Biomedicine & Pharmacotherapy 149 (2022) 112863

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Cannabidiol improves glucose utilization and modulates glucose-induced

dysmetabolic activities in isolated rats’ peripheral adipose tissues
Ochuko L. Erukainure a, Motlalepula G. Matsabisa a, *, Veronica F. Salau a,
Kolawole A. Olofinsan b, Sunday O. Oyedemi a, d, Chika I. Chukwuma c, Adeline Lum Nde a,
Md. Shahidul Islam b
a
Department of Pharmacology, School of Clinical Medicine, Faculty of Health Sciences, University of the Free State, Bloemfontein 9300, South Africa
b
Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, (Westville Campus), Durban 4000, South Africa
c
Center for Quality of Health and Living, Faculty of Health Sciences, Central University of Technology, Bloemfontein 9301, South Africa
d
Department of Pharmacology, School of Science and Technology, Nottingham Trent University, Nottingham, UK

A R T I C L E I N F O A B S T R A C T

Keywords: Reduced glucose uptake and utilization, with concomitant lipolysis in adipose tissues has been linked to the
Adipose tissue pathogenesis of obesity and its complications. The present study investigated the effect of cannabinoid-
Cannabidiol stimulated glucose uptake on redox imbalance, glucose and lipid metabolisms, as well as cholinergic and puri­
Glucose-lipid homeostasis
nergic dysfunctions in isolated rats’ adipose tissues. Freshly Isolated rats’ adipose tissues were incubated with
Obesity
glucose and different concentrations of cannabidiol for 2 h at 37 ◦ C. The negative control consisted of incubation
without cannabidiol, while normal control consisted of incubations without glucose and/or cannabidiol and
Metformin served as the standard drug. Cannabidiol caused an increase in adipose-glucose uptake, with
concomitant elevation of glutathione, triglyceride level, superoxide dismutase, catalase and 5′ nucleoidase ac­
tivities. It also caused suppression in malondialdehyde and cholesterol levels, acetylcholinesterase, ENTPDase,
fructose-1,6-biphosphatase, glucose 6-phosphatase, glycogen phosphorylase, and lipase activities. In silico studies
revealed a strong molecular interaction of cannabidiol with adipose triglyceride lipase, hormone-sensitive lipase,
and monoglyceride lipase. These results indicate that cannabidiol-enhanced glucose uptake in adipose tissues is
associated with enhanced antioxidative activities, concomitant modulation of cholinergic and purinergic dys­
functions, and improved glucose – lipid homeostasis.

1. Introduction energy production [8,9]. A process described as Randle’s hypothesis

The role of adipose tissue in glucose uptake and utilization have been
reported to be of importance in energy metabolism vis-à-vis glucose and
lipid homeostasis [1]. Aside the liver and skeletal muscles, excess
glucose from postprandial spikes is transported to the adipose tissue via
glucose transporter 4, GLUT4 which is triggered by pancreatic insulin
secretion [1,2]. Glucogenesis leading to cellular levels of glucose have
been implicated in impaired lipogenesis and glucose uptake [3]. This is
depicted by elevated activities of glucogenic enzymes vis-à-vis
fructose-1,6-biphoshatase and glucose 6-phosphatase [3,4].
Excess glucose is utilized in the adipose tissue as a lipogenic sub­
strate, leading to storage of lipids in the form of triglyceride [5–7]. In
fasting states, adipose glucose uptake and lipogenesis are diminished
thus triggering lipolysis of stored lipids to free fatty acids (FFAs) for
[10,11]. Impaired adipose glucose uptake, with concomitant lipolysis
characterized by insulin resistance and pancreatic β-cell dysfunction has
been implicated in the pathophysiology of obesity [12,13].
Obesity ranks among the world’s global epidemics, with 13% of the
world’s adult population being obese [14,15]. It also contributes to 8%
of global mortality [14]. It is a major risk factor for several diseases
including cardiovascular diseases, type 2 diabetes, renal disease, and
cancers such as those of breast, prostate, ovarian, colon, liver and gall­
bladder. It is estimated that over 1 billion of the world’s population will
be affected by obesity by 2025 [16]. The costs of treating obesity and its
complications are of major concern, particularly in developing countries
with underdeveloped health infrastructures. Thus, the search for more
affordable and alternative treatments for obesity and its complication.
Cannabidiol (Fig. 1) is among the phytocannabinoids of Cannabis

* Corresponding author.
E-mail address: MatsabisaMG@ufs.ac.za (M.G. Matsabisa).

https://doi.org/10.1016/j.biopha.2022.112863
Received 1 February 2022; Received in revised form 16 March 2022; Accepted 23 March 2022
Available online 30 March 2022
0753-3322/© 2022 University of the Free State, Bloemfontein 9300, South Africa. Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
O.L. Erukainure et al.
Fig. 1. Chemical structure of cannabidiol.
sativa, with reported antioxidant, anti-inflammatory, antipsychotic,
hepatoprotective and anxiolytic properties [17,18]. Cannabidiol is one
of the major pharmacologically active phytocannabinoid in C. sativa
plant, with non-psychoactive properties [19]. It has been reported for its
therapeutic effect on diabetes, cardiovascular diseases, cancers, and
neurodegenerative diseases [19,20]. These properties have been
attributed to its potent antioxidant properties which consists of free
radical quenching and mopping, activation of antioxidant enzymes,
chelation of transition metals, and antiperoxidative activities [19,21,
22]. Its chemical structure consists of 21 carbon atoms, a cyclohexene
ring, phenolic ring, pentyl side chain, hydroxyl groups at the C-10 and
C-50 positions, pentyl chain at the C-30 of the phenolic ring, and methyl
group at the C-1 position of the cyclohexene ring, with an empirical
chemical formula of C21H30O2 [19,23,24]. The potency of cannabidiol
as an antioxidant has been attributed to the presence and positions of the
hydroxy and methyl groups, and pentyl chain in its chemical structure.
Cannabidiol has been reported for its antiobesogenic and therapeutic
potentials in the treatment and management of obesity. This is depicted
by its ability modulate food intake and weight gains in rats fed on high
fat diet as well as suppress fat accumulations in adipocytes [25,26].
Despite the reported antiobesogenic effect of cannabidiol, there is
still a dearth on the biochemical mechanism by which it modulates
metabolic switches in adipose tissues. Thus, the present study was car­
ried out to investigate the effect of cannabinoid stimulated glucose up­
take on redox imbalance, glucose and lipid metabolisms, and cholinergic
(acetylcholinesterase activity) and purinergic (purinergic enzymes ac­
tivities) dysfunctions in isolated rats’ adipose tissues.
2. Materials and methods
2.1. Cannabidiol
Cannabidiol was obtained from Sigma-Aldrich, Johannesburg, South
Africa.
2.2. Permit approval
This research has been undertaken under the permit approval
(Permit No. POS 014/2021/2022) from the South African Health
Products Regulatory Authority to import, conduct, collect, posses,
transport and store cannabis plant, plant parts and products for research
purposes.
2.3. Animals
Six male Sprague Dawley rats (7–8 weeks old) weighing about 220 g
were obtained from the Biochemical Research Unit of the University of
KwaZulu-Natal, Durban, South Africa. The rats were fasted overnight
(8 h) before humanely sacrificed. They were sacrificed by placing in a
euthanizing chamber containing isoform. After euthanizing, their blood
was collected via cardiac puncture with the aid of a syringe. The rats
were subsequently dissected, and their adipose tissues around the kid­
ney were harvested and utilized immediately for ex vivo studies.
The present study was carried out in accordance with the approved
guidelines of the Animal Ethics Committee of the University of KwaZulu-
Natal, Durban, South Africa (Protocol approval number: AREC/
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Biomedicine & Pharmacotherapy 149 (2022) 112863
00002325/2021).
2.4. Determination of glucose uptake in isolated adipose tissues
Glucose uptake was carried out according to a published protocol
[27], with slight modofications. 0.5 g of each freshly isolated adipose
tissues was incubated with 8 mL of Krebs buffer containing 200 mg/dL
glucose and different concentrations of cannabidiol (30–240 µg/mL)
under a 5% CO2, 95% oxygen and 37 ◦ C conditions for 2 h. Normal
control consisted of incubation without cannabidiol, and metformin
served as the standard drug at a dose of 240 µg/mL. The glucose con­
centrations were measured before and after the 2 h incubation with an
Automated Chemistry Analyzer (Labmax Plenno, Labtest Inc., Lagoa
Santa, Brazil).
Glucose uptake was calculated using the formula:
GC1− GC2
Glucose uptake per g of rat adipose tissue =
Weight of adipose tissue (g)
Where GC1 and GC2 are glucose concentrations (mg/dL) before and
after incubation, respectively.
After glucose uptake determination, the adipose tissues were
collected from the buffer and homogenized in cold 50 mM phosphate
buffer containing 1% triton X-100 (pH 7.5) and centrifuged at
15,000 rpm for 10 mins at 4 ◦ C temperature. The supernatants were
decanted into 2 mL Eppendorf tubes and stored at − 20 ◦ C for subse­
quent biochemical analyses.
2.5. Determination of oxidative stress markers
Oxidative stress parameters were analysed in the adipose tissues by
deteminining the reduced glutathione (GSH) level, superoxide dismut­
ase (SOD) and catalase activities, and malondialdehyde (MDA) level.
i. GSH level
The Ellman’s method was used in determining the GSH levels
of the tissues [28]. Briefly, 200 μL of the adipose tissue super­
natant was deproteinized with 10% TCA before centrifuging at
3500 rpm for 5 mins (25 ◦ C). 150 μL of the resulting supernatant
was mixed with 50 μL of Ellman’s reagent and incubated in a 96
well plate for 5 mins. Absorbance was measured at 415 nm and
GSH level was extrapolated using a standard GSH curve. GSH
level was presented as µmol/mL.
ii. SOD activity
This was carried out using a previously established method
[29]. Briefly, 15 μL of the adipose tissue supernatant was pipetted
into a 96 well plate containing 170 μL of 0.1 mM diethylene­
triaminepentaacetic acid (DETAPAC). 15 μL of 1.6 mM 6-hydrox­
ydopamine (6-HD) was added to the reaction mixture.
Absorbance was immediately read at 492 nm wavelength for
3 min at 1 min interval. The SOD activity was presented as
µmol/min/mL.
iii. Catalase activity
This was carried out using a previously published method [30].
Briefly, 100 μL of the adipose tissue supernatant was incubated
with 1000 μL of H2O2 (65 μM) in 6.0 mM sodium phosphate
buffer (pH 7.4) at 37 ◦ C for 2 min 4 mL of 32.4 mM ammonium
molybdate was added to the reaction mixture to terminate the
reaction. Absorbance was read at 347 nm against the blank. The
catalase activity was presented as µmol/min/mL.
iv. MDA level
This was carried out using a previously published method [31].
Briefly, 200 μL of the adipose tissue supernatant was mixed with 200 μL
of 8.1% SDS solution, 750 μL of 20% acetic acid, 2 mL of 0.25% thio­
barbituric acid (TBA), and 850 miliQ water. The reaction mixture was
O.L. Erukainure et al.
boiled for 1 h. After cooling, a 200 μL aliquot of the reaction mixture
was collected into a 96 well plate and absorbance was read at 532 nm. A
standard MDA curve was used to extrapolate the TBARS concentrations
from which lipid peroxidation was estimated. The MDA level was pre­
sented as µmol/mL.
2.6. Determination of acetylcholinesterase activity
The Ellman’s method was used in determining the acetylcholines­
terase activity of the adipose tissue as previously described [32]. Briefly,
200 μL of the adipose tissue supernatant was mixed with 100 μL of
3.3 mM Ellman’s reagent (pH 7.0) and incubated for 20 min at 25 ◦ C.
100 μL of 0.05 M acetylcholine iodide was then added to the reaction
mixture, and absorbance was immediately read at 412 nm at 3 min
intervals.
2.7. Determination of purinergic enzymes activities
Purinergic activities of the adipose tissues were determined by
assaying for ectonucleotidase (E-NTPDase) and 5′ nucleotidase activities.
i. ENTPDase activity
This was carried out using a previously published method with
slight modifications [33,34]. Briefly, 20 μL of the adipose tissue su­
pernatant was incubated with 200 μL of the reaction buffer (1.5 mM
CaCl2, 5 mM KCl, 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose
and 45 mM Tris-HCl) for 10 mins at 37 ◦ C. 20 μL of 50 mM ATP was
added to the reaction mixture and further incubated in a shaker for
20 mins at 37 ◦ C. 200 μL of 10% TCA was used in terminating the
reaction. 200 μL of 1.25% ammonium molybdate and a freshly pre­
pared 9% ascorbic acid were added to the reaction mixture. The
reaction mixture was allowed to stand on ice for 10 mins. Absor­
bance was read at 600 nm.
ii. 5′ nucleotidase activity
This was carried out using a previously published method, with
slight modifications [35]. Briefly, 20 μL of the adipose tissue superna­
tant was incubated with 50 μL of 0.1 M MgCl2 and 50 μL of 0.1 M
Tris-HCl at 37 ◦ C for 10 mins. This was followed by the addition of 20 μL
of 50 mM ATP to the mixture and further incubated for another 20 mins
at 37 ◦ C. The reaction was terminated by the adding 200 μL of 10% TCA.
The reaction mixture was allowed to stand on ice for 10 mins, and the
absorbance was measured at 600 nm.
2.8. Determination of glucogenic enzymes activities
The Glucogenic enzyme activities of the adipose tissues were deter­
mined by assaying for fructose-1,6-bisphosphatase, glucose 6 phospha­
tase and glycogen phosphorylase activities.
i. Fructose 1,6-bisphosphatase activity
This was carried out using a previously published method with
slight modifications [36,37]. Briefly, 100 μL of the adipose tissue
supernatant was incubated with 100 μL of 0.05 M fructose,
1200 μL of 0.1 M Tris–HCl buffer (pH 7.0), 250 μL 0.1 M MgCl2,
100 μL 0.1 M KCl, and 250 μL 1 mM EDTA at 37 ◦ C for 15 mins.
10% TCA was used in halting the reaction. The reaction mixture
was subjected to centrifugation for 10 mins at 3000 rpm (4 ◦ C),
and the supernatant collected. 100 μL of the resulting superna­
tant was pipetted into a 96 well plate containing 50 μL of 1.25%
ammonium molybdate and freshly prepared 9% ascorbic acid in a
96 well plate. The reaction mixture was incubated for 20 mins at
room temperature. Absorbance was measured at 680 nm.
ii. Glucose 6-phosphatase activity
This was carried out using a previously published method [38,
39] with slight modifications. Briefly, 200 μL of the adipose tissue
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Biomedicine & Pharmacotherapy 149 (2022) 112863
supernatant was incubated with 100 μL of 0.25 M glucose 6-phos­
phate, 200 μL of 5 mM KCl, 1300 μL of 0.1 M Tris-HCl buffer and
40 μL of 50 mM ATP for 30 mins at 37 ◦ C in a shaker. The reac­
tion was stopped with 1 mL of distilled water and 1.25%
ammonium molybdate. The reaction mixture was incubated with
1 mL of a freshly prepared 9% ascorbate for 30 mins. Absorbance
was then measured at 660 nm.
iii. Glycogen phosphorylase activity
This was carried out using a previously published method [40].
Briefly, 100 μL of the adipose tissue supernatant was incubated with
64 mM glucose-1-phosphate and 4% glycogen for 10 min at 30 ◦ C. The
reaction was terminated with 20% ammonium molybdate in concen­
trated H2SO4. The reaction mixture was further incubated with a
mixture of Elon reducer and distilled water for 45 min at 30 ◦ C.
Absorbance was read at 340 nm.
2.9. Determination of lipase activity
The lipase activity of the adipose tissues was determined using a
previously published protocol [41]. Briefly, 100 μL of the tissue super­
natant was incubated with 169 μL of Tris buffer (100 mM Tris–HC1 and
5 mM CaCl2, pH 7.0) for 15 min at 37 ◦ C. 5 μL of 10 mM p-NPB
(p-nitrophenyl butyrate in dimethyl formamide) was then added to the
reaction mixture and further incubated for 15 min at 37 ◦ C. Absorbance
was read at 405 nm at 1 min interval. The activity was expressed as the
rate of reaction (ΔA/min).
2.10. Determination of lipid contents
The lipid contents of the adipose tissues were determined by assaying
for cholesterol and triglyceride levels in the tissue supernatant using an
Automated Chemistry Analyzer (Labmax Plenno, Labtest Co. Ltd., Lagoa
Santa, Brazil) with commercial assay kits according to manufacturer’s
manual [41].
2.11. Molecular docking studies
Molecular docking analysis was carried out to determine the mo­
lecular interaction and binding free energy of cannabidiol with adipose
triglyceride lipase (ATGL; P0C548), hormone-sensitive lipase (HSL;
P15304) and monoglyceride lipase (MGL; Q8R431) proteins. The amino
acids sequences of the proteins were obtained from the Universal Protein
Resource (UniProt) (https://www.uniprot.org/) and modelled using the
Swiss Model online tools (https://swissmodel.expasy.org/) [42]. The
generated 3D structures of the proteins were prepared with the auto­
mated Dock prep tool of UCFS Chimera software V. 1.14 [43] which
added hydrogen atoms and AMBER 94 force field gasteiger charges [44].
The 3D structure of cannabidiol was retrieved from PubChem in SDF
format and prepared with the same software employed for the proteins.
Molecular docking of the compound ligands with the modelled proteins
was done with the Lamarck genetic algorithm of AutodockVina within a
search grid covering the whole protein [45]. The search volume centre
dimension for ATGL was 80 × 35 × 80, while HSL was 32 × 32 × 45
and Q8R431 was − 3 × 8 × − 3. The inspection of 2D and 3D images of
the docking pose with the highest binding affinity was done with BIOVIA
Discovery Studio [46].
2.12. Data analysis
Data was analyzed using one-way analysis of variance (ANOVA) and
presented as mean ± SD. The Tukey’s HSD-multiple range post-hoc test
was used to determine significant difference at p < 0.05. Statistical
analyses were done using IBM Statistical Package for the Social Sciences
(SPSS) for Windows, version 23.0 (IBM Corp., Armonk, NY, USA).
O.L. Erukainure et al. Biomedicine & Pharmacotherapy 149 (2022) 112863

Fig. 2. Effect of cannabidiol on adipose glucose uptake. Data = mean ± SD; n = 3. abcdValues with different letter above the bars are significantly different (p < 0.05)
from each other. Control = adipose tissue incubated in glucose only.

Fig. 3. Effect of cannabidiol on (A) GSH level, (B) SOD, (C) catalase and (D) MDA level in glucose treated adipose tissue. Data = mean ± SD; n = 3. *Statistically
significant (p < 0.05) compared to Glucose-only treated tissues; #Statistically significant (p < 0.05) compared to control. Control = adipose tissue with no glucose.

3. Results levels and activities dose-dependently and compared favorably with

There was a significant (p < 0.05) increase in adipose glucose uptake
on incubation of adipose tissues with cannabidiol in the presence of
glucose as shown in Fig. 2. The uptake was dose-dependent and
compared favorably with metformin.
Incubation of adipose tissues with only glucose led to significant
(p < 0.05) depletion in GSH level, SOD and catalase activities, while
significantly (p < 0.05) elevating MDA level as shown in Fig. 3A–C.
Incubation with cannabidiol significantly (p < 0.05) reversed these
metformin.
As shown in Fig. 4, there was a significant (p < 0.05) elevation of
acetylcholinesterase activity in adipose tissues incubated with only
glucose. This activity was significantly (p < 0.05) suppressed on incu­
bation with cannabidiol and compared favorably with metformin.
There was a significant (p < 0.05) elevation in the activity of
ENTPDase, while suppressing 5′ nucleotidase activity in adipose tissues
incubated with only glucose as shown in Fig. 5A–C. These activities were
significantly (p < 0.05) reversed dose-dependently in adipose tissues

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O.L. Erukainure et al. Biomedicine & Pharmacotherapy 149 (2022) 112863

Fig. 4. Effect of cannabidiol on acetylcholinesterase activity in glucose treated adipose tissue. Data = mean ± SD; n = 3. *Statistically significant (p < 0.05)
compared to Glucose-only treated tissues; #Statistically significant (p < 0.05) compared to control. Control = adipose tissue with no glucose.

Fig. 5. Effect of cannabidiol on (A) ENTPDase and (B) 5′ nucleotidase activities in glucose treated adipose tissue. Data = mean ± SD; n = 3. *Statistically significant
(p < 0.05) compared to Glucose-only treated tissues; #Statistically significant (p < 0.05) compared to control. Control = adipose tissue with no glucose.

incubated with cannabidiol. dependently suppressed on incubation with cannabidiol.

Incubation of adipose tissue with only glucose led to significant
(p < 0.05) elevation in fructose-1,6-biphosphatase, glucose 6-phospha­
tase and glycogen phosphorylase activities as shown in Fig. 6A–C.
These activities were significantly (p < 0.05) suppressed dose-
dependently on incubation with cannabidiol. These activities
compared favorably with those of metformin.
As shown in Fig. 7, incubation of adipose tissue with only glucose
significantly (p < 0.05) elevated lipase activity. The activity was dose-
There was a significant (p < 0.05) elevation in cholesterol level, with
concomitant suppressed triglyceride level in adipose tissues incubated
with only glucose as shown in Fig. 8A and B. These levels were signifi­
cantly (p < 0.05) reversed on incubation with cannabidiol.
Molecular docking studies revealed strong molecular interactions of
cannabidiol with ATGL, HSL and MGL as shown in Fig. 9A–F. This is
further depicted by the respective binding energies of − 7.4,− 6.4 and
− 7.5 kcal/mol for ATGL, HSL and MGL.

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Fig. 6. Effect of cannabidiol on (A) fructose-1,6-biphosphatase, (B) glucose 6-phosphatase and (C) glycogen phosphorylase activities in glucose treated adipose
tissue. Data = mean ± SD; n = 3. *Statistically significant (p < 0.05) compared to Glucose-only treated tissues; #Statistically significant (p < 0.05) compared to
control. Control = adipose tissue with no glucose.

4. Discussion lipolytic and cholinergic activities in adipose tissues.

The role of adipose tissues in energy metabolism has been well
established and its dysfunctions have been implicated in perturbed
glucose and lipid homeostasis which predisposes the body to insulin
resistance, hyperglycemia and lipotoxicity [47]. These features have
also been reported as major pathophysiology of obesity and type 2
diabetes [48,49]. Cannabidiol is among the non-psychotic cannabinoids
from C. sativa, with reported modulatory effect on the adipose functions
and obesity [25,26]. The present study reports the effect of cannabidiol
enhanced adipose glucose uptake on key metabolism implicated in
obesity. To the best of our knowledge, this study also reports for the first
time the ability of cannabidiol to modulate glucogenic, purinergic,
Adipose glucose uptake has been reported as one of the mechanisms
by which the body store excess glucose following consumption of a
carbohydrate diet [1]. Impaired adipose glucose uptake has been
implicated in the pathogenesis of lipotoxicity, a major pathophysiology
of obesity [9,50]. This arises from exacerbated and incessant lipolysis of
adipose-stored triglycerides to FFAs [51] and predisposes the body to
insulin resistance and hyperglycemia [52]. Thus, the increased glucose
uptake in cannabidiol-incubated adipose tissues (Fig. 2) indicates its
ability to promote glucose utilization in adipose tissues. This corrobo­
rates previous reports on the ability of the studied cannabinoid to
stimulate glucose uptake in fat cells [26].
Adipose oxidative stress has been implicated in the pathogenesis of

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O.L. Erukainure et al. Biomedicine & Pharmacotherapy 149 (2022) 112863

Fig. 7. Effect of cannabidiol on lipase activity in glucose treated adipose tissue. Data = mean ± SD; n = 3. *Statistically significant (p < 0.05) compared to Glucose-
only treated tissues; #Statistically significant (p < 0.05) compared to control. Control = adipose tissue with no glucose.

Fig. 8. Effect of cannabidiol on (A) cholesterol and (B) triglyceride levels in glucose treated adipose tissue. Data = mean ± SD; n = 3. *Statistically significant
(p < 0.05) compared to Glucose-only treated tissues; #Statistically significant (p < 0.05) compared to control. Control = adipose tissue with no glucose.

obesity and its complications. This has been attributed to increased
generation and accumulation of ROS in the tissues’ mitochondria arising
from imbalance in glucose-lipid homeostasis [53,54]. Further, the high
lipid content of the tissues makes it highly susceptible to peroxidative
attack [54,55]. The depleted GSH level, SOD and catalase activities as
well as exacerbated MDA level in adipose tissues incubated with only
glucose (Fig. 3A–D) depict an occurrence of adipose oxidative stress. The
increased MDA level further depicts peroxidation of the lipid contents.
This correlates with previous reports on the induction of oxidation of
oxidative stress in tissues incubated with only glucose [3,9]. Adipose
oxidative stress increases proliferation, differentiation and size of adi­
pocytes as well as alter intracellular signaling in adipocytes [56,57].
Targeting adipose oxidative stress has been reported to be a therapeutic
strategy in managing adipose dysfunction and obesity [58]. Thus, the
exacerbated GSH level, SOD and catalase activities, with concomitant
depleted MDA level in adipose tissues incubated with cannabidiol depict

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Fig. 9. In silico (A) 2D and (B) 3D molecular interaction of adipose triglyceride lipase; (C) 2D and (D) 3D molecular interactions of hormone-sensitive lipase; (E) 2D
and (F) 3D molecular interactions of monoglyceride lipase – with cannabidiol in silico.

8
O.L. Erukainure et al.
an antioxidant therapeutic effect. This can be attributed to the reported
potent antioxidant properties of cannabidiol [19]. Therefore, corrobo­
rating previous reports on the potentials of antioxidants in mitigating
adipose oxidative stress [59,60].
The non-neuronal choline system has been shown to be an important
part of the adipose tissue, where acetylcholine secreted by macrophages
aids the adaptation of adipocytes during thermogenesis [60]. The
exacerbated acetylcholinesterase activity in adipose tissues incubated
with only glucose (Fig. 4) indicates a depleted cellular level of acetyl­
choline, as the enzyme catalyzes its hydrolysis to choline and acetate.
Altered adipocyte levels of acetylcholine has been implicated in
diminished adaptive response of the adipocytes to thermogenesis, which
increases their susceptibility to lipid overaccumulation. The depleted
acetylcholinesterase activities in cannabidiol-incubated adipose tissues
therefore indicates an increased cellular level of acetylcholine, and
therefore suggests an improved adaptive response to thermogenesis.
The roles of the extracellular signaling molecules, adenosine and
adenosine triphosphate (ATP) in the physiological activities of adipose
tissues are well reported [61,62]. They bring about their activities by
acting on purinergic receptors expressed in adipose tissues [63]. Alter­
ations in their activities have been implicated in the pathology of adi­
pose dysfunction as they alter cellular availability adenosine which in
turn suppresses glucose uptake and lipogenesis [64,65]. ATP is hydro­
lyzed to adenosine by a reaction catalyzed by ecto-nucleotidase
(ENTPDase and 5′ nucleotidase). Thus, the exacerbated ENTPDase ac­
tivities in adipose tissues incubated with only glucose (Fig. 5A) indicates
a suppressed cellular level of ATP and adenosine. Adenosine has been
reported for their anti-lipolytic effect, with concomitant increased
lipogenesis and leptin production [66]. Low adipose level of ATP has
been reported for its inhibitory effect on insulin-mediated adipose
glucose uptake [67]. This corroborates the low glucose uptake in adi­
pose tissue incubated with only glucose (Fig. 2). Targeting purinergic
dysfunctions in adipose tissues has been suggested as therapeutic
strategy in the management of adipose dysfunctions and obesity.
Therefore, the suppressed ENTPDase activities, and elevated
5′ nucleotidase activity (Fig. 5B) in cannabidiol-incubated adipose tis­
sues indicate an increased cellular availability of ATP and adenosine.
Thus, suggesting an improved lipid and glucose metabolism.
Glucose metabolism in adipose tissues is important in energy ho­
meostasis. Glucose is also a lipogenic substrate in adipose tissues via the
glycolytic generation of acetyl-CoA for de novo synthesis of lipids [6,7].
The exacerbated activity of fructose − 1,6-biphosphatase in adipose
tissues incubated with only glucose (Fig. 6A–C) indicate an arrest of
glycolysis, and concomitant activation of gluconeogenesis. Thus, sug­
gesting depleted cellular levels of acetyl-CoA for de novo synthesis of
lipids. This is further corroborated by the increased activities of glucose
6-phosphatase and glycogen phosphorylase (Figs. 6B and C) which de­
pict glycogenolysis. Increased glycogenolysis and gluconeogenesis have
been correlated with increased lipolysis in adipose tissues [64,65]. The
activation of these pathways may be in response to glucose uptake in
adipose tissues incubated with only glucose (Fig. 2). Incessant glyco­
genolysis and gluconeogenesis would lead to cellular accumulation of
glucose and reduced ATP levels. The latter corroborating with the
altered purinergic activities (Fig. 5A–C). Accumulated cellular glucose
in its enediol form can undergo oxidation to generate ROS [66]. The
suppressed activities of these enzymes in adipose tissues incubated with
cannabidiol therefore indicate reactivation of glycolysis and glycogen­
esis. This suggests availability of acetyl-CoA for lipogenesis, ATP for
purinergic signaling, and increased adipocyte glycogen level. The
exacerbated activities may be attributed to the cannabidiol enhanced
glucose-uptake (Fig. 2).
Increased adipose lipase activity characterized by hydrolysis of adi­
pose triglycerides to FFAs has been implicated in adipocyte lipolysis.
The enzyme plays an important role in maintaining metabolic balance in
glucose-lipid homeostasis. In normal adipose physiology, it is activated
in response to suppressed cellular level of glucose as seen in fasting
9
Biomedicine & Pharmacotherapy 149 (2022) 112863
states to generate FFAs for energy production [67,68]. Thus, the
increased activity in adipose tissues incubated with only glucose (Fig. 7)
may be in response to suppressed glucose-uptake (Fig. 2), which cor­
roborates the exacerbated glucogenic enzyme activities (Fig. 6A–C). It
also correlates with the depleted triglyceride level of adipose tissue
incubated with only glucose (Fig. 8B). Thus, depicting an increased lipid
metabolism. Incessant activation of the enzyme will lead to increased
cellular levels of FFAs, causing lipotoxicity. Lipotoxicity has been
implicated in the pathophysiology of adipose dysfunction and obesity
[69]. It has also been implicated in the pathogenesis of pancreatic β-cell
dysfunction and insulin insensitivity, leading to hyperglycemia [47].
The reduced lipase activity in cannabidiol-incubated adipose tissues
indicate an antiobesogenic potential of the cannabinoid. This is further
corroborated by the reduced cholesterol level (Fig. 8A) as well as the
strong molecular interactions and binding energies of cannabidiol with
ATGL, HSL and MGL (Fig. 9). This correlates with the enhanced glucose
uptake, suppressed activities of the studied glucogenic enzymes, and
elevated triglyceride level in cannabidiol-incubated adipose tissues.
Thus, insinuating the potential of cannabidiol to maintain glucose-lipid
homeostasis.
The limitations of the present study are lack of in vivo studies and
expression levels of the studied enzymes.
5. Conclusion
Taken together, these results indicate that cannabidiol-enhanced
glucose uptake in adipose tissues is associated with enhanced anti­
oxidative activities, concomitant modulation of cholinergic and puri­
nergic dysfunctions, and improved glucose-lipid homeostasis. However,
further in vivo studies are recommended to elucidate the molecular
transcription of the studied effects by the cannabinoid in normal and
obese rats.
CRediT authorship contribution statement
OLE and MGM: Conceptualization. OLE, VFS, KAO, CIC, MSI, and
ALN: Methodology. SOO: Tissue culture. OLEO: original draft. All au­
thors: Reviewing and Editing. MGM: Supervision.
Conflict of interest statement
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Acknowledgements
Dr. Erukainure OL is thankful to the University of the Free State,
Bloemfontein, South Africa for Incentives for Rated Researchers
(2019060769); and the National Research Foundation (NRF) for Scarce
Skills Postdoctoral Research Grant (UID: 132822). Prof. Matsabisa MG is
thankful to the IKS Based Technology Innovation Unit of DSI South
Africa, for financial support (Grant contracts: DST/CON 0162/201 and
DST/CON 0206/2019/2020).
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