoński, Bartosz Fotschki, and Jerzy Juśkiewicz

Department of Biological Function of Food, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn,
Poland

ABSTRACT
Background: Hemp seeds are rich in PUFAs and other bioactives that can attenuate the development of obesity-related
disorders; however, the extent to which their lipid fraction is responsible for this effect is unknown.
Objective: We hypothesized that hemp seed or hemp oil supplementation can attenuate genetically determined
disorders and that the former are more effective in doing so.
Methods: Lean and obese male Zucker rats, aged 8 wk, weighing 174 ± 4.2 g and 223 ± 3.8 g, respectively, were
allocated to 4 groups. The lean (LC) and obese controls (OC) were fed a standard diet, whereas the other 2 obese
groups were fed a modified diet in which hemp oil (4% diet; O + HO) or hemp seeds (12% diet; O + HS) were included.
All diets had the same proportions of protein (18%), fat (8%), and fiber (5%) and a similar carbohydrate proportion
(∼52%). Diets fed to O + HO and O + HS had similar fatty acid profiles. After 4 wk, markers of gut and liver function,
antioxidant status, and lipid metabolism were measured.
Results: The total SCFA concentration in the cecal digesta was lower in OC (64.8 ± 4.21 mol/g) compared with LC
(78.1 ± 2.83 mol/g) (P ≤ 0.05), whereas it was greater in O + HS (89 ± 4.41 mol/g) compared with LC, OC, and
O + HO (69.7 ± 2.68 mol/g) (P ≤ 0.05). Plasma total cholesterol was greater in OC (6.20 ± 0.198 mmol/L) and O + HO
(5.60 ± 0.084 mmol/L) compared with LC (2.71 ± 0.094 mmol/L) (P ≤ 0.05); in O + HS, the concentration did not differ
from the other groups (5.16 ± 0.278 mmol/L). The liver cholesterol concentration was greater in OC (1.79 ± 0.379 mg/g)
compared with the other groups (1.28–1.43 mg/g) (P ≤ 0.05). Hepatic expression of peroxisome proliferator-activated
receptor γ was lower in OC (11.9 ± 0.93 units) compared with LC (17.3 ± 1.3 units) (P ≤ 0.05), whereas it was greater
in O + HS (19.2 ± 1.04 units) compared with OC and O + HO (14.0 ± 1.33 units) (P ≤ 0.05).
Conclusions: Dietary hemp seeds more effectively attenuate metabolic disorders in genetically obese rats than the oil
extracted from them, which suggests that the lipid fraction is only partly responsible for these effects. J Nutr 2020;00:1–
9.

Keywords: hemp seeds, hemp seed oil, gut, metabolic disorders, genetic obesity

Introduction including the most studied cannabinoids; however, only traces


of these compounds are present in the seeds of industrial hemp,
Cannabis sativa L., commonly called industrial hemp, can
but they contain significant amounts of lignanamides (7, 8).
play an important role in the human diet, providing both
These phenolic amides are claimed to have cytotoxic, anti-
nutrients and bioactive compounds (1). Hemp seeds contain
inflammatory, antineoplastic, and cardioprotective activity (7).
∼26% protein rich in arginine, aspartic acid, and glutamic acid,
Nevertheless, hemp seeds also contain some antinutritional
whereas lysine is the limiting amino acid (1–3). Antioxidative
components, especially phytic acid (∼63 mg/g), which can
and antihypertensive peptides from hemp seeds have already
reduce the absorption of mineral elements (9).
been isolated (4, 5), and some oligopeptides exhibited α-
Hemp seeds contain a high percentage of oil (∼36%), which
glucosidase inhibitory activity, which suggests their use as
is usually extracted by cold pressing, and have a characteristic
potential antihyperglycemic agents (6). Hemp seeds are also rich
green color and fresh nutty taste and smell (10). Hemp seed
in dietary fiber, the content of which is ∼28%, including its
oil has a high percentage of PUFAs, including linoleic acid
mainly insoluble fraction (22%) that can beneficially affect an
(56%), α-linolenic acid (20%), γ -linolenic acid (4%), and
organism, especially gut function (1).
stearidonic acid (2%); the latter 2 are rare in nature (1, 11).
A distinctive feature of the Cannabis genus, especially the C.
Moreover, hemp seed oil is characterized by the optimal ratio
indica variety, is that it contains over 480 bioactive compounds,

Copyright  C The Author(s) 2020.


Manuscript received October 8, 2019. Initial review completed January 16, 2020. Revision accepted March 9, 2020.
First published online 0, 2020; doi: https://doi.org/10.1093/jn/nxaa081. 1
of n–6 to n–3 PUFAs (3:1), which is especially important for company (Panków). The chemical composition of hemp seeds and
cardiovascular health (12). Furthermore, Montserrat-de la Paz hemp seed oil was determined by an accredited research laboratory
et al. (13) reported some amounts of other bioactive compounds (Nuscana, Mrowino, Poland). In the seeds, the dry matter (DM) and
in the oil, such as sterols and their derivatives (β-sitosterol, ash content were determined using the gravimetric method after drying
at 105◦ C and ashing at ∼580◦ C. Total dietary fiber was determined
campesterol, cycloartenol) and γ -tocopherol (14). Interestingly,
by the enzymatic-gravimetric method, crude protein was determined
Al-Khalifa et al. (15) demonstrated that dietary hemp seeds can
by the Kjeldahl method, crude fat was determined by the Soxhlet
provide significant cardioprotective effects during postischemic extraction method and then the nitrogen-free extract was calculated.
reperfusion. The authors suggested that it is due to its high PUFA The fatty acid profile of the oil and oil fraction extracted from the
content; however, the extent to which the oil is responsible for seeds was determined by GC with flame-ionization detection after

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such effects remains unknown. previous conversion of the fatty acids into their respective methyl esters.
Among various animal models of diseases, Zucker rats Supplementary Table 1 details the basic chemical composition of the
deserve special attention as an example of the best known and seeds and fatty acid profile of the seeds and oil.
most widely used animal model of genetic obesity (16). Obesity
in Zucker rats is inherited as an autosomal recessive trait caused Animals and experimental design
by a mutation in the leptin receptor gene (17). The mutation The feeding experiment was conducted on 30 lean (Fa/?) and obese
relies on a single adenine to cytosine change at nucleotide (fa/fa) Zucker male rats aged 8 wk, which were allocated to 4 groups
880, which results in a glutamine to proline substitution at (1 lean and 3 obese). Initial body weight (BW) of the rats is shown in
residue 269 (18). Leptin is produced by adipose tissue and Table 1. The rats were individually housed in plastic cages and
affects the brain via the leptin receptor, which leads to decreased a controlled environment (12-h light-dark cycle, a temperature of
21 ± 1◦ C, relative humidity of 50% to 70%, and 20 air changes
food intake and increased energy expenditure (19). As a result
per hour). For 29 d, each group was fed a modified version of the
of the mutation, obese Zucker (fa/fa) rats are insensitive to semipurified casein diet recommended for rodents by Reeves (21). The
leptin circulating in their body (20). They are characterized lean control (LC) and obese control (OC) groups were fed a diet
by food overconsumption, disturbed energy homeostasis, and containing casein, cellulose, and rapeseed (canola type) and palm oil
drastic weight gain. An additional aspect of their phenotype is (4% of each oil) as the source of protein, fiber, and fat, respectively. The
disorders similar to those seen in human metabolic syndrome, other 2 obese groups were fed a modification of the standard diet in
such as fatty liver, dyslipidemia, insulin resistance, chronic which hemp seed oil was added at the expense of palm oil (4% diet; O
inflammation, and some endocrinological abnormalities (19). + HO group) or ground hemp seeds were added at the expense of palm
Most of the available studies have focused either on the oil, cellulose, and casein (12.05% diet; O + HS group). Hemp seeds were
antioxidative and antihypertensive activities of peptides isolated ground for 1 min at a temperature below 37◦ C prior to their inclusion in
the diet. All diets had the same proportion of protein (18%), fat (8.3%),
from hemp seeds (4, 5) or on the cardioprotective properties of
and fiber (5%) and a similar carbohydrate proportion (∼52%). Diets
hemp seed oil (12). However, to date, the extent to which the fed to the O + HO and O + HS groups also had similar fatty acid
lipid fraction can contribute to the health effects of hemp seed profiles, including a >2-fold increase in the proportion of PUFAs that
consumption has not been studied. At the same time, a similar originated either from hemp seeds or hemp seed oil. This experimental
lack of knowledge is seen when considering the extent to which design allowed us to assess the extent to which the lipid fraction of
dietary factors can affect the development of genetic obesity in hemp seeds can contribute to the health effects of their consumption.
general. Therefore, the aim of this study was to compare the The detailed composition of the diets, which were freely available to
effect of dietary supplementation with hemp seeds and hemp rats for the entire experimental period, is shown in Supplementary
seed oil on the development of metabolic disorders in genetically Table 2. All diets were in a powdery form and during preparation,
obese Zucker rats. We hypothesized that hemp seed or hemp powdered ingredients of commercial origin, such as casein preparation,
corn starch preparation, vitamin mixture, etc. were mixed together in an
seed oil supplementation can attenuate genetically determined
ordinary mixer and under standard conditions. After preparation, the
metabolic disorders and that the former is more effective due to diets were stored at 4◦ C. The experiment was conducted in compliance
a wider range of potentially bioactive compounds present in the with the European guidelines for the care and use of laboratory animals,
seeds. and the animal protocol employed in this study was approved by the
local Institutional Animal Care and Use Committee in Olsztyn, Poland
(permission number: 37/2017).
Methods
Chemical composition of hemp seeds and hemp seed Analysis of body composition in rats
oil At the beginning and end of the feeding experiment, the body lean and
This study used whole hemp seeds (Cannabis sativa L.), which were a fat masses of the rats were determined by time-domain NMR using the
certified organic food product from the Ekogram company (Zielonki), Minispec LF 90II analyzer (Bruker). The method relies on transmitting
whereas unrefined, cold-pressed hemp seed oil was from the Ol’Vita various radio frequency pulses into soft tissues to reorient the nuclear
magnetic spins of the hydrogen and then detects radio frequency signals
generated by the hydrogen spins from these tissues. The contrast in
This research was financially supported by the National Science Centre, Poland
relaxation times of the hydrogen spins found between adipose tissue
(project number: 2016/23/B/NZ9/01012).
and water-rich tissues is used to estimate fat and lean body masses.
Author disclosures: The authors report no conflicts of interest.
Supplemental Tables 1 and 2 are available from the “Supplementary data” link
in the online posting of the article and from the same link in the online table of Collection of biological material and analytical
contents at https://academic.oup.com/jn. procedures
Address correspondence to AJ (e-mail: a.jurgonski@pan.olszty.pl).
After 4 wk of experimental feeding, rats were anesthetized with a
Abbreviations used: Actb, β-actin; ALP, alkaline phosphatase; ALT, alanine
mixture of xylazine and ketamine in physiological salt (10 mg and
transaminase; AST, aspartate transaminase; DM, dry matter; GSH, glutathione;
GSSG, glutathione disulfide; LC, lean control; OC, obese control; O + HO, obese 100 mg/kg BW, respectively). Each animal was then weighed, and the
fed a diet containing hemp seed oil; O + HS, obese fed a diet containing hemp abdomen was cut open. Blood was subsequently collected from the vena
seeds; Pparα, peroxisome proliferator-activated receptor α; Pparγ , peroxisome cava into heparinized tubes, centrifuged for 10 min at 380 × g and
proliferator-activated receptor γ ; Srebp1c, sterol regulatory element-binding 4◦ C, and the obtained plasma was then frozen until analysis. Next, the
protein 1c; TBARS, thiobarbituric acid-reactive substances; TFA, trans fatty acid. small intestine, cecum, colon, kidneys, liver, and epididymal fat were

2 Opyd et al.
TABLE 1 Diet intake, body weight, and body composition of lean and obese Zucker rats fed a diet containing hemp seed oil or hemp
seeds for 4 wk1

Group2
Index LC OC O + HO O + HS ANOVA P value
Initial body weight, g 174 ± 4.2b 224 ± 5.1a 227 ± 8.8a 219 ± 6.6a ≤0.001
Initial body fat, % 16.3 ± 0.54b 47.1 ± 0.58a 47.7 ± 2.33a 46.4 ± 1.25a ≤0.001
Initial body lean, % 77.6 ± 0.59a 45.3 ± 0.54b 45.5 ± 2.80b 46.3 ± 1.17b ≤0.001
± 0.248b ± 0.854a ± 0.580a ± 0.591a ≤0.001

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Diet intake, g/d 15.2 23.9 23.7 24.0
Final body weight, g 266 ± 4.23b 367 ± 10.7a 373 ± 8.62a 350 ± 8.68a ≤0.001
Final body fat, % 22.3 ± 0.854b 68.0 ± 0.869a 65.6 ± 0.610a 67.6 ± 1.95a ≤0.001
Final body lean, % 67.1 ± 1.11a 24.2 ± 1.07c 27.4 ± 0.6851b,c 29.4 ± 2.05b ≤0.001
Body weight gain, g 91.8 ± 4.07b 143.6 ± 6.69a 145.6 ± 7.692a 132.2 ± 7.10a ≤0.001
Body fat gain, g 31.0 ± 2.01b 145.0 ± 8.28a 137.0 ± 5.123a 133.1 ± 5.77a ≤0.001
Body lean gain, g 43.4 ± 3.69a − 13.2 ± 3.18c − 0.270 ± 5.85b,c 2.97 ± 5.25b ≤0.001
1
Values are means ± SEMs, n = 7–8. Labeled means in a row without a common letter (a, b, c) differ at P ≤0.05 (Duncan’s or Dunn’s post hoc test).
2
LC, lean control; OC, obese control; O + HO, obese fed a diet containing hemp seed oil; O + HS, obese fed a diet containing hemp seeds.

removed, weighed, and frozen using liquid nitrogen or were used for analyzed using Single Tube TaqManVR Gene Expression Assays (Life
further treatment. Technologies). Amplification was performed using a 7900HT Fast Real-
Disaccharidase activity (lactase, maltase, and sucrase) was assayed in Time PCR System. The mRNA expression levels of Pparα, Pparγ , and
jejunal mucosal samples collected using a microscope slide according to Srebp1c were normalized to Actb and multiplied by 10.
the method of Dahlqvist with previously described modifications (22).
The disaccharidase activity was expressed as mol of glucose liberated
Statistical analysis
from disaccharide per min per gram of protein. The mucosal protein
The results are expressed as the mean ± SEM: however, the chemical
content was determined using the Bradford method with BSA as the
composition of hemp seeds and hemp seed oil is expressed as the
standard.
mean ± SD. One-factor ANOVA and Duncan’s multiple range post
Samples of fresh ileal, cecal, and colonic digesta were collected, and
hoc test were used to determine significant differences between groups
the pH values were measured using a microelectrode and pH/ION meter
(P ≤ 0.05). If the variance was not homogenous, the Kruskal–Wallis
(model 301, Hanna Instruments). The ammonia concentration in the
1-factor ANOVA by ranks was used followed by Dunn’s post hoc test
fresh cecal digesta was determined in Conway dishes according to the
(P ≤ 0.05). All calculations were performed using STATISTICA version
method described by Hofirek and Haas (23). The SCFA concentration
12 (StatSoft Corp.).
was determined in cecal digesta after storage at −20◦ C using GC (Shi-
madzu Co.) and a capillary column (SGE BP21, 30 m × 0.53 mm; SGE
Europe Ltd.) as previously described (24). Microbial glycolytic activity
in the colonic digesta (α- and β-glucosidase, α- and β-galactosidase,
and β-glucuronidase) was measured spectrophotometrically using the
Results
rate of p- or o-nitrophenol release from nitrophenylglucosides (Sigma- The chemical composition of hemp seeds and hemp seed oil is
Aldrich) according to the method described by Cholewińska et al. (25). shown in Supplementary Table 1. The main component of the
The enzymatic activity (α- and β-glucosidase, α- and β-galactosidase,
seeds was fat, the proportion of which equaled 33.2% DM.
and β-glucuronidase) was expressed as micromoles of product formed
per hour per gram of digesta.
Hemp seeds were also a good source of crude protein and
The glutathione (GSH) and glutathione disulfide (GSSG) content dietary fiber; the proportions of these components were 26.3%
in the liver tissue was determined spectrophotometrically using the and 27.5% DM, respectively. The fatty acid content of the seeds
method of Rahman et al. (26). Thiobarbituric acid-reactive substances and oil was similar and expressed as the percentage of total
(TBARS) were determined in the kidney and liver tissue after storage fatty acids. Both hemp seeds and hemp seed oil were rich in
at −20◦ C using a procedure developed by Botsoglou et al. (27). The PUFAs (76.4% and 76.1%, respectively), especially in linoleic
TBARS content was determined spectrophotometrically at 532 nm and acid (52.3% and 52.8%, respectively), α-linolenic acid (18.1%
expressed as micrograms of malondialdehyde per gram of tissue. Liver and 17.5%, respectively), and γ -linolenic acid (4.4% and 4.3%,
lipids were extracted according to the method of Folch et al. (28) respectively). The total content of MUFAs equaled 9.37% and
with previously described modifications (29). The liver cholesterol and
10.0% for the seeds and oil, respectively, including oleate as the
triglyceride concentrations were determined spectrophotometrically in
the extracted lipid phase using reagents from Alpha Diagnostics Ltd.
main MUFA (7.9% and 8.6%, respectively). The overall content
The plasma concentration of cholesterol (total and its HDL and of SFAs in the seeds and oil was 9.34% and 9.02%, respectively,
LDL fractions), triglycerides, urea, uric acid, creatinine and the plasma including palmitate as the main SFA (5.80% and 5.46% of total
activities of aspartate transaminase (AST), alanine transaminase (ALT), fatty acids, respectively). Interestingly, the fatty acid fraction of
and alkaline phosphatase (ALP) were determined using an automatic the seeds and oil also contained a small proportion of a trans
biochemical analyzer (Pentra C200, Horiba Ltd.). fatty acid (TFA, cis-9, trans-12-octadecadienoic acid, 0.11%
and 0.12%, respectively).
The effect of dietary supplementation with hemp seeds and
Gene expression analysis in the liver hemp seed oil on rat BW and body composition is shown in
qRT-PCR was performed according to the previously described method
Table 1. The initial BW and body fat percentage were similarly
(29). Briefy, RNA was extracted from the liver using TRI Reagent
solution (Thermo Fisher Scientific) according to the manufacturer’s higher, whereas the initial body lean percentage was similarly
instructions. β-actin (Actb) was selected as a reference gene. The levels lower in all 3 obese groups compared with the LC group
of peroxisome proliferator-activated receptor α (Pparα), peroxisome (P ≤ 0.05). The initial body fat percentage was almost 3 times
proliferator-activated receptor γ (Pparγ ), sterol regulatory element- lower in the LC group compared with the obese groups, which
binding protein 1c (Srebp1c), and Actb mRNA expression were did not differ from one another. Moreover, the dietary intake
Effects of dietary hemp seeds and hemp seed oil 3
TABLE 2 Physiological indices of the small intestine, cecum, and colon in lean and obese Zucker rats fed a diet containing hemp
seed oil or hemp seeds for 4 wk1

Group2
Index LC OC O + HO O + HS ANOVA P value
Small intestine
Mass with digesta, g/100 g BW 3.13 ± 0.037b 8.68 ± 0.391a 7.29 ± 0.249a 7.15 ± 0.361a,b ≤0.001
pH of digesta 7.06 ± 0.064a 7.06 ± 0.127a 7.02 ± 0.110a 6.57 ± 0.058b <0.01
Mucosal disaccharidase activity, mol · min−1 · g protein−1

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Sucrase 8.22 ± 0.627b 13.1 ± 0.993a 12.4 ± 0.984a 11.9 ± 1.05a <0.005
Maltase 62.4 ± 3.75 66.7 ± 4.91 62.2 ± 6.64 59.7 ± 4.48 NS
Lactase 2.42 ± 0.199 2.82 ± 0.200 2.55 ± 0.188 2.67 ± 0.260 NS
Cecum
Tissue mass, g/100 g BW 0.154 ± 0.007a 0.107 ± 0.005c 0.106 ± 0.004c 0.125 ± 0.004b ≤0.001
Digesta mass, g/g tissue 3.73 ± 0.279 3.40 ± 0.299 3.45 ± 0.193 3.08 ± 0.256 NS
pH of digesta 7.31 ± 0.065 7.55 ± 0.105 7.38 ± 0.091 7.29 ± 0.028 NS
Ammonia, mg/g digesta 0.431 ± 0.019 0.391 ± 0.035 0.428 ± 0.027 0.408 ± 0.009 NS
Microbial enzyme activity, mol · h−1 · g digesta−1
α-glucosidase 13.9 ± 1.25 14.2 ± 1.43 16.8 ± 1.68 15.2 ± 1.12 NS
β-glucosidase 1.93 ± 0.470a,b 1.21 ± 0.177b 1.59 ± 0.124a,b 3.17 ± 0.391a <0.01
α-galactosidase 14.9 ± 1.65a 7.93 ± 1.93b 8.86 ± 1.72b 15.1 ± 1.05a <0.005
β-galactosidase 23.9 ± 2.45b 24.1 ± 3.09b 28.2 ± 3.67b 52.8 ± 5.17a ≤0.001
β-glucuronidase 19.8 ± 3.94 17.3 ± 2.49 22.8 ± 4.46 24.8 ± 1.59 NS
Colon
Tissue mass, g/100 g BW 0.299 ± 0.023a 0.248 ± 0.011b 0.227 ± 0.011b 0.235 ± 0.007b <0.01
Digesta mass, g/g tissue 1.12 ± 0.188 1.59 ± 0.062 1.45 ± 0.107 1.24 ± 0.069 NS
pH of digesta 7.42 ± 0.047a 7.31 ± 0.042a 7.37 ± 0.051a 7.14 ± 0.062b <0.005
1
All values are expressed as the mean ± SEM (n = 7–8). Values not sharing the same superscript (a, b, c) within a row are different at P ≤ 0.05. BW, body weight; NS,
nonsignificant, P >0.05.
2
LC, lean control; OC, obese control; O + HO, obese fed a diet containing hemp seed oil; O + HS, obese fed a diet containing hemp seeds.

calculated on a daily basis was also similarly higher in the obese was comparable among all groups (P > 0.05). Moreover, both
groups compared with the LC group (P ≤ 0.05). As a result, after the endogenous and exogenous intestinal enzymatic activities
4 wk of experimental feeding, obese rats, irrespective of the diet were affected by the rat phenotype and/or dietary factors
type, gained similarly more body weight and >4 times the body (Table 2). As for the endogenous enzymes of the small intestinal
fat mass compared with the LC group (P ≤ 0.05). Interestingly, mucosa, the sucrase activity was similarly increased in all obese
obese rats from the OC and O + HO groups had negative lean groups (compared with the LC group; P ≤ 0.05), whereas
body gain, whereas dietary hemp seeds significantly increased the maltase and lactase activity did not differ among all
body lean gain (compared with the OC group; P ≤ 0.05) but not groups (P > 0.05). In the cecum, the microbial α-galactosidase
to the level of the LC group where it was still >14 times higher. activity was decreased in the OC and O + HO groups
The final body fat percentage was almost 3 times higher whereas (compared with the LC group; P ≤ 0.05), whereas dietary
the final body lean percentage was >2 times lower in the obese hemp seeds increased the activity to the level of the LC group.
groups compared with those in the LC group. Nevertheless, the When compared with the OC group, supplementation with
final body lean percentage was significantly increased in the hemp seeds increased β-glucosidase and β-galactosidase activity
O + HS group compared with the percentage in the OC group (P ≤ 0.05).
(P ≤ 0.05). The concentration, profile, and pool of SCFAs, being the
The effects of hemp seed and hemp seed oil supplementation main microbial fermentation products in the cecum, are shown
on physiological indices of the small intestine, cecum, and colon in Table 3. The total SCFA concentration was decreased in
in rats are shown in Table 2. In the OC and O + HO groups, the OC group compared with the LC group (P ≤ 0.05).
an increase in the mass of the small intestine with digesta Dietary supplementation with hemp seeds caused a significant
calculated per 100 g of BW was observed (compared with (compared with the LC and OC groups; P ≤ 0.05) increase in
the LC group; P ≤ 0.05), whereas hemp seed supplementation the total SCFA concentration. This was mainly a result of the
prevented a significant increase (P ≤ 0.05). The cecum and increased acetate concentration in the O + HS group compared
colon relative tissue masses were decreased in all 3 obese with that in the other groups, which did not differ from one
groups (compared with the LC group; P ≤ 0.05); dietary another, as well as the increased propionate concentration in
hemp seeds significantly increased the cecum tissue mass, but the O + HS group compared with the concentration in the
not to the level of the LC group (P ≤ 0.05). Neither the rat OC and O + HO groups (all at P ≤ 0.05). The acetate,
phenotype nor the experimental factors influenced the relative propionate, and butyrate percentage was comparable among
cecal and colon digesta masses (P = 0.925 and P = 0.095, all groups (P > 0.05). When compared with both control
respectively). However, dietary hemp seed supplementation groups, dietary hemp seeds decreased the SCFA proportion of
decreased the small intestinal and colonic digesta pH compared putrefactive origin (PSCFA; P ≤ 0.05), which was calculated as
with the pH values of the other groups (P ≤ 0.05), which the sum of isobutyrate, isovalerate, and valerate. The SCFA pool
did not differ from one another, but the cecal digesta pH was decreased in the OC group compared with the pool in the

4 Opyd et al.
TABLE 3 SCFA production in the cecal digesta of lean and obese Zucker rats fed a diet containing hemp seed oil or hemp seeds for
4 wk1

Group2
Index LC OC O + HO O + HS ANOVA P value
SCFA concentration, mol/g digesta
Acetate 48.9 ± 2.40b 41.2 ± 3.14b 44.9 ± 2.49b 59.6 ± 4.10a <0.005
Propionate 15.3 ± 0.554a,b 13.3 ± 1.35b 13.7 ± 0.705b 17.6 ± 0.750a <0.05
± ± ± ±

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Butyrate 8.56 0.674 5.72 1.097 6.98 0.827 7.29 0.509 NS
PSCFA3 5.24 ± 0.226 4.59 ± 0.364 4.17 ± 0.232 4.76 ± 0.243 NS
Total 78.1 ± 2.83b 64.8 ± 4.21c 69.7 ± 2.68b,c 89.3 ± 4.41a ≤0.001
SCFA profile, %
Acetate 62.5 ± 1.19 63.3 ± 1.61 64.2 ± 1.64 66.4 ± 1.32 NS
Propionate 19.7 ± 0.319 20.6 ± 1.86 19.7 ± 1.03 19.8 ± 0.492 NS
Butyrate 11.1 ± 0.885 8.91 ± 1.57 10.1 ± 1.26 8.37 ± 0.803 NS
PSCFA 6.74 ± 0.255a 7.20 ± 0.538a 6.01 ± 0.372a,b 5.45 ± 0.436b <0.05
SCFA pool, mol/total digesta mass 119 ± 9.38a 85.2 ± 8.4b 94.6 ± 6.21a,b 117 ± 7.46a <0.05
1
All values are expressed as the mean ± SEM (n = 7–8). Values not sharing the same superscript (a, b, c) within a row are different at P ≤ 0.05. NS, nonsignificant, P >0.05;
PSCFA, SCFA of putrefactive origin.
2
LC, lean control; OC, obese control; O + HO, obese fed a diet containing hemp seed oil; O + HS, obese fed a diet containing hemp seeds.
3
The sum of isobutyrate, isovalerate, and valerate.

LC group (P ≤ 0.05), and dietary hemp seeds prevented this the other groups; P ≤ 0.05). The antioxidant status of the
decrease (P ≤ 0.05). liver was comparable between the LC, OC, and O + HO
Markers of liver function, the antioxidant status of the liver, groups (P > 0.05). In contrast, the liver TBARS content,
and the plasma and liver markers of lipid metabolism are shown as a marker of lipid peroxidation, was significantly reduced
in Table 4. The relative liver mass was almost 4 times higher by dietary hemp seeds compared with that of the LC group
in the OC and O + HO groups than the LC group, whereas (P ≤ 0.05). Moreover, dietary supplementation with hemp seeds
dietary hemp seeds decreased it to a level comparable with that increased the liver GSH and the sum of reduced and oxidized
of the LC group. The liver fat and triglyceride contents were glutathione (GSH + GSSG) compared with those of the LC
>3 times higher in all obese groups compared with content group (P ≤ 0.05). The plasma ALT, AST, and ALP activities
in the LC group, whereas the liver cholesterol content was were increased in the OC and O + HO groups compared
significantly increased only in the OC group (compared with with activities in the LC group (P ≤ 0.05), and dietary hemp

TABLE 4 Liver and blood plasma markers of lipid metabolism, antioxidant status, and liver function in lean and obese Zucker rats fed
a diet containing hemp seed oil or hemp seeds for 4 wk1

Group2
Marker LC OC O + HO O + HS ANOVA P value
Liver
Mass, g/100 BW 6.88 ± 0.446b 26.7 ± 2.05a 25.9 ± 2.65a 21.8 ± 1.57a,b ≤0.001
Fat, % 6.89 ± 0.629b 24.7 ± 1.63a 26.0 ± 2.02a 22.8 ± 1.53a ≤0.001
Triglycerides, mg/g 5.92 ± 0.565b 27.5 ± 1.51a 28.7 ± 1.53a 26.1 ± 1.90a ≤0.001
Cholesterol, mg/g 1.43 ± 0.093b 1.79 ± 0.379a 1.28 ± 0.142b 1.28 ± 0.118b <0.05
Antioxidant status
TBARS, g/g 539 ± 22.8a 486 ± 25.6a,b 491 ± 15.7a,b 454 ± 10.8b <0.05
GSH, mol/g 5.84 ± 0.226b 6.00 ± 0.180b 6.34 ± 0.280a,b 6.99 ± 0.262a <0.05
GSSG, mol/g 0.363 ± 0.021 0.363 ± 0.015 0.388 ± 0.025 0.403 ± 0.018 NS
GSH + GSSG, mol/g 6.20 ± 0.237b 6.36 ± 0.189b 6.72 ± 0.297a,b 7.39 ± 0.264a <0.05
GSH/GSSG, mol/g 1.64 ± 0.091 1.66 ± 0.059 1.66 ± 0.080 1.76 ± 0.095 NS
Plasma
Enzyme activity, U/L
AST 72.3 ± 5.12b 255 ± 61.4a 216 ± 34.7a 113 ± 15.1a,b ≤0.001
ALT 41.0 ± 1.96b 263 ± 56.2a 149 ± 12.6a 130 ± 18.5a,b ≤0.001
ALP 235.6 ± 10.9b 593 ± 116.0a 718 ± 80.9a 364 ± 23.2a,b ≤0.001
Lipid profile, mmol/L
Total cholesterol 2.71 ± 0.094b 6.20 ± 0.198a 5.60 ± 0.084a 5.16 ± 0.278a,b ≤0.001
HDL cholesterol 0.984 ± 0.028c 2.19 ± 0.099a 1.71 ± 0.053b 1.62 ± 0.065b ≤0.001
Non-HDL cholesterol 1.73 ± 0.076b 4.01 ± 0.120a 3.89 ± 0.086a 3.54 ± 0.250a,b ≤0.001
Triglycerides 2.42 ± 0.322b 5.14 ± 0.692a 3.42 ± 0.679b 4.01 ± 0.372a,b <0.05
1
All values are expressed as the mean ± SEM (n = 7–8). Values not sharing the same superscript (a, b, c) within a row are different at P ≤ 0.05. BW, body weight; GSH,
glutathione; GSH/GSSG, glutathione to glutathione disulfide ratio; GSSG, glutathione disulfide; NS, nonsignificant, P >0.05; TBARS, thiobarbituric acid-reacting substances.
2
LC, lean control; OC, obese control; O + HO, obese fed a diet containing hemp seed oil; O + HS, obese fed a diet containing hemp seeds.

Effects of dietary hemp seeds and hemp seed oil 5


the cultivar, the environment, including soil conditions, and
the grade of postharvest processing, which causes variability
in the available study results (2, 30). Nevertheless, our findings
confirm that hemp seeds are a good source of protein, fat, and
fiber. The protein content was slightly higher in the present
study (26.3% DM) than in the literature, where it ranged from
24.0 to 25.6% (1, 2, 31). The fiber content was similar to that
reported by Callaway et al. (1) and was ∼6% lower than in
the study by Mattila et al. (3). The fat content in hemp seeds

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reportedly ranged from 29.2 to 35.5% DM (1–3, 31), which
was in accordance with this study, where it was 33.3% DM. Of
importance, from the study design perspective, was that the fatty
acid profile of hemp seed oil and hemp seeds was very similar,
which may indicate sufficient stability of fatty acids during oil
extraction. Moreover, da Porto et al. (11) showed that PUFAs
can comprise over 80% of the total fatty acids in hemp seed
FIGURE 1 qRT-PCR analysis of Pparα, Pparγ , and Srebp1c mRNA oil, which was higher than the proportions found for hemp
expression in the liver of lean and obese Zucker rats fed a diet seed oil (76.1%) and hemp seeds (76.4%) in the present study.
containing hemp seed oil or hemp seeds for 4 wk. Values are
The main PUFAs determined in similar quantities in the seeds
means ± SEMs, n = 7–8. Labeled means without a common letter
and oil were linoleic acid, α-linolenic acid, and γ -linolenic acid
(a, b, c) differ at P ≤0.05 (Duncan’s or Dunn’s post hoc test). Actb,
β-actin; LC, lean control; OC, obese control; O + HO, obese fed a (∼52%, 18%, and 4%, respectively), which was generally in
diet containing hemp seed oil; O + HS, obese fed a diet containing accordance with the literature (1, 11, 32), although the linoleic
hemp seeds; Pparα, peroxisome proliferator-activated receptor α; acid percentage was 4–7% lower in the present study. Linoleic
Pparγ , peroxisome proliferator-activated receptor γ ; Srebp1c, sterol acid and α-linolenic acid are 2 essential fatty acids belonging to
regulatory element-binding protein 1c. the n–6 and n–3 fatty acid families, respectively. Consumption
of n–6 and n–3 fatty acids is associated with an improvement in
blood lipid profile and the regulation of inflammatory processes
seeds slightly decreased the activity; however, the activity was in the body through the production of eicosanoids (33). γ -
comparable to that of the LC group (Table 4). The plasma Linolenic acid is rarely found in nature, but it occurs in human
cholesterol concentration, including the HDL and non-HDL milk and has anti-inflammatory and anticancer activities (34).
fractions, and the plasma triglyceride concentrations were at Hemp seeds also contained a small amount of TFA (cis-9, trans-
least twice as high in the OC group compared with the LC 12-octadecadienoic acid), which can potentially be detrimental
group. Both dietary oil and seeds decreased the HDL cholesterol to the body because TFAs are known to raise the risk of
concentration (compared with the OC group; P ≤ 0.05), but not atherosclerosis and coronary heart disease by inhibiting the
to the level of the LC group (P >0.05). The total cholesterol synthesis of PUFAs in the phospholipids of arterial cells (35).
and non-HDL cholesterol concentrations were decreased by Obesity in Zucker (fa/fa) rats begins manifesting in the
dietary hemp seeds to the level of the LC group. The triglyceride first month of their life (19). At the beginning of the present
concentrations were significantly decreased only by dietary experiment, obese rats aged 8 wk weighed ∼30% more than
hemp seed oil (compared with the OC group; P ≤ 0.05), but their lean counterparts, and their body fat percentage was
their concentrations both in the O + HO and O + HS groups almost 3 times higher (Table 1). As a result of leptin resistance,
corresponded with those of the LC group. obese Zucker rats develop hyperphagia 17 d after birth(19). In
The effects of dietary hemp seed and hemp seed oil the present study, an increased diet intake in obese rats was
supplementation on the mRNA expression of Pparα, Pparγ , also observed, which in turn led to the increase in BW and
and Srebp1c in rat livers are shown in Figure 1. Pparα and fat percentage and to the decrease in lean body percentage by
Srebp1c expression was similarly decreased in all 3 obese groups the end of the experiment. Dietary supplementation with hemp
compared with expression in the LC group (P ≤ 0.05). Pparγ seeds slightly, but statistically significantly, increased the lean
expression was decreased in the OC group compared with the body content, which might be due to some favorable changes in
LC group (P ≤ 0.05), whereas dietary supplementation with the amino acid profile of the diet. It is known that the quality,
hemp seeds significantly increased Pparγ expression (P ≤ 0.05) digestibility, and amino acid profile of consumed protein can
to a level comparable with that of the LC group. trigger a rise in muscle protein synthesis (36). Hemp seed protein
is highly digestible and the overall pattern of amino acid supply
positions it as a good source of vegetable-based protein (2, 3).
It is thought that a single genetic mutation in a mammalian
Discussion
gene responsible for regulating food intake can affect the
The aim of this study was to compare the effect of dietary composition of the gut microbiota within the body (37). An in
supplementation with hemp seed oil and hemp seeds on vivo experiment on Zucker rats has shown that the obesogenic
metabolic disorders in genetically obese rats. The study required phenotype is characterized by significantly lower bacterial
a precise design of semipurified diets to assess the extent to counts in the hindgut than the lean phenotype (38). This finding
which the lipid fraction could contribute to the health effects of seems to be in accordance with the present study because both
hemp seed supplementation. Thus, the first stage of the study the cecal α-galactosidase activity and SCFA production were
was to analyze, in detail, the chemical composition of hemp decreased in the OC group, indicating that the metabolic activity
seeds and hemp seed oil, so that the experimental diets would of the microbiota was reduced (Table 2 and 3). Interestingly,
be adequately formulated (results in Supplementary Table 1). dietary hemp seed supplementation increased the microbial
The chemical composition of hemp seeds can be affected by glycolytic activity (β-glucosidase and α- and β-galactosidase),
6 Opyd et al.
which was apparently due to the fiber fraction of hemp seeds. the aforementioned disorders were accompanied by reduced
This fraction constituted 66% of the dietary fiber in the O + HS hepatic expression of Pparα, Pparγ , and Srebp1c (Figure 1),
group, and although mostly insoluble, it was apparently a good which are transcription factors involved in the regulation of
substrate for the gut microbiota as the cecal SCFA production lipid metabolism. Genes activated by Pparα or Pparγ stimulate
was also considerably increased in this group. Fermentation of β-oxidation of fatty acids or adipogenesis, respectively, whereas
dietary fiber within the hindgut affects the microbiota and its genes activated by Srebp1c stimulate de novo lipogenesis (44,
activity. The main product of fiber fermentation is SCFAs, which 45). This suggests that the lipid metabolism in Zucker rats is
play an important role in the regulation of energy metabolism heavily disturbed at the molecular level as well. Interestingly, it
and fulfill a number of important metabolic functions within has been shown that Pparγ -deficient mice develop severe fatty

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the body (39). Moreover, the present study shows that the liver associated with a failure to store and process triglycerides
endogenic enzyme activity is also changed in obese Zucker rats, (46, 47), which seems to be in accordance with our results.
namely, the mucosal sucrase activity in the small intestine was Nevertheless, both dietary hemp seed oil and hemp seeds were
considerably increased. One of the explanations for this change able to affect lipid metabolism, although the effectiveness of
is overconsumption of the diet, including sucrose, which is an the seeds was better. To the best of our knowledge, this is the
important dietary source of carbohydrates. first study examining the effect of hemp seed supplementation
GSH is a tripeptide involved in the elimination of reactive on lipid metabolism, whereas the effect of hemp seed oil
oxygen species from the organism, especially from the liver. has already been studied to some extent. Sargolzaie et al.
In our study, dietary hemp seeds significantly elevated the (48) showed that an 8-d long hemp seed oil intraperitoneal
content of total glutathione (GSH + GSSG) and its reduced injection decreased total and HDL cholesterol and triglyceride
form (GSH) in the liver (Table 4). This might be partly due concentrations in the serum of rats. Schwab et al. (49) observed
to a relatively high content of cysteine in hemp seed protein the lipid-lowering effects of hemp seed oil consumed by healthy
compared with its 5 times lower content in casein, which was humans. In the present study, both dietary supplements reduced
used as the main source of protein in all experimental diets. the hepatic cholesterol content and plasma HDL cholesterol
Cysteine is 1 of the primary amino acids necessary for GSH concentration, whereas seed supplementation also decreased
synthesis, and the intracellular pool of cysteine is relatively the plasma concentration of total cholesterol and its non-HDL
small compared with the metabolically active pool of GSH, fraction (Table 4). On the other hand, the plasma triglyceride
so it is generally considered the limiting amino acid for GSH concentration was not significantly decreased by the seeds,
synthesis (40). Moreover, in the present study, dietary hemp as it was by the oil. All the aforementioned effects can be
seeds also decreased the TBARS content in the liver as a marker considered favorable for the organism, except for the reduction
of lipid peroxidation (Table 4), which is in accordance with the in plasma HDL cholesterol observed after hemp seed and hemp
study by Girgih et al. (5), who showed that hemp seed peptides seed oil supplementation. Since rats in the OC group had
were effective inhibitors of oxidative stress in spontaneously >2 times higher plasma HDL cholesterol concentrations, this
hypertensive rats. Nevertheless, the amino acid profile of protein detrimental effect could have been strain related. Indeed, our
is not the only factor that may be responsible for the antioxidant latest study comparing the effects of hemp seed oil in lean
activity of hemp seeds. For example, Chen et al. (41) reported a and obese Zucker rats indicated that a decrease in the plasma
high antioxidant activity of hemp seed hull extract and amides HDL cholesterol concentration was observed only in obese
and lignanamides responsible for it (N-trans-caffeoyltyramine animals (data not shown). Nevertheless, the modest presence of
and cannabisin B). Moreover, an in vitro study by Yan et al. cis-9, trans-12-octadecadienoic acid in the lipid fraction might
(8) showed that lignanamides, which are 1 of the primary also have been a factor, since it has been shown that TFAs
phenolic compounds in hemp seeds, exhibited powerful radical can considerably decrease HDL cholesterol in rat blood (50).
scavenging activity. Moreover, our study suggests that the lipid-lowering effects of
In addition to obesity, Zucker (fa/fa) rats are also character- hemp seeds were not strictly associated with PUFAs and that
ized by liver disorders, including liver enlargement and steatosis other components present in the seeds also played a role. One
(17). In our study, these disorders are exhibited by the several- of the factors could be the fiber fraction and, thus, the increased
fold increased liver fat percentage and plasma aminotransferase cecal formation of SCFAs, especially propionate, which after
activities (ALP and AST) as well as the >2-fold increased plasma absorption, can decrease hepatic lipogenesis and improve the
ALP activity (Table 4). This indicates liver damage and problems blood lipid profile (51). Interestingly, Al-Lahham et al. (52)
with bile outflow to the gut. Interestingly, 4-wk feeding with suggested that propionic acid can activate the expression of
dietary hemp seeds reduced ALT, AST, and ALP activity to levels Pparγ , and in the present study, both the hepatic expression of
comparable with those of the LC group. Pparγ and the cecal production of propionate were increased
In the present study, obese rats were characterized by >2-fold after dietary hemp seed supplementation. However, further
increased plasma concentrations of triglycerides and choles- studies on the PPARG protein concentration and expression
terol, including the HDL and non-HDL fractions (Table 4). of genes activated by this transcription factor are necessary to
These results are in agreement with the study by Liao et al. (42), confirm this finding.
who observed elevated plasma concentrations of cholesterol Obese Zucker rats are characterized by a number of
and triglycerides in obese Zucker rats. De Artinano and Castro metabolic disorders, including those found within the gut and
(19) showed that an increase in plasma lipid and lipoprotein mainly associated with reduced metabolic activity of the micro-
concentrations is 1 of the first abnormalities that can be biota, as well as many others, such as hyperlipidemia, fatty liver,
observed in obese Zucker rats. In this study, obese Zucker signs of liver injury, and decreased hepatic mRNA expression
rats were also characterized by liver enlargement, which was of transcription factors that regulate lipid metabolism. Dietary
mainly due to the >3-fold higher fat content (Table 4). The liver supplementation with hemp seeds can improve the fermentation
triglyceride and cholesterol contents were also considerably efficiency of the gut microbiota and liver functions, including
increased in the OC group, which is in accordance with previous its antioxidant status, and has lipid-lowering effects; however,
studies on this animal model (43). In the present study, all of it is not able to attenuate the development of obesity
Effects of dietary hemp seeds and hemp seed oil 7
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Effects of dietary hemp seeds and hemp seed oil 9

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