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Article
Olive Leaf Extract of Olea europaea Reduces Blood Glucose
Level through Inhibition of AS160 in Diabetic Rats
Abd Al-Rahman Al-Shudiefat 1, *,† , Hadeel Alturk 1,† , Hamzeh J. Al-Ameer 2,3,4 , Malek Zihlif 2
and Maha Alenazy 5

1 Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, The Hashemite University,
Zarqa 13133, Jordan; [email protected]
2 Department of Pharmacology, Faculty of Medicine, The University of Jordan, Amman 11942, Jordan;
[email protected] or [email protected] (H.J.A.-A.); [email protected] (M.Z.)
3 Department of Biology and Biotechnology, Faculty of Science, American University of Madaba,
Madaba 11821, Jordan
4 Department of Biological Sciences, Faculty of Science, Yarmouk University, Irbid 21163, Jordan
5 Department of Physiology, College of Medicine, King Saud University, Riyadh 4545, Saudi Arabia;
[email protected]
* Correspondence: [email protected]
† These authors contributed equally to this work.

Featured Application: Olive leaf extract could be a natural good alternative to the drug metformin
for the management of blood glucose in diabetic patients, with few side effects.

Abstract: Introduction: It has been shown that olive leaf extract exerts (OLE) a positive effect on
lipid and blood glucose levels; however, the mechanism remains poorly understood. This study
aimed to examine the mechanism behind this effect by evaluating the proteins related to glucose
metabolism, including glucose transporter 4 (Glut4), Akt Substrate of 160 kDa (AS160), and adenosine
monophosphate-activated protein kinase (AMPK α2). Methods: Eighty-four male Sprague–Dawley
rats were divided into three major groups: group one (control); group two, which was treated with
OLE or metformin (Met.) before streptozotocin (STZ) injection; and group three, which was treated
Citation: Al-Shudiefat, A.A.-R.; with OLE or Met. after STZ injection. The body weights, fasting blood sugar, postprandial sugar
Alturk, H.; Al-Ameer, H.J.; Zihlif, M.; levels, insulin levels, and lipid profile were assessed. Western blot was used to measure the Glut4,
Alenazy, M. Olive Leaf Extract of Olea AS160, and AMPKα 2 levels. Results: Treatments with (1% and 3% OLE) significantly decreased the
europaea Reduces Blood Glucose glucose level, AS160 expression level, and STZ toxicity; additionally, insulin levels were maintained
Level through Inhibition of AS160 in within the normal range and similar to Met. treatment. Conclusions: These findings indicated that
Diabetic Rats. Appl. Sci. 2023, 13,
OLE exerted antihyperglycemic effects via AS160 inhibition and it could be used as an alternative to
5939. https://doi.org/10.3390/
Met. treatment. Further studies on the long-term effects of OLE on diabetes are warranted.
app13105939

Academic Editor: Marco G. Alves Keywords: diabetes; streptozotocin; Olea europaea leaves extract; glucose metabolism; AS160; metformin

Received: 11 April 2023

Revised: 28 April 2023
Accepted: 1 May 2023
Published: 11 May 2023
1. Introduction
Presently, diabetes mellitus (DM) is one of the most widely spread diseases [1]. It is a
chronic metabolic disorder that results from elevated levels of blood glucose and may be
inherited or caused by other lifestyle factors [1]. It is characterized by a defect in the beta
Copyright: © 2023 by the authors. cells of the pancreas; consequently, the pancreas either does not produce sufficient amounts
Licensee MDPI, Basel, Switzerland. of insulin, the utilization of insulin is impaired, or both [1].
This article is an open access article In 2019, according to the World Health Organization (WHO) [2], approximately 422 mil-
distributed under the terms and
lion people worldwide were estimated to have diabetes, and approximately 1.6 million
conditions of the Creative Commons
people died of this condition each year. By 2045, this figure is estimated to reach 629 million
Attribution (CC BY) license (https://
people globally [3]. Recent studies conducted among different population groups reported
creativecommons.org/licenses/by/
prevalence rates of up to 20% in the United Arab Emirates, 16% in Qatar, 24% in Saudi
4.0/).

Appl. Sci. 2023, 13, 5939. https://doi.org/10.3390/app13105939 https://www.mdpi.com/journal/applsci

Appl. Sci. 2023, 13, 5939 2 of 17

Arabia, and 8% in Oman [4]. According to the WHO, the number of people suffering from
diabetes in Jordan was 195,000 in 2000, and this number is predicted to increase to 680,000
in 2030 [4].
DM can be classified as type 1 diabetes (T1D), which is characterized by autoimmune
damage of the beta cells in the pancreas. Autoimmune destruction usually occurs via
autoantibodies mediated by T cells against beta-cell antigens, leading to a decrease in the
level of insulin production [1]. The frequency of T1D is reported to range from 5% to 10%
of all cases of diabetes [5]. Some environmental factors such as toxins, viral infection, and
dietary components can stimulate an autoimmune reaction against beta cells in individuals
who are genetically susceptible to T1D. Patients with T1D require exogenous insulin to
manage the disease.
Type 2 diabetes (T2D) is the result of insulin resistance and impaired beta cell function,
which accounts for approximately 90% of all diabetes cases [1,5,6]. In the case of insulin
resistance, muscles, fat, and liver cells do not respond properly to insulin, resulting in
the accumulation of glucose in the bloodstream and a decrease in the amount of glucose
entering the cells. This leads to an increase in the production of insulin from the beta cells
in the pancreas to compensate for the decreased levels of glucose in the cells [5,7]. The
suggested reasons for insulin resistance include insulin receptor mutation, excess weight,
physical inactivity, older age, and smoking [5,8]. Insulin resistance causes alterations in
glucose levels from normal to low (impaired beta cells function) or high (insulin resistance)
in patients suffering from T2D. T2D typically occurs in obese individuals and those with a
family history of diabetes [5,9].
Although lifestyle and diet modifications are the basis for the treatment and man-
agement of diabetes, most patients will require pharmacotherapy, such as biguanides,
sulfonylureas, metformin, and thiazolidinediones. Patients with both types of diabetes
suffer from polyuria, thirst, blurred vision, and general fatigue [10].
Nevertheless, the use of diabetes medications is usually accompanied by different
side effects, including gastrointestinal anomalies, weight gain, hypoglycemia, edema,
anemia, and congestive heart failure [11,12]. Therefore, it is necessary to look for alternative
medications with fewer or no side effects.
Complementary and alternative medicine approaches that include herbs may prove
to be promising. Several medicinal herbs have been used in traditional medicine for
many years to improve or prevent diabetes. Bitter melon, aloe vera [9], ginseng [10],
fenugreek [12], and fig leaf [11] are some of the commonly used herbs to manage diabetes.
Several studies have shown that Olea europaea leaf extract is involved in multiple bio-
logical activities, such as the reduction of free radicals during tissue injury, normalization
of the lipid profile, increase in immune function, lowering of the blood glucose level and
blood pressure, decrease in arrhythmia, and prevention of intestinal muscle spasms, antiox-
idant and antimicrobial effects [13–16]. These important bioactivities could be attributed to
its bioactive components, which include oleuropein, hydroxytyrosol, tyrosol, tocopherol,
elenolic acid derivatives, caffeic acid, polyphenols (verbascoside, apigenin-7-glucoside, and
luteolin-7-glucoside), and flavonoids (rutin and diosmin) [13–15].
There are no detailed studies on the efficiency of olive leaf extract (OLE) in managing
and treating diabetes; additionally, the role of OLE in modulating the metabolic markers
associated with insulin resistance and diabetes is poorly understood. Therefore, the objec-
tive of this study was to investigate and confirm the possible antihyperglycemic effects
of Olea europaea leaf extract on streptozotocin (STZ)-induced diabetic rats. Additionally,
the possible mechanism/(s) by which the leaf extracts attenuate or prevent diabetes were
evaluated. The rats were rendered diabetic by a single intraperitoneal injection of STZ,
which is a chemical agent that can damage the beta cells in the pancreas [17]. Subsequently,
insulin sensitivity was restored using different concentrations of Olea europaea leaf extract,
and the effects were compared with those after treatment with metformin, which is a drug
used to manage diabetes by suppressing glucose production in the liver [17].
Appl. Sci. 2023, 13, 5939 3 of 17

We hypothesized that OLE reduces glucose and lipid levels through cell signals and
glucose metabolism-related molecules, such as glucose transporter 4 (glut4), 160 kDa Akt
substrate (AS160) and adenosine monophosphate (AMP) protein kinase (AMPK)2. Both
AS160 and AMPK2 participate in the transfer of the glucose transporter [18–20]. AMPK
is an enzyme that plays an important role in cell energy homeostasis. It is activated by
AMPK kinase by the phosphorylation of Thr172 in the alpha subunit. The activation of
AMPK catalytic activity accelerates the ATP-generating catabolic pathways, including
glycolysis, and suppresses the anabolic pathways, including cholesterol synthesis, while
consuming ATP [21]. In addition, activation of AMPK improves glucose absorption by
regulating GLUT4 translocation to cell membranes and genetic expression [22–24]. Previous
studies have shown that homocysteine sulfinic acid and curcumin have an antidiabetic
effect that can improve glucose absorption through the AMPK pathway [25,26]. 6-gingerol
stimulates glucose uptake and GLUT4 translocation in L6 myotube cells [27]. An in vitro
study showed that the activation of AMPK is associated with 6-gingerol-mediated glucose
uptake [28].

2. Materials and Methods

2.1. Animals
Eighty-four male Sprague–Dawley rats were purchased from Jordan University of
Science and Technology and treated according to a protocol approved by the Institute
Review Board at The Hashemite University in Jordan. The animals were acclimatized for
a week before the commencement of the experiment and maintained under controlled
temperature (22 ± 2 ◦ C) with a constant 12 h light/dark cycle. All animals were allowed free
access to food and water. The average body weight of the animals was 200 ± 20 g, which is
consistent with previous similar studies [29–33]. The rats were fed, treated, and sacrificed
after being anesthetized intra-peritoneally with a mixture of 9:1 ketamine (90 mg/kg)
to xylazine (10 mg/kg) in the animal house at the Faculty of Medicine, University of
Jordan. The measurements and analysis of different parameters (The body weights, fasting
blood sugar, postprandial sugar levels, insulin levels, lipid profile, and Western blot) were
performed in the research laboratory at The Hashemite University.

2.2. OLE
The OLE purchased from Nature’s Care Manufacture (Sydney; Australia; www.
healthycare.com.au, accessed on 22 April 2022) consisted of water and glycerin (no alcohol
or sugar was added). Every 5 mL of OLE contained Olea europaea leaf extract equivalent
to 5 g of dry leaf and 22 mg of oleuropein. A reverse phase High-performance liquid
chromatography (HPLC) method was conducted to analyze the concentration of Oleu-
ropein [34], HP 1100 HPLC-UV with an MZ Perfect Aqua C18 column (50 × 2.1 mm, 5 µm
particle size + precolumn 10 × 2.1 mm). Oleuropein was analyzed from the Olea europaea
leaf extract (OLE) to detect the validity of the OLE. An 80% (v/v) of Acetonitrile: water
solution was used as a solvent and 10 mg of OLE was dissolved in 1 mL of the solvent.
The resultant mixture was filtered through a 0.45 µm membrane filter into 2 mL HPLC
vials before injecting it into the HPLC device. The mobile phase used was 20% (v/v) of
Acetonitrile: water [34].

2.3. Protocol
After 1 week of acclimatization, the animals were divided into three major groups
(n = 28 each) as follows: group 1 (control; G1), which did not receive STZ injection; group
2, which received treatments with OLE and Metformin (Met.) 2 weeks before STZ injection
and for 3 weeks after the injection (G2); group 3, which received treatments with OLE and
Met. 1 week after STZ injection for 2 weeks (G3). Two forms of olive leaf extracts, water or
alcohol based, can be used medically [35,36]. All the animals in the present study received
a dose of 1 mL (1%, 3% v/v) of OLE and 100 mg/kg of Met. daily via oral gavage. At
the beginning of the third week, 56 rats from groups 2 and 3 were injected with a single
Appl. Sci. 2023, 13, 5939 4 of 17

intraperitoneal dose of STZ (AppliChem GmbH in Darmstadt, Germany) at a concentration

of 35 mg/kg body weight, as described previously [37]. The rats in group 1 were injected
with 0.1 M of citrate buffer only, which was used to dissolve the STZ.
As shown in Table 1, the rats in the three groups were further divided into four
subgroups (n = 7 rats each) based on the treatment received: distilled water (D.W); 1% OLE;
3% OLE; and Met. (100 mg/kg).

Table 1. Groups of rats in the study. n = 7 rats in each subgroup.

Week G1 G2 G3
1% 3% 1% 3%
1 D.W Met. D.W Met. N.T. N.T. N.T. N.T.
OLE OLE OLE OLE
1% 3% 1% 3%
2 D.W Met. D.W Met. N.T. N.T. N.T. N.T.
OLE OLE OLE OLE
1% 3%
3 D.W Met. STZ treatment + previous treatment. STZ treatment only.
OLE OLE
1% 3% 1% 3% 1% 3%
4 D.W Met. D.W Met. D.W Met.
OLE OLE OLE OLE OLE OLE
1% 3% 1% 3% 1% 3%
5 D.W Met. D.W Met. D.W Met.
OLE OLE OLE OLE OLE OLE
G1: control group. G2: received OLE treatment before and after injection with streptozotocin. G3: received OLE
treatment after injection with streptozotocin. D.W, distilled water; OLE, olive leaf extract; Met., metformin; N.T.:
no treatment.

2.4. Biochemical Tests

2.4.1. Measurement of Fasting Blood Sugar (FBS) and Postprandial Sugar (PPS) Levels
The glucose level was measured every day after 8 h of fasting to diagnose diabetes [38].
The postprandial sugar level was determined 2 h after eating to determine glucose tolerance
in their bodies. A glucose meter (Prodigy, Prodigy Diabetes Care Inc., London, UK) was
used to measure the glucose concentration in one drop of tail blood from each rat in all
the groups.

2.4.2. Measurement of Serum Insulin Level

The rats in G2 and G3, which received STZ, were tested for T1D during the third week
by measuring the glucose level, observing the symptoms of polydipsia and polyuria, and
checking the insulin level using a commercially available kit (sandwich enzyme-linked
immunosorbent assay kit; Abcam Plc, Cambridge, UK). The insulin levels in rats belonging
to G1 were measured.
Blood was collected from the rats before (at the beginning of the third week) and after
(at the end of the third week) STZ injection to measure the insulin levels in all groups. The
rats were anesthetized with ether, following which 5 mL of whole blood was collected
from the retro-orbital capillary vessels (before and after STZ injection). The blood was
centrifuged at 3000 rpm for 5 min to obtain the serum, which was then stored at −20 ◦ C for
future analysis.

2.4.3. Serum Lipid Profile

The lipid concentration was measured and analyzed using Humalyzer 3500 and kits
from human diagnostic worldwide (Germany) according to the manufacturer’s instruc-
tions. Blood serum samples were used to measure the total cholesterol (T.Ch.), HDL, and
triglyceride (T.G) levels, which were expressed as mg/dL. Subsequently, the low-density
lipoprotein–concentration (LDL-C) was calculated from the concentrations of T.Ch., HDL,
and T.G using the Friedwald equation as follows [39]:
Low Density Lipoprotein Concentration = Total Cholesterol. − High Density Lipopro-
tein − (Triglyceride/5)
Appl. Sci. 2023, 13, 5939 5 of 17

2.5. Muscle Biopsies

At the end of the experiment, two soleus muscles were obtained from each rat and
stored at −80 ◦ C for analysis. This muscle was used because of its rich microvasculature
and susceptibility to the pathophysiological effects of T1D [40,41].

2.6. Western Blot Analysis

The soleus muscle (0.4 g) was homogenized in 1 mL of cold lysis buffer (0.01 mL of
1 M Tris pH 7.5, 0.02 mL of 0.1 M EGTA, 0.01 mL of 1 M DTT, 0.2 mL of 10% SDS, 0.01 mL of
NaF, and 0.05 mL deionized water) for 5 s using a homogenizer, and the concentrations of
the proteins were estimated using the filter paper assay [42]. Briefly, grids (1.5 cm × 1.5 cm)
were drawn on circular Whatman filter papers. Protein standards (1–5 µg/mL egg albumin)
and protein samples (volume, 1 µL) were applied to the center of each square of the grid and
air dried. The filter paper was rinsed in methanol for 20 s and air dried. The samples were
stained in Coomassie blue (0.5% Coomassie blue G, 7% acetic acid) for 10 min, destained
in 7% acetic acid until the background was evenly pale with no spots, and air dried. The
squares were cut and each one was placed in a 1.5 mL centrifuge tube; 1 mL of extraction
buffer (66% methanol, 33% water, and 1% ammonium hydroxide) was added to each tube,
and vortexed on a rack for 1 min. Then, 300 µL of each sample was added to a well in a
96-well plate and read at an absorbance of 600–405 nm using a microplate reader.
Subsequently, the cell lysates were heated at 95 ◦ C for 5 min and solubilized with 4×
sample preparation buffer (glycerol, 2-mercaptoethanol [Sigma, St. Louis, MO, USA], 10%
SDS, and 0.5 M Tris/HCL; pH 6.8). The proteins were separated by SDS-PAGE using either
7% or 10% polyacrylamide gels, according to the molecular weights of the proteins, and
transferred to a nitrocellulose membrane for 2 h at 200 mA.
The membranes were blocked for 1 h with Tris-buffered saline (TBS; pH 7.5) containing
1% bovine serum albumin (BSA) and incubated overnight at 4 ◦ C with the following
specific primary antibodies: AMPK α2 (dilution, 1:500; rabbit polyclonal antibody; Abcam),
AS160 (dilution, 1:100; goat polyclonal antibody; Santa Cruz Biotechnology), and Glut 4
(dilution, 1:500; rabbit polyclonal antibody; Abcam). The antibodies were diluted in 1%
BSA in TBS-tween20 (TBST). To verify the increase or decrease in protein expression, the
GAPDH antibody (dilution, 1:500; purified mouse monoclonal antibody; R&D systems,
Minneapolis, MN, USA) was used as a control. The membranes were incubated overnight
at 4 ◦ C with continuous shaking, washed thrice with TBST for 1 h, and incubated with
horseradish peroxidase-conjugated antirabbit/goat secondary antibody (1:4000 in 1% BSA
in TBST) for 1 h at 25 ◦ C. Finally, the membranes were washed three times for 1 h with
TBST and subjected to Western blotting using a Pierce ECL substrate (Thermo Scientific,
Waltham, MA, USA). The bands were visualized using X-ray films (AGFA) or 3,3,5,5
tetramethylbenzene (Sigma, USA), and quantified using image analysis software (Quantity
One, Bio-Rad Laboratories).

2.7. Statistical Analysis

Differences between groups were considered significant at a p-value of <0.05. Standard
deviation was used to express variations in the data compared with the mean values. The
groups were compared using the one-way analysis of variance test (ANOVA) followed
by Bonferroni’s test. All statistical analyses were performed using the Graph Pad Instant
program (Sacramento, CA, USA).

3. Results
3.1. Body Weight
The body weights of all the rats in the various groups ranged from 180 to 220 g and
were not significantly different at the beginning of the experiment. A significant increase
(p < 0.05) in weight was observed in the 1% OLE subgroup in G1 when compared with
the 1% OLE and 3% OLE subgroups in G3. Similarly, a significant increase (p < 0.05) was
observed in the 3% OLE G1 subgroup compared with the 3% OLE G3 subgroup (Table 2).
Appl. Sci. 2023, 13, 5939 6 of 17

There was a significant increase (p < 0.05) in body weight changes in all subgroups of G1
(D.W, 1% OLE, 3% OLE, Met.) compared with 1% OLE G3 as seen in Table 2.

Table 2. Effect of various treatments on the body weights of the rats.

Experimental Initial Body Final Body Weight

Groups Weight (g) Weight (g) Changes (g)
D.W G1 199.28 ± 15.39 255 ± 37.74 55.71 ± 27.14 e
1% OLE G1 258.33 ± 18.34 319.16 ± 10 ef 60.833 ± 21.77 e
G1
3% OLE G1 220 ± 18.70 291 ± 28.80 f 71 ± 16.73 e
Met. G1 244.5 ± 30.97 280.83 ± 35.55 36.33 ± 10.80 e
D.W G2 254 ± 51.64 252 ± 77.91 −2 ± 50.81
1% OLE G2 265 ± 14.71 287.5 ± 45.73 22.5 ± 55.45
G2
3% OLE G2 283 ± 19.55 279 ± 63.38 −4 ± 56.94
Met. G2 268.75 ± 11.81 285 ± 26.77 16.25 ± 18.90
D.W G3 254 ± 51.64 252 ± 77.91 −2 ± 50.81
1% OLE G3 245 ± 14.71 196 ± 26.31 b −49 ± 25.83 abcd
G3
3% OLE G3 226.66 ± 36.56 235 ± 40.49 bc 8.33 ± 26.39
Met. G3 258 ± 43.09 246 ± 32.86 −12 ± 59.85
G1: control group; G2: received treatments with olive leaves extract (OLE) and Metformin (Met.) 2 weeks before
injection with STZ and for another 3 weeks after STZ injection; G3: received treatments with OLE and Met. 1 week
after STZ injection and continued for 2 weeks. a p < 0.05 vs. D.W G1, b p < 0.05 vs. 1% OLE G1, c p < 0.05 vs. 3%
OLE G1, d p < 0.05 vs. Met. G1, e p < 0.05 vs. 1% OLE G3, and f p < 0.05 vs. 3% OLE G3.

3.2. Effects of Various Treatments (1% OLE, 3% OLE, and Met.) on Glucose Levels
The blood glucose concentration was measured daily throughout all 5 weeks to assess
the effects of various treatments on the FBS and PPS levels in all the groups. In the fifth
week, significant increases (p < 0.05) in both FBS and PPS were observed in D.W G2 and
the D.W and 1% OLE subgroups from G3 when compared with those in G1; the values in
the other subgroups from G2 (1% OLE, 3% OLE, and Met.) and subgroups (3% OLE and
Met.) of G3 were comparable with those from G1 (Figure 1).

3.3. STZ Induces T1D

3.3.1. Protective Effect of OLE and Met. against STZ Toxicity in the Beta Cells
In week 3, the rats in G2 and G3 were injected with STZ. A significant decrease in the
development of diabetes (p < 0.05) was observed in G2 compared with G3 (Table 3).

Table 3. Streptozotocin toxicity.

% of Diabetic Rats % of Nondiabetic Rats

G2 35% a 65% a
Subgroups (1% OLE, 3% OLE) Five rats were not protected Nine rats were protected from
n = 14 from STZ toxicity STZ toxicity
G3 57% b 43% b
Subgroups (1% OLE, 3% OLE) Eight rats were not protected Six rats were protected from
n = 14 from STZ toxicity STZ toxicity
G2: received the treatments with OLE and Met. treatments 2 weeks before and 3 weeks after STZ injection. G3:
received treatments with OLE and Met. 1 week after STZ injection for 2 weeks. ANOVA test; a p < 0.05 vs. G2
without treatment (D.W); b p < 0.05 vs. G3 without treatment (D.W).

3.3.2. Effect of STZ on Insulin Levels

The insulin levels were similar among the groups of rats on week 3 before the STZ
injection was provided to the G2 rats. After STZ treatment, the levels in most of the
subgroups in G2 and G3 were significantly decreased (p < 0.05) when compared with those
in G1 (Figure 2). However, the levels in the 1% OLE and 3% OLE subgroups in G2 were not
significantly different from those in the 3% OLE and Met. subgroups in G1. Furthermore,
the insulin levels in the 1% OLE and 3% OLE subgroups from G2 were significantly higher
Appl. Sci. 2023, 13, 5939 7 of 17

(p < 0.05) than those in the D.W and Met. subgroups in the same group. Similarly, significant
Appl. Sci. 2023, 13, x FOR PEER REVIEW
increases 7 of 18
(p < 0.05) in insulin levels were observed in the 1% OLE and 3% OLE subgroups
in G2 when compared with all untreated subgroups in G3 (Figure 2).

Figure1.1.Blood
Figure Bloodglucose
glucoselevels
levelsin
inall
allgroups
groupsduring
duringthe thefifth
fifthweek.
week.G1:
G1:(control),
(control),G2:
G2:received
receivedOLE
OLE
and Met. 2 weeks before and 3 weeks after STZ, G3: received treatments with OLE
and Met. 2 weeks before and 3 weeks after STZ, G3: received treatments with OLE and Met. 1 week and Met. 1 week
afterSTZ
after STZinjection
injection and
and continued
continued for
for 22 weeks.
weeks. a,
a, pp << 0.05
0.05 vs.
vs. D.W
D.WG1;G1;b,b,pp<<0.05
0.05vs.
vs.1%
1%OLE
OLEG1;
G1;c,c,p
< 0.05 vs. 3% OLE G1; d, p < 0.05 vs. Met.G1; e, p < 0.05 vs. D.W G2; f, p < 0.05 vs. D.WG3; g, p <0.05
p < 0.05 vs. 3% OLE G1; d, p < 0.05 vs. Met. G1; e, p < 0.05 vs. D.W G2; f, p < 0.05 vs. D.W G3; g,
vs. 1% OLE G3. Data are presented as mean ± standard deviation (SD). n = 5–7 animals in each
p < 0.05 vs. 1% OLE G3. Data are presented as mean ± standard deviation (SD). n = 5–7 animals in
subgroup. FBS: Fast blood sugar, PPS: Postprandial sugar.
each subgroup. FBS: Fast blood sugar, PPS: Postprandial sugar.
3.3.Effects
3.4. STZ Induces T1DTreatments on the Lipid Profile Levels
of Various
3.3.1.Figures
Protective Effect of OLE
3 and 4 show the and Met.
serum against
T.Ch., T.G,STZ Toxicity
HDL, in thelevels
and LDL Beta Cells
in the rats in all
groups,In week
which3,were
the rats in G2 and
measured G3 were
at the injected
end of with STZ. Aperiod
the experimental significant
(afterdecrease
week 5).inNo
the
development
significant of diabetes
differences (p < 0.05) was
in cholesterol observed
levels in G2 compared
were observed between with G3 (Table
the groups 3). 3A).
(Figure
A significant increase (p < 0.05) in the triglyceride level was observed in the 1% OLE G1
Table 3. Streptozotocin
subgroup compared with toxicity.
the 1% OLE G3 subgroup (Figure 3B). No significant differences in
HDL levels were noted among the groups (Figure 4A). Nevertheless, a significant increase
% of Diabetic Rats % of Nondiabetic Rats
in the LDL level (p < 0.05) was observed in the 1% OLE G3 subgroup compared with all
G2 the subgroups in G1; similarly, a significant increase in LDL was noted65%
35% a a
in the 3% OLE G3
Subgroups (1% OLE, 3% OLE) compared
subgroup Fivewith
rats the
were 3%not
OLEprotected
and Met.from STZ Nine
subgroups in G1rats were 4B).
(Figure protected from
n = 14 toxicity STZ toxicity
G3 57% b 43% b
Subgroups (1% OLE, 3% OLE) Eight rats were not protected from STZ Six rats were protected from STZ
n = 14 toxicity toxicity
G2: received the treatments with OLE and Met. treatments 2 weeks before and 3 weeks after STZ
injection. G3: received treatments with OLE and Met. 1 week after STZ injection for 2 weeks.
ANOVA test; a p < 0.05 vs. G2 without treatment (D.W); b p < 0.05 vs. G3 without treatment (D.W).
 groups in G2 and G3 were significantly decreased (p < 0.05) when compared with those in
G1 (Figure 2). However, the levels in the 1% OLE and 3% OLE subgroups in G2 were not
significantly different from those in the 3% OLE and Met. subgroups in G1. Furthermore,
the insulin levels in the 1% OLE and 3% OLE subgroups from G2 were significantly higher
(p < 0.05) than those in the D.W and Met. subgroups in the same group. Similarly, signifi‐
Appl. Sci. 2023, 13, 5939 8 of 17
cant increases (p < 0.05) in insulin levels were observed in the 1% OLE and 3% OLE sub‐
groups in G2 when compared with all untreated subgroups in G3 (Figure 2).

Figure2.2. Insulin
Figure Insulin concentration
concentrationafter
afterinjection
injectionwith
withSTZ
STZonon week
week3. G1: (control),
3. G1: G2: received
(control), treat‐
G2: received
ments withwith
treatments OLEOLEandand
Met.Met.
2 weeks before
2 weeks and and
before 3 weeks afterafter
3 weeks STZ injection, G3 (four
STZ injection, subgroups
G3 (four with‐
subgroups
out treatment and injected with STZ). a, p < 0.05 vs. D.W G1; b, p < 0.05 vs. 1% OLE G1; c, p < 0.05
without treatment and injected with STZ). a, p < 0.05 vs. D.W G1; b, p < 0.05 vs. 1% OLE G1; c, p < 0.05
vs. 3% OLE G1; d, p < 0.05 vs. Met. G1; e, p < 0.05 vs. D.W G2; f, p < 0.05 vs. 1% OLE G2; g, p < 0.01
vs. 3% OLE G1; d, p < 0.05 vs. Met. G1; e, p < 0.05 vs. D.W G2; f, p < 0.05 vs. 1% OLE G2; g, p < 0.01
vs. 3% OLE G2; h, p < 0.05 vs. Met. G2; i, p < 0.05 vs. G3; l, p < 0.01 vs. G3.
vs. 3% OLE G2; h, p < 0.05 vs. Met. G2; i, p < 0.05 vs. G3; l, p < 0.01 vs. G3.
3.4.AMPK
3.5. Effects α2
of Various Treatments on the Lipid Profile Levels
Expression
Figures
The results3 and 4 showα2the
of AMPK serum T.Ch.,
expression T.G.,no
revealed HDL, and LDL
significant levels in in
differences the
therats in all
AMPK
groups,
α2 whichlevels
expression wereamong
measured at groups
all the the end(Figure
of the experimental
5). period (after week 5). No
significant differences in cholesterol levels were observed between the groups (Figure
3.6.
3A).AS160 Expression
A significant increase (p < 0.05) in the triglyceride level was observed in the 1% OLE
G1 subgroup
The expression levelswith
compared the 1%were
of AS160 OLEnot G3 comparable
subgroup (Figure
among3B). No significant
all groups differ‐
at the end of
ences in HDL levels were noted among the groups (Figure 4A). Nevertheless,
the experiments (5 weeks). Significant differences were observed among all groups (Table 4 a significant
increase
and Figurein6).
theTheLDL level trend
general (p < 0.05)
was was observed
a decrease in the in
in AS160 1%groups
OLE G3 subgroup
2 and compared
3 compared with
with all
group 1. the subgroups
Treatment withinboth
G1; Met.
similarly,
and OLEa significant increase
significantly in LDLthe
decreased was noted in the
expression 3%
levels
OLE
of G3 subgroup
AS160 when comparedcomparedwithwith
thosethe 3% OLEinand
observed theMet. subgroups in G1 (Figure 4B).
controls.

Table 4. Expression levels of AS160 in all the groups (a.u) normalized over the expression level
of GAPDH.

Experimental Groups AS160

D.W G1 1.60 ± 0.11 bcfghjkl
D.W G2 1.58 ± 0.033 bcfghjkl
1% OLE G1 0.824 ± 0.025 acdefghi
1% OLE G2 1.097 ± 0.0005 abcdeghil
3% OLE G1 1.20 ± 0.017 abdefghikl
3% OLE G2 0.98 ± 0.0038 abcdefhil
Met. G1 1.46 ± 0.055 bcfghjkl
Met. G2 0.57 ± 0.082 abcdefgil
Appl. Sci. 2023, 13, 5939 9 of 17

Table 4. Cont.

Experimental Groups AS160

D.W G3 1.66 ± 0.08 bcfghjkl
1% OLE G3 0.92 ± 0.17 adehi
3% OLE G3 0.79 ± 0.153 acdefi
Met. G3 0.62 ± 0.06 acdefgi
G1: (control), G2: received OLE and Met. 2 weeks before and 3 weeks after STZ injection, G3: received treatments
with OLE and Met. 1 week after STZ injection for 2 weeks. a p < 0.05 vs. D.W G1; b p < 0.05 vs. 1% OLE G1;
c p < 0.05 vs. 3% OLE G1; d p < 0.05 vs. Met. G1; e p < 0.05 vs. D.W G2; f p < 0.05 vs. 1% OLE G2; g p < 0.01 vs. 3%

OLE G2; h p < 0.05 vs. Met. G2; i p < 0.05 vs. D.W G3; j p < 0.05 vs. 1% OLE G3; k p < 0.05 vs. 3% OLE G3, l p < 0.05
vs. Met. G3. a.u, arbitrary units.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 9 of 18

Figure3.3.Serum
Figure Serum lipid
lipid profiles
profiles in
inall
allthe
thegroups
groupsafter
after5 weeks. (A)(A)
5 weeks. Cholesterol (T.Ch);
Cholesterol (B) Triglyceride
(T.Ch.); (B) Triglyceride
(T.G). G1: (control), G2: received OLE and Met. 2 weeks before and 3 weeks after STZ injection, G3:
(T.G). G1: (control), G2: received OLE and Met. 2 weeks before and 3 weeks after STZ injection, G3:
received treatments with OLE and Met. 1 week after STZ injection for 2 weeks. a, p < 0.05 vs. 1%
received treatments with OLE and Met. 1 week after STZ injection for 2 weeks. a, p < 0.05 vs. 1%
OLE G1; b, p < 0.05 vs. 1% OLE G3. Data are presented as mean ± SD in four to seven animals per
OLE G1; b, p < 0.05 vs. 1% OLE G3. Data are presented as mean ± SD in four to seven animals
subgroup.
per subgroup.
Appl. Sci. 2023, 13, 5939 10 of 17
Appl. Sci. 2023, 13, x FOR PEER REVIEW 10 of 18

Figure4.4.Serum
Figure Serum lipid
lipid profiles
profiles of of
thethe
ratsrats in the
in all all the groups
groups afterafter 5 weeks.
5 weeks. (A) HDL
(A) HDL cholesterol.
cholesterol. (B)
(B) LDL
LDL cholesterol.
cholesterol. G1: (control),
G1: (control), G2: received
G2: received OLE and OLE and
Met. Met. 2before
2 weeks weeksand
before and after
3 weeks 3 weeks
STZafter STZ
injection,
injection, G3: received treatments with OLE and Met. 1 week after STZ injection for 2 weeks. a, p <
G3: received treatments with OLE and Met. 1 week after STZ injection for 2 weeks. a, p < 0.05 vs.
0.05 vs. D.W G1; b, p < 0.05 vs. 1% OLE G1; c, p < 0.05 vs. 3% OLE G1; d, p < 0.05 vs. Met. G1, j, p <
D.W G1; b, p < 0.05 vs. 1% OLE G1; c, p < 0.05 vs. 3% OLE G1; d, p < 0.05 vs. Met. G1, j, p < 0.05 vs.
0.05 vs. 1% OLE G3, k, p < 0.05 vs. 3% OLE G3. Data are presented as mean ± SD. n = 4–7 rats per
1% OLE G3, k, p < 0.05 vs. 3% OLE G3. Data are presented as mean ± SD. n = 4–7 rats per subgroup.
subgroup.
3.7. GLUT4 Expression
3.5. AMPK α2 Expression
No significant differences (p < 0.05) in the expression levels of GLUT4 were observed
The results of AMPK α2 expression revealed no significant differences in the AMPK
among the groups (Figure 7).
α2 expression levels among all the groups (Figure 5).
Appl.
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11 18

Figure 5.5. Expression

Figure Expression levels
levels of
ofAMPK
AMPKα2 α2ininallallthe
thegroups
groupsat at
thethe
end of of
end week 5. G1:
week (control),
5. G1: G 2:
(control),
received OLE and Met. 2 weeks before and 3 weeks after STZ injection, G3: received treatments
G2: received OLE and Met. 2 weeks before and 3 weeks after STZ injection, G3: received treatments with
OLE and Met. 1 week after STZ injection for 2 weeks. Data are presented as mean ± SD. n = 4–7 rats
with OLE and Met. 1 week after STZ injection for 2 weeks. Data are presented as mean ± SD. n
Appl. Sci. 2023, 13, x FOR PEER REVIEW 12=of4–7
18
per subgroup. a.u, arbitrary units.
rats per subgroup. a.u, arbitrary units.

3.6. AS160 Expression

The expression levels of AS160 were not comparable among all groups at the end of
the experiments (5 weeks). Significant differences were observed among all groups (Table
4 and Figure 6). The general trend was a decrease in AS160 in groups 2 and 3 compared
with group 1. Treatment with both Met. and OLE significantly decreased the expression
levels of AS160 when compared with those observed in the controls.

Table 4. Expression levels of AS160 in all the groups (a.u) normalized over the expression level of
GAPDH.

Experimental Groups AS160

D.W G1 1.60 ± 0.11 bcfghjkl
D.W G2 1.58 ± 0.033 bcfghjkl
1%OLE G1 0.824 ± 0.025 acdefghi
1% OLE G2 1.097 ± 0.0005 abcdeghil
3% OLE G1 1.20 ± 0.017 abdefghikl
3% OLE G2 0.98 ± 0.0038 abcdefhil
Met. G1 1.46 ± 0.055 bcfghjkl
Met. G2 0.57 ± 0.082 abcdefgil
D.W G3 1.66 ± 0.08 bcfghjkl
1%OLE G3 0.92 ± 0.17 adehi
Figure6.6.AS160
Figure 3% OLEinG3
AS160expression
expression inall
allthe
thegroups
groupsatatthe
theend week5.5.0.79
endofofweek ±(control),
0.153 acdefi
G1:(control),
G1: G2:received
G2: receivedOLE
OLE
and Met. 2 weeks Met.
before G3
and 3 weeks after STZ injection, G3: 0.62
received ± 0.06 acdefgi with OLE and
treatments
and Met. 2 weeks before and 3 weeks after STZ injection, G3: received treatments with OLE and Met.
Met.(control),
1 week after STZ injection for
and2Met.
weeks. Data are presented as mean ± SD. n injection,
= 4–7 animalsre‐
in
1G1:
week after STZ G2:injection
received OLE
for 2 weeks. 2 weeks
Data before
are presented and 3 weeks
as mean after
± SD. n =STZ
4–7 animals inG3:
each
each subgroup.
ceived One‐way
treatments with OLEanalysis of variance
and Met. is used.
1 week after STZa.u, arbitrary
injection forunits.
2 weeks. a p < 0.05 vs. D.W G1;
subgroup. One-way analysis of variance is used. a.u, arbitrary units.
b p < 0.05 vs. 1% OLE G1; c p < 0.05 vs. 3% OLE G1; d p < 0.05 vs. Met. G1; e p < 0.05 vs. D.W G2; f p <

0.05 vs. 1% OLE G2; g p < 0.01 vs. 3% OLEG2; h p < 0.05 vs. Met. G2; i p < 0.05 vs. D.W G3; j p < 0.05 vs.
1% OLE G3; k p < 0.05 vs. 3% OLE G3, l p < 0.05 vs. Met. G3. a.u, arbitrary units.
Appl.
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13 of 17
18

Figure7.7. Expression
Figure Expression levels
levels of
of GLUT4
GLUT4ininthe
thegroups
groupsatatthe
theend
endofof
week
week5. G1: (control
5. G1: group),
(control G 2:
group),
received OLE and Met. 2 weeks before and 3 weeks after STZ injection, G3: received treatments
G2: received OLE and Met. 2 weeks before and 3 weeks after STZ injection, G3: received treatments with
OLE and Met. 1 week after STZ injection for 2 weeks. Data are presented as mean ± SD in 4–7 animals
with OLE and Met. 1 week after STZ injection for 2 weeks. Data are presented as mean ± SD in
per subgroup. a.u, arbitrary units.
4–7 animals per subgroup. a.u, arbitrary units.

4.4.Discussion
Discussion
Oleaeuropaea
Olea europaealeaves
leaveshavehavebeen
beenused
usedto totreat
treatdiabetes
diabetesowing
owingto totheir
theirability
abilitytotodecrease
decrease
glucose,triglyceride,
glucose, triglyceride, cholesterol,
cholesterol, andandLDL LDLlevels levelsand andincrease
increasehigh-density
high‐densitylipoprotein
lipoprotein
(HDL)levels.
(HDL) levels. Many studies studies ononthethetoxicity
toxicityofofOLE OLEreported
reported that
thatit is
it generally
is generally safesafe
for for
use
even
use at high
even doses
at high [34,35].
doses The purpose
[34,35]. The purpose of thisofstudy was towas
this study explore the effect
to explore theofeffect
OLE of on
glucose and lipid metabolism in diabetic rats. The mechanism
OLE on glucose and lipid metabolism in diabetic rats. The mechanism involved in the involved in the manage‐
ment of hyperglycemia
management of hyperglycemiaby OLEby in OLE
diabetic animals animals
in diabetic has not has beennot explored in previous
been explored in
studies [29–32].
previous studies Two [29–32].concentrations of OLE (1%
Two concentrations of OLEand(1% 3%)andwere used
3%) wereto used
determine the pro‐
to determine
the protective
tective effect oneffect
theon the animals
animals in thein the present
present study.study. Although
Although several several
drugsdrugs
havehave beenbeen
used
used
for thefortreatment
the treatment of diabetes,
of diabetes, their their highand
high cost costadverse
and adverse
effectseffects have resulted
have resulted in the in the
search
search for alternative
for alternative therapies.
therapies. Several Several
typestypes
of herbs of herbs
havehavebeenbeenusedused to manage
to manage diabetes,
diabetes, and
and approximately 80% of people with diabetes
approximately 80% of people with diabetes are reported to depend are reported to depend on medicinal plants
medicinal plants
for
forsuccessful
successfultreatment
treatment[33]. [33].
In
Inagreement
agreementwith withthe findings
the findings of previous
of previous studies, no significant
studies, no significant effecteffect
of OLE of or
OLE Met.
or
was
Met.observed
was observed on theon body
the weights of the of
body weights ratstheinrats
the inpresent study study
the present [29,43].[29,43].
Alternatively,
Alterna‐
significant increases
tively, significant in both FBS
increases andFBS
in both PPSand were PPS observed in the D.W
were observed in thesubgroups in groups
D.W. subgroups in
2groups
and 3 when compared with all the subgroups in G1 at the
2 and 3 when compared with all the subgroups in G1 at the end of week 5, indi‐ end of week 5, indicating
the effect
cating theof STZofinSTZ
effect these animals
in these (Figure
animals (Figure1). Furthermore,
1). Furthermore, thethe1%1% OLEOLEG3 G3subgroup
subgroup
presented with significantly higher levels of FBS and PPS
presented with significantly higher levels of FBS and PPS than the other subgroups in G1,than the other subgroups in
G1, thus indicating that the low dose used (1% OLE) was
thus indicating that the low dose used (1% OLE) was not effective. A previous study not effective. A previous study
showed
showedthat thatapproximately
approximately 2–62–6weeks
weeks of of
treatment
treatment with OLE
with OLEwere required
were to observe
required the
to observe
desired effects [29,32,44]. The glucose levels in the 1% OLE
the desired effects [29,32,44]. The glucose levels in the 1% OLE G2 and 3% OLE G2 sub‐ G2 and 3% OLE G2 subgroups
and the 3%
groups andOLE the G3 3%subgroup
OLE G3 were similar
subgroup wereto G1; therefore,
similar to G1; OLE might prove
therefore, OLE beneficial
might prove in
reducing STZ toxicity, as reported previously [29,32,45]. Additionally,
beneficial in reducing STZ toxicity, as reported previously [29,32,45]. Additionally, the the STZ toxicity in
the
STZ 1% OLE G2
toxicity in subgroup
the 1% OLE wasG2lower than that
subgroup was in the 1%
lower thanOLEthatG3in subgroup,
the 1% OLE indicating
G3 subgroup, that
treatment with OLE before and after STZ injection might be
indicating that treatment with OLE before and after STZ injection might be more effective more effective in preventing
STZ toxicity and thereby preventing diabetes (Figure 1).
in preventing STZ toxicity and thereby preventing diabetes (Figure 1).
The Met. subgroups in both groups 2 and 3 showed comparable results to those in
The Met. subgroups in both groups 2 and 3 showed comparable results to those in
the G1 subgroups, indicating the protective effect of Met. against diabetic hyperglycemia,
the G1 subgroups, indicating the protective effect of Met. against diabetic hyperglycemia,
as reported previously [43,46]. Moreover, decreased glucose levels were observed in
as reported previously [43,46]. Moreover, decreased glucose levels were observed in the
the 1% OLE, 3% OLE, and Met. subgroups in G2 when compared with their negative
1% OLE, 3% OLE, and Met. subgroups in G2 when compared with their negative controls
controls (D.W-treated); similarly, decreased levels of FBS and PPS were observed in the
(D.W‐treated); similarly, decreased levels of FBS and PPS were observed in the 3% OLE
Appl. Sci. 2023, 13, 5939 13 of 17

3% OLE G3 and Met. G3 subgroups when compared with their relative negative controls,
statistical significance was notwithstanding. This may be explained by the increase in the
duration and concentration required to lower the glucose levels, as reported in previous
studies [29,32,47], which remained for 2–6 weeks when treated with OLE and may reach
6% OLE and 1–3 months when treated with Met. [43,46].
A previous study showed that STZ can partially damage beta cells and induce T1D
(type 1 diabetes (<50 mg/kg) or completely damage the beta cells in the pancreas leading to
T1D (type 1 > 50 mg/kg), based on the dose used [37]. In the present study, the protective
effect of 1% and 3% OLE against STZ toxicity was higher in G2 than in G3; 35% of the rats
in G2 developed diabetes compared with 57% in G3. This difference in response to STZ
toxicity may be attributed to the OLE treatment provided to the G2 rats before STZ injection.
The insulin levels in the 1% and 3% OLE G2 subgroups were similar to those in the 3%
OLE and Met. G1 subgroups, which might be due to the protective effect of OLE against
STZ cytotoxicity in the beta cells (Figure 2). Furthermore, significant increases (p < 0.05) in
insulin levels in the G2 subgroups treated with OLE were observed when compared with
those in the D.W and Met. subgroups of the same group, thus indicating that OLE treatment
was more effective than the Met. treatment in terms of enhancement of the insulin level.
This enhancement might be due to the protective effect of OLE against STZ toxicity in the
beta cells (Figure 2). A significant increase (p < 0.05) in insulin levels was observed in the
G2 subgroups treated with OLE compared with those in the G3 subgroups. Alternatively,
treatment with Met. did not lead to any significant enhancement in the insulin level in G2
compared with G3 (Figure 2).
The induction of T1D in the rats was confirmed by the elevated levels of fasting plasma
glucose and decreased levels of insulin. Similar results have been reported, wherein OLE
was effective in increasing insulin sensitivity, reducing the fasting blood glucose level, and
improving the glucose-induced release of insulin [14,48]. Additionally, previous studies
showed that metformin increased hepatic insulin sensitivity and insulin dependence in
the muscles and reduced blood glucose levels in humans [49,50]. One study reported that
metformin was responsible for the release of insulin from beta cells [44], whereas another
showed that it enhanced glucose uptake without any increase in insulin secretion [43].
Previous studies have shown that OLE reduces T.Ch., T.G, and LDL, and increase
HDL in diabetic animals due to the antioxidant effect of phenolic compounds such as
oleuropein, aglycone, and hydroxytyrosol [29,32,45,51]. In the present study, OLE did
not appear to alter the levels of T.Ch., T.G, HDL, and LDL; except increase in LDL in
1% OLE G3 and in 3% OLE G3 this may be due to the short duration of 5 weeks used
in this study. Another explanation of LDL increase in G3 treated with 1% and 3% OLE
could be treatment with OLE were after two weeks of streptozotocin injection not like G2
in which treatment with OLE was before streptozotocin injection, while G1 was control
group without streptozotocin injection. The reason for the increase of LDL in G3 treated
with 1% or 3% OLE is streptozotocin treatment, not OLE treatment, since G1 control
without streptozotocin and treated with 1% and 3% OLE showing normal comparable
LDL levels to control treated with distilled water in Figure 4. These results are supported
by previous studies, which have been shown that streptozotocin treatment significantly
increased LDL in rats [52,53]. Long-term use of OLE or alcoholic extract with higher OLE
concentrations has been reported to decrease the levels of cholesterol, triglyceride, and LDL,
and increase the HDL level [29,32,51]. Nonetheless, consistent with the findings of previous
studies [31,54], Met. treatment did not appear to have any effect on the lipid profile.
No significant changes in the expression of AMPK α2 were observed in any of the
groups, thus indicating that both OLE and Met. did not affect AMPK α2 expression;
additional studies are required to assess the long-term effects (Figure 5). One study reported
an increase in AMPK α2 expression after 10 weeks of Met. treatment, which may have
caused an improvement in the glucose level [51,54,55].
Treatment with both OLE and Met. resulted in a significant decrease in AS160 levels
in groups 2 and 3 when compared with their relative negative controls (D.W treatment;
Appl. Sci. 2023, 13, 5939 14 of 17

Table 4, Figure 6). This result agrees with a previous finding, wherein the knockdown
or loss of AS160 increased the level of surface GLUT4 in adipocytes, whereas activation
of AS160 led to the accumulation of GLUT4 in the cytosol [18]. Therefore, decreased
phosphorylation or deactivation of AS120 is required for the increase in GLUT4 expression
on the cell membrane. Another study showed that Met. stimulated the phosphorylation of
AS160 [51]; alternatively, Met. unaltered insulin-stimulated AS160 phosphorylation after
Met. treatment [56]. This finding is different from that observed in the present study, which
may be due to differences in the type of cell and concentration of metformin used; moreover,
the previous study was an in vitro study. Both OLE and Met. treatment decreased the
AS160 expression level in the rats in this study. This might suggest that both treatments
have the same mechanism in diabetes management via glucose transporter translocation
onto the surface of the skeletal muscle cell.
No significant effects of 1% OLE, 3% OLE, and Met. on GLUT 4 expression were
observed in all the groups in this study. The level of the signaling protein GLUT 4 in G2
and G3 was not altered when compared with that in G1. Our results agreed with that of a
previous study [50], which reported no significant effects of OLE on Glut 4 translocation
when a specific concentration was used. Conversely, this result was not in agreement with a
recent study, which reported that olive leaf polyphenols stimulated GLUT4 expression and
translocation in skeletal muscles in diabetic rats dose-dependently. This disagreement may
be due to the use of a higher concentration of OLE in their study; at low concentrations, the
translocation of GLUT 4 was comparable with that observed in diabetic animals without
OLE treatment in the study [57]. Three proteins in the soleus muscles of rats (Rab8A, Rab13,
and Rab14) were activated dynamically and expressed more with a high concentration of
olive leaf extract than the lower concentrations, in a dose dependent manner in which they
are colocalized with Glut4 and suggested to be responsible for insulin-stimulated Glut4
translocation [57]. Additional time may be required for Met. to increase the translocation
of GLUT 4, and this might explain why the results of the present study were different from
those of previous studies [51,54].

5. Conclusions
OLE treatment protected the rats from developing hyperglycemia and maintained the
insulin at levels that were comparable with those of the controls; these effects were found to
be better than those observed after treatment with metformin. These findings may suggest
the use of olive leaf extract as an alternative treatment to metformin in the treatment of
diabetes. The antihyperglycemic effect of olive leaf extract may be due to a decrease in
the phosphorylation of AS160. There was no effect of OLE on the lipid profile or on the
levels of AMPK and Glut 4, which are needed for glucose metabolism and the prevention of
hyperglycemia. This may be due to the short duration of this study, or the low concentration
of the OLE used. Hence, additional long-term studies using various concentrations of OLE
are required to assess the possible beneficial effects on glucose metabolism. We suggest
to increase the duration of the treatments to at least 10 weeks, which could probably give
better results. Moreover, a molecular approach might prove useful in evaluating the effects
of OLE on glucose metabolism at the genetic and protein levels.

Author Contributions: A.A.-R.A.-S.: Conceptualization, methodology, supervision, Writing—original

draft, correspondence. H.A.: Data curation, investigation, formal analysis, Writing—original draft,
H.J.A.-A.: Data curation, Resources, Investigation. M.Z.: Writing—reviewing editing, validation
M.A.: Writing—reviewing editing, validation. Authors A.A.-R.A.-S. and H.A. contributed equally to
the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The animal study protocol was approved by Institutional
Review Board in the Hashemite University/Jordan approval number 1/1/2014, date 1 January 2014.
Informed Consent Statement: Not applicable (animal study).
Appl. Sci. 2023, 13, 5939 15 of 17

Data Availability Statement: Data used to support the findings of this study may be released upon
application to the authors, namely, Abd Al-Rahman Al-Shudiefat via [email protected].
Acknowledgments: We would like to thank the Hashemite University for supporting the project, our
colleagues in the Department of Biology, and our colleagues at Jordan University for their assistance.
Conflicts of Interest: The authors declare no conflict of interest.

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