Hindawi Publishing Corporation

Evidence-Based Complementary and Alternative Medicine

Volume 2011, Article ID 538671, 9 pages
doi:10.1155/2011/538671

Research Article
The In Vivo Antidiabetic Activity of Nigella sativa Is Mediated
through Activation of the AMPK Pathway and Increased Muscle
Glut4 Content

Ali Benhaddou-Andaloussi,1, 2 Louis Martineau,1, 2 Tri Vuong,1, 2 Bouchra Meddah,3

Padma Madiraju,1, 2 Abdellatif Settaf,3 and Pierre S. Haddad1, 2
1
Department of Pharmacology and Montreal Diabetes Research Center, Université de Montréal, Montréal, QC, Canada H3C 3J7
2 Institut
des Nutraceutiques et des Aliments Fonctionnels, Laval University, Quebec City, QC, Canada G1K7P4
3
Research Team in Pharmacokinetics, Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy,
Mohammed V University, Rabat-Souissi, Morocco

Correspondence should be addressed to Pierre S. Haddad, pierre.haddad@umontreal.ca

Received 5 July 2010; Revised 26 January 2011; Accepted 15 February 2011

Copyright © 2011 Ali Benhaddou-Andaloussi et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

The antidiabetic effect of N. sativa seed ethanol extract (NSE) was assessed in Meriones shawi after development of diabetes.
Meriones shawi were divided randomly into four groups: normal control, diabetic control, diabetic treated with NSE
(2 g eq plant/kg) or with metformin (300 mg/kg) positive control, both administered by daily intragastric gavage for 4 weeks.
Glycaemia and body weight were evaluated weekly. At study’s end, an Oral Glucose Tolerance Test (OGTT) was performed
to estimate insulin sensitivity. Upon sacrifice, plasma lipid profile, insulin, leptin, and adiponectin levels were assessed. ACC
phosphorylation and Glut4 protein content were determined in liver and skeletal muscle. NSE animals showed a progressive
normalization of glycaemia, albeit slower than that of metformin controls. Moreover, NSE increased insulinemia and HDL-
cholesterol, compared to diabetic controls. Leptin and adiponectin were unchanged. NSE treatment decreased OGTT and tended
to decrease liver and muscle triglyceride content. NSE stimulated muscle and liver ACC phosphorylation and increased muscle
Glut4. These results confirm NSE’s previously reported hypoglycaemic and hypolipidemic activity. More significantly, our data
demonstrate that in vivo treatment with NSE exerts an insulin-sensitizing action by enhancing ACC phosphorylation, a major
component of the insulin-independent AMPK signaling pathway, and by enhancing muscle Glut4 expression.

1. Introduction disease; over a thousand plants being reported to combat

diabetes or its major symptoms [2–4]. The hypoglycaemic
Diabetes is a chronic disease that occurs when the pancreas action of these plants is exerted by several mechanisms, such
does not produce enough insulin, and/or when the body as stimulation of insulin production [5], enhancement of
cannot effectively use the insulin it produces. Hypergly- insulin sensitivity [6], or inhibition of intestinal amylase [4].
caemia, or high blood sugar, is a common effect of uncon- N. sativa is a herbaceous plant growing to about 20–
trolled diabetes and over time leads to serious damage to 30 cm in height, commonly known as black seed because
many of the body’s systems, especially the nerves, kidney, and of the small triangular black seeds it generates. The plant
blood vessels [1]. The World Health Organization (WHO) is also known as Blessed Seed (Arab: Habbat ul Baraka, or
estimates that more than 180 million people worldwide have Habbat ul Sauda). It has been consumed for more than 2000
diabetes. This number is likely to more than double by years, is used extensively in the traditional medicine of many
2030 [1]. Therapeutic interventions for patients with type southern Mediterranean and Middle Eastern countries, and
II diabetes include diet, exercise, oral hypoglycemic agents, has been shown to produce multisystemic beneficial actions
and/or insulin. For centuries or even millennia, medicinal [7], including hypocholesterolemic [8], antioxidant [9, 10],
plants around the world have also been used to treat the and anti-inflammatory effects [11].
2 Evidence-Based Complementary and Alternative Medicine

The hypoglycaemic and antidiabetic effect of N. sativa insulin resistance (AUC greater than 1500) were considered
has been reported by numerous in vivo and in vitro scientific diabetic. This selection has enabled us to have a yield of 25%
studies [9, 12–25]. In a recent study, we have demonstrated diabetic animals.
that N. sativa seed ethanol extract (NSE) exhibits the
remarkable ability in vitro to concomitantly increase insulin 2.4. Experimental Procedure. Twenty-four diabetic and eight
secretion, induce proliferation of pancreatic β cells, and normal Meriones shawi were divided into four groups of 8
stimulate glucose uptake in skeletal muscle and fat cells animals each as follows: normal control animals, diabetic
[24]. On the other hand, most of the in vivo studies of the control animals, diabetic NSE treated animals, and diabetic
antidiabetic effect of N. sativa were carried out on models metformin-treated animals. Treatment was given by daily
of type I diabetes. We therefore investigated the effects of intragastric gavage at a dose 48 mg/kg/day of N. sativa
NSE on the diabetic Meriones shawi that represents a model extract (equivalent to 2 g plant/kg/day) and 300 mg/kg/day
of type II diabetes associated with hyperinsulinemia and metformin for four weeks in one milliliter of 0.5% methyl
dyslipidemia. We have also attempted to determine some of cellulose suspension. Control animals received equal volume
the mechanisms of action through which NSE may exert its of vehicle (1 mL). Glycaemia and body weight were measured
antidiabetic effect, notably adipokines, AMP-kinase (AMPK) every week. At the end of the experimental period (4
dependent signalling, and Glut4 protein content. weeks), the animals were fasted overnight, anaesthetized
with an intraperitoneal injection of sodium pentobarbital
2. Material and Methods (60 mg/kg), and sacrificed for obtaining blood and tissues
samples (liver, soleus muscle). The study was conducted
2.1. Reagents and Antibodies. Antibodies against pan-specific in accordance with the accepted principles outlined in
and phosphorylated acetyl CoA carboxylase (ACC) (Ser the “Guide for the Care and Use of Laboratory Animals”
79), as well as Glut4 antibodies were purchased from prepared by the National Academy of Sciences and published
Cell Signaling Technology (Danvers, MA, USA). Secondary by the National Institutes of Health and all efforts were made
HRP-conjugated antibodies were purchased from Jackson to minimize animal suffering and the number of animals
Immunoresearch (Cedarlane Laboratories, Hornby, ON). used. Ethics approval was obtained from Mohammed V
Protein assay kit was purchased from Pierce (Brockville, University.
ON). Rat Insulin-specific RIA kit, Rat Leptin RIA kit, and
Mouse Adiponectin RIA kit-125T were purchased from 2.5. Oral Glucose Tolerance Test (OGTT). One day before the
Linco Research Inc (Saint Charles, MO). Sodium pentobar- beginning and the end of the experiment, an Oral Glucose
bital, triglyceride, and free glycerol reagents as well as D- Tolerance Test (OGTT) was performed to assess glucose
glucose were purchased from Sigma-Aldrich (Saint Louis, tolerance. For this purpose, overnight fasted rats were fed
MO). D-glucose (3 g/kg body weight) by intragastric gavage, and
then blood was collected after 0, 15, 30, 60, and 120 min
2.2. Plant Material. Seeds of N. sativa were obtained from intervals from the tail vein. Plasma glucose concentrations
an herbalist in Rabat, Morocco in August 2005 and were were determined by the glucose oxydase method using a
authenticated by an experienced botanist (Professor A. glucometer (One Touch Ultra, LifeScan Inc, Milpitas, CA).
Oulyahya, Institut Scientifique, Rabat, Morocco). A voucher The areas under the curve (AUC) of changes in the blood
specimen has been deposited in the herbarium of the Institut glucose were calculated by using Origin software (Microcal
Scientifique of Rabat (no. 10359). Seeds were washed, dried, Inc, Northampton, MA).
and then powdered with an electric microniser. Powder was
extracted three times with 80% ethanol and the solvent 2.6. Plasma Lipid Assays. After 4 weeks of the treatment,
was evaporated at 40◦ C under reduced pressure. This Meriones shawi were sacrificed and fasting blood sam-
procedure resulted in a two-phased extract. The oily and the ples were collected for plasma chemical analysis. Total
solid phases were recombined in proportion to their yield amount of cholesterol, LDL-cholesterol, HDL-cholesterol,
(typically 70% and 30%, resp.). The extract was conserved serum triglyceride, and blood glucose were measured by
at 4◦ C and protected from light and humidity. an automated analyzer (Cobas-Mira Plus, Hoffman-LaRoche
Diagnostics, Germany).
2.3. Animals. One hundred Meriones shawi of both sexes
were captured in the semiarid area of Boulmane in the 2.7. Radioimmunoassay. Plasma insulin, leptin, and adi-
Middle Atlas region of Morocco. Appropriate traps were used ponectin levels were determined by radioimmunoassay
for catching the animals. They were then transported to the (RIA). Rat Insulin-specific RIA kit, Rat Leptin RIA kit, or
Faculty of Medicine and Pharmacy of Mohamed V University Mouse Adiponectin RIA kit-125T were used. Generally, the
in Rabat, Morocco. Animals were allowed to adapt to the samples were incubated in 12 × 75 mm polypropylene RIA
laboratory environment, numbered and placed in individual tubes with rat 125 I-insulin, 125 I-leptin, or 125 I-adiponectin
cages. All animals received a standard laboratory diet, ad and a primary antibody against rat insulin, rat leptin, or
libitum, which represents a hypercaloric food source for mouse adiponectin, respectively, at 4◦ C overnight in the
them. After three months of such diet, Meriones shawi having dark. The tubes were then incubated with the precipitating
blood glucose greater than 8 mmol/L and having developed reagent for 20 min at 4◦ C and centrifuged at 5350 g for
Evidence-Based Complementary and Alternative Medicine 3

15 min. Radioactivity in the pellet was measured using using StatView software (SAS Institute Inc, Cary, NC), with
a gamma counter (Wallac Wizard 1470, Perkin Elmer, posthoc analysis as appropriate. Statistical significance was
Waltham, MA). Human insulin, rat leptin, or mouse set at P ≤ .05.
adiponectin were used as respective standards.
3. Results
2.8. Triglyceride Assay in Liver and Skeletal Muscle Tis-
sues. Tissue was homogenized and extracted with a 2 : 1 3.1. Body Weight Is Not Significantly Affected by NSE. The
chloroform-methanol mixture and washed by addition of results of body weight are presented in Table 1. At the begin-
50 mM NaCl solution, resulting into two phases. The lower ning of the trial period (day 0), the diabetic Meriones shawi
phase contained the total lipid extract. A fixed volume of this weighed between 177 g–187 g as compared to approximately
extract was dried, resuspended in isopropanol and an aliquot 158 g for non-diabetic controls. At the end of four weeks
was used for triglyceride measurement using triglyceride and of treatment, an increment of weight of about 6 to 10 g for
free glycerol reagents. Absorbance was measured at ambient each Meriones shawi group was observed, but no significant
temperature at 540 nm using a Wallac Victor 2 plate reader difference was apparent between them, albeit the weight gain
(Perkin-Elmer, Waltham, MA). Triglyceride content of the of NSE-treated animals had a tendency to be lower.
tissue was expressed as mg/g of wet weight of tissue.
3.2. NSE Improves Blood Glucose of Diabetic Meriones
2.9. Western Blot for Proteins Involved in Glucose Homeostasis. shawi. Daily NSE treatment for four weeks resulted in a
Samples of liver and skeletal muscle tissues were ground in gradual decrease in glycemia that reached values similar
liquid nitrogen and subsequently lysed. For ACC western to normal non-diabetic controls animals by the end of
blot analysis, 1 mL of RIPA lysis buffer was used (25 mM the treatment period (reduction from 8.2 ± 0.5 mmol/L
Tris-HCl pH 7.4, 25 mM NaCl, 0.5 mM EDTA, 1% Triton- to 6.4 ± 0.3 mmol/L, P < .05, n = 8, Figure 1). By
X-100, 0.1% SDS), whereas sucrose lysis buffer (20 mM comparison, metformin reduced blood glucose from 9.2 ±
Tris-HCl pH 7.4, 255 mM sucrose, 1 mM EDTA) was used 0.4 mmol/L to 5.4 ± 0.4 mmol/L (P < .05, n = 8, Figure 1)
for Glut4. For all samples, a protease inhibitor cocktail within the first week and glycemia remained stable until
was added (Roche, Mannheim, Germany) as well as 1 mM the end of the study. In contrast, both diabetic and non-
phenylmethanesulfonyl fluoride and phosphatase inhibitors diabetic control animals displayed stable glycaemia during
(1 mM sodium orthovanadate, 10 mM sodium pyrophos- the four weeks of treatment; normal animals demonstrated
phate, 10 mM sodium fluoride). Cells were allowed to lyse normal blood sugar levels and control diabetic animals were
for 30 min on ice and were then centrifuged at 12000 × g hyperglycaemic throughout the study period (Figure 1).
for 10 min. Supernatants were then stored at −80◦ C until
analysis. Protein content was assayed by the bicinchoninic 3.3. NSE Reduces Insulin Resistance in the Diabetic Meri-
acid method standardized to bovine serum albumin (Roche, ones shawi. An oral glucose tolerance test (OGTT) was
Laval, QC). performed at the beginning of the study to identify the
Lysates were diluted to a concentration of 1.25 mg/mL Meriones shawi that developed diabetes after three months
total protein and boiled for 5 min in reducing sample buffer of relatively hypercaloric diet. Another OGTT was also
(62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% β- carried out on all selected study animals at the end of the
mercaptoethanol and 0.01% bromophenol blue). 20 μL of treatment to determine the effect of NSE- and metformin-
each sample was separated on 10% polyacrylamide mini- positive control on insulin resistance. Figure 2 presents the
gels and transferred to nitrocellulose membrane (Millipore, changes in blood sugar observed during the two hours
Bedford, MA). Membranes were blocked for 2 h at room following administration of the glucose load. As can be
temperature with Tween-20 and 5% skim milk in TBS seen, normal (non-diabetic) control animals had a small
(20 mM Tris-HCl, pH 7.6 and 137 mM NaCl). Membranes response to the glucose load as compared to control diabetic
were then incubated overnight at 4◦ C in blocking buffer Meriones, indicating glucose intolerance/insulin resistance
with appropriate phospho-specific or pan-specific antibodies in the latter group. Treatment of diabetic animals for
against ACC and Glut4 at 1 : 1000. Membranes were washed four weeks with the positive control metformin succeeded
5 times and incubated 1.5 h at room temperature in TBS in completely restoring the OGTT response to that of
plus Tween 20 with anti-rabbit HRP-conjugated secondary non-diabetic congeners. In contrast, NSE-treated animals
antibodies at 1 : 100000 to 1 : 50000. Revelation was per- displayed a significant improvement in insulin sensitivity,
formed using the enhanced chemiluminescence method and but the glyceamic response to the OGTT was intermediate
luminescence captured to blue-light-sensitive film (Amer- between normal and diabetic Meriones controls. The AUC
sham Biosciences, Buckinghanshire, England). Lysates from of the glyceamic response to the glucose load over time
all experimental conditions were separated and transferred confirmed and quantified the interpretation of Figure 2.
simultaneously to a single membrane. Indeed, all diabetic animals showed similarly elevated AUC
values at the beginning of the experimental protocol as
2.10. Statistical Analysis. Data are reported as the mean compared to non-diabetic controls (Table 2). In diabetic
± SEM of the indicated number of experiments. Results Meriones shawi treated for four weeks with metformin, the
were analysed by one-way analysis of variance (ANOVA) values of the AUC fell significantly to reach values seen in
4 Evidence-Based Complementary and Alternative Medicine

Table 1: Evolution of body weight over the course of the 4-week treatment.

Time (week) N. sativa (g ± SEM) Metformin (g ± SEM) Diabetic control (g ± SEM) Normal control (g ± SEM)
0 178 ± 17 177 ± 11 187 ± 15 158 ± 14
1 172 ± 16 166 ± 11 178 ± 15 153 ± 12
2 183 ± 19 180 ± 10 186 ± 15 161 ± 13
3 185 ± 18 183 ± 9 192 ± 16 166 ± 13
4 184 ± 16 184 ± 9 197 ± 15 170 ± 12
No statistically significant differences were observed between experimental groups at any of the time points (n = 8 per group, N.S.).

10 20

18
9
16
8

Blood glucose (mmol/L)

14
Blood glucose (mmol/L)

∗
7 ∗
12

6 ∗ 10

8
5 ∗
∗
∗ 6
4
4
3 2

2 0

0 1 2 3 4 5 0 30 60 90 120
Time (weeks) Time (min)
NSE
NSE
Metformin
Metformin
DC
DC
NC
NC
Figure 2: Effect of NSE on oral glucose tolerance test (OGTT)
Figure 1: Changes of plasma glucose in diabetic Meriones shawi
in diabetic Meriones shawi. OGTT (glucose 2 g/kg) was performed
treated with NSE. Meriones shawi received a daily oral administra-
on fasted animals and blood glucose measured at the onset of
tion of NSE of 48 mg/kg or 300 mg/kg metformin (positive control)
glucose challenge (0 min) and at various time points afterwards
whereas normal control group (NC) and diabetic control (DC)
(15–120 min). Results are expressed as mean ± SEM (n = 8).
groups received the methyl-cellulose vehicle. Results are expressed
Significantly different from diabetic control (DC) ∗ P < .05.
as mean ± SEM (n = 8). Significantly different from diabetic
control (DC) group at the same time point, ∗ P < .05.

to be higher. Plasma LDL-cholesterol concentration was

non-diabetic controls. By comparison, NSE treatment for the
equivalent in all groups of Meriones shawi (Table 3), whereas
same period significantly reduced the levels of AUC to values
metformin induced a significant rise in plasma triglycerides,
slightly greater than those of non-diabetic animals.
as compared to control diabetic animals (Table 3).

3.4. Plasma Lipid Profile: Differential Modulation by Met-

formin and NSE. In the diabetic Meriones shawi, treatment 3.5. NSE Increases Insulinemia but Does Not Affect Circulating
for 4 weeks with 48 mg/kg/day NSE or 300 mg/kg/day Leptin or Adiponectin. Untreated diabetic Meriones shawi
metformin increased total plasma cholesterol concentration exhibited a significant increase in plasma insulin as com-
by 49% and 38%, respectively, and the HDL-cholesterol pared to their non-diabetic congeners (P < .05, n = 8,
concentration by 142% and 92%, respectively, as compared Figure 3). Treatment for four weeks with metformin reduced
to the diabetic control group (Table 3). These increases insulinemia back to levels observed in normal controls.
brought levels of total cholesterol and HDL-cholesterol close In contrast, NSE treatment significantly increased plasma
to values observed in the normal control group, although insulin levels beyond those observed in the diabetic control
HDL-cholesterol in NSE-treated animals had a tendency group (P < .05, n = 8, Figure 3).
Evidence-Based Complementary and Alternative Medicine 5

Table 2: Area-under-the-curve (glyceamic (mmol/L)∗time (min)) for OGTT before and after 4-week treatment.

Treatment
Time (week)
N. sativa Metformin Diabetic control Normal control
0 1648 ± 107 1685 ± 84 1666 ± 82 956 ± 61§
4 1163 ± 81∗§ 819 ± 54∗§ 1514 ± 83 941 ± 41§
∗
Significantly different from week 0; P < .05; § Significantly different from Diabetic controls; P < .05.

Table 3: Effect of NSE on blood biochemistry and tissue lipid parameters.

Normal control Diabetic control N. sativa Metformin

Plasma total cholesterol (mmol/L) 0.86 ± 0.09 0.65 ± 0.08 0.97 ± 0.08∗ 0.90 ± 0.10
Plasma LDL-cholesterol (mmol/L) 0.13 ± 0.04 0.10 ± 0.06 0.16 ± 0.05 0.08 ± 0.06
Plasma HDL-cholesterol (mmol/L) 0.48 ± 0.07 0.26 ± 0.05 0.63 ± 0.04∗ 0.50 ± 0.08∗
Plasma triglyceride (mmol/L) 0.55 ± 0.08 0.50 ± 0.04 0.41 ± 0.08 0.72 ± 0.09∗
Plasma leptin (ng/mL) 8.60 ± 2.00 3.50 ± 1.20 3.20 ± 0.50 5.00 ± 1.70
plasma adiponectin (μg/mL) 8.59 ± 1.86 8.50 ± 1.34 5.51 ± 1.32 6.35 ± 1.86
Liver triglyceride (mg/g of tissue) 35.1 ± 18.0 83.3 ± 43.2 28.1 ± 13.5 79.8 ± 31.6
Muscle triglyceride (mg/g of tissue) 5.23 ± 1.10 8.92 ± 3.50 4.23 ± 0.78 10.5 ± 4.39
∗
Significantly different from Diabetic controls; P < .05.

2 3.6. NSE Tends to Reduce Liver and Skeletal Muscle Triglyc-

eride Content. As detailed in Table 3, liver and skeletal mus-
cle triglyceride content appeared to be elevated in control
$∗
diabetic versus non-diabetic Meriones shawi and metformin
Plasma insulin (ng/mL)

treatment did not change this tendency. In contrast, NSE had

a tendency to decrease the triglyceride content of both tissues
1 $ (Table 3). Because of the large data variability, however, none
of these changes reached statistical significance.

3.7. NSE Increases Muscle ACC Phosphorylation and Glut4

∗ Protein Content. In order to begin understanding the mech-
anisms through which the effects of NSE are mediated,
0
NC DC NSE Metformin we assessed some key intracellular components involved in
glucose homeostasis. We first evaluated the phosphorylation
Figure 3: Effect of NSE on plasma insulin levels in Meriones shawi. of ACC, a major component of the AMPK pathway. As
The plasma insulin was measured after 4 weeks of treatment in shown in Figure 4, NSE treatment significantly increased the
diabetic control group (DC), normal control group (NC), and
phosphorylation of ACC in liver (panel (a)) and skeletal
animals receiving 48 mg/kg/day N. sativa ethanolic extract (NSE)
or 300 mg/kg/day of the reference oral antidiabetic drug metformin. muscle (panel (b)) in comparison with control diabetic
Results are expressed as mean ± SEM (n = 8). Significantly different Meriones shawi. Secondly, we probed the soleus muscle
from diabetic control animals (DC) ∗ P < 0.05. Significantly tissue of control and NSE-treated diabetic animals for their
different from normal (non-diabetic) control animals (NC) $ P < total content in Glut4 protein content. As illustrated in
.05. Figure 4(c), NSE substantially increased the amount of Glut4
present in the soleus muscle of diabetic Meriones shawi
animals.

The determination of plasma leptin in different groups 4. Discussion

of diabetic Meriones shawi yielded very similar values irre-
spective of NSE or metformin treatment. However, plasma N. sativa seeds are used in the traditional medicine of
leptin in normal Meriones shawi was found to be significantly numerous Middle Eastern and North African countries for
higher than values observed in control diabetic animals their antihyperglycemic activity [26–28]. Most studies to
(Table 3). In contrast, the results of plasma adiponectin date have reported results from either normal animals or
showed no significant differences between groups, although models of Type I diabetes [9, 12–15, 17–20], or from cells
NSE and metformin treated animals had a tendency to have in culture [21, 24]. Only a single study presented limited
lower adiponectin values (Table 3). evidence for an antidiabetic effect of N. sativa in an animal
6 Evidence-Based Complementary and Alternative Medicine

DC NSE (kDa) several studies using animal models of type I [9, 10, 15, 17–
20] and on similar models of Type II diabetes [8, 16]. In
Phospho-ACC 280 our experimental conditions, NSE decreased blood sugar
starting from the 3rd week of treatment whereas metformin
normalized glycaemia within first week. It is known that
(a) thiazolidinediones (TZD) also decrease blood sugar after 3 or
DC NSE
(kDa) 4 weeks of treatment [29]. In vitro studies in our laboratory
have confirmed that NSE stimulates PPAR-γ in cultured
adipocytes as do TZD [25]. Part of the action of NSE on
Phospho-ACC 280
the regulation of blood glucose in vivo may therefore be
similar to TZDs. However, the lack of effect of NSE on plasma
(b) leptin and adiponectin indicates that the action of NSE on
(kDa)
adipose tissue may not implicate the modulation of these two
DC NSE
adipokines in the Meriones shawi model.
Moreover, the decrease of blood glucose by NSE was
Glut4 45 associated with a significant reduction in insulin resistance,
as revealed by the pattern of the OGTT response in diabetic
animals and the resulting significant decrease in AUC that
(c)
clearly indicate an improvement in glucose tolerance. This
Figure 4: Effect of NSE on the phosphorylation of ACC in skeletal improvement in insulin sensitivity is fully consistent with
muscle and liver tissues and on Glut4 expression in skeletal muscle our previous results showing increases of basal and insulin
tissue. Samples of soleus muscle (a) and (c) and liver (b) tissues stimulated phospho-Akt in hepatocytes isolated from normal
were obtained from diabetic Meriones shawi treated with NSE rats treated with the petroleum ether extract of N. sativa
or vehicle (DC) and analysed by immunoblotting with antibody [22].
specific to phospho-ACC (a) and (b) and Glut4 (c). Immunoblots NSE treatment also modulated the lipid profile of dia-
are representatives of results obtained from four animals of each betic Meriones shawi, most notably by significantly increasing
group.
HDL-cholesterol, an effect that we have previously observed
in normal rats with a different N. sativa extract [22]. We also
observed a tendency towards a decrease in triglycerides in the
NSE-treated group. Labhal et al. [16] observed a significant
model of Type II diabetes [16]. The present study focussed decrease in blood triglycerides in the same animal model
on antidiabetic and hypolipidemic effects in the Meriones using an aqueous extract of N. sativa administered for a
shawi model of Type II diabetes and aimed to elucidate the period of three months. Once again, differences in extract
mechanisms of action of NSE in skeletal muscle and liver type and treatment period may explain discrepancies with
tissues (Figure 5). our results at the level of blood lipid profile.
Meriones shawi are rodents from semi-arid regions of However, our studies went further by assessing triglyc-
Morocco that can gain weight and become insulin resistant eride content in insulin-sensitive tissues, notably skeletal
and diabetic if kept in captivity (reduced physical activity) muscle and liver. Although the apparent NSE induced
and fed normal laboratory chow (hypercaloric relative to decrease in these parameters failed to reach statistical signif-
their natural diet) [16]. This was confirmed in our study by icance, such an action could participate in the improvement
the greater body weight of the Meriones shawi animals that of systemic insulin sensitivity observed in our studies.
also demonstrated insulin resistance as assessed by an OGTT, Indeed, it is known that high levels of intracellular triglyc-
as compared to non-diabetic animals maintaining normal erides can increase some lipid metabolites such as ceramides,
glucose tolerance. Treatment with NSE or metformin for four diacylglycerol, and long-chain acyl-coenzyme A [30]. The
weeks failed to significantly affect body weight, although a latter play a key role in attenuating insulin signaling by
tendency for smaller weight gain was seen in NSE-treated increasing intracellular serine-threonine phosphorylation of
animals as compared to untreated diabetic controls. This IRS protein, with a resultant reduction in insulin signal
contrasts with the results of Labhal and collaborators who transduction that underlies insulin resistance [30].
reported a decrease in body weight among Meriones shawi More importantly, the NSE-treated Meriones shawi also
treated with an aqueous extract of N. sativa [16]; similar showed a large increase in insulinemia after 4 weeks of
results having been obtained with the sand rat Psammomys treatment that can contribute to the antihyperglycemic
obesus [8]. Aside from the difference in N. sativa extract type, effect of NSE. Such an increase was previously observed
the treatment period of these studies lasted three months. It by our group in normal rats [22] and is also observed
is therefore possible that our shorter treatment regimen and in streptozotocin-nicotinamide hamsters [18]. In addition,
use of an ethanol extract may explain the lack of an effect on treatment of pancreatic β cells in culture with N. sativa was
body weight in the present studies. found to increase insulin secretion as a result of an improved
In contrast, our studies confirmed the oral hypoglycemic secretagogue capacity of these cells [21]. On the other hand,
action of N. sativa in an animal model of Type II diabetes. As N. sativa increases regeneration of pancreatic β cells [20] and
mentioned, such a decrease in blood glucose was reported in protects the same from streptozotocin [10]. Our own recent
Evidence-Based Complementary and Alternative Medicine 7

NSE

Muscle
Liver
Pacncreas
Activation of AMPK
↑ Insulin secretion Activation of AMPK
↑ β cell proliferation
↓ ACC activity
↑ Glut4 translocation ↑ Glut4 synthesis
↑ Fatty acid oxidation
↓ Gluconeogenesis
↑ Glucose uptake
↓ Fatty liver
↑ Hepatic insulin sensitivity

↓ Glyceamia

Figure 5: NSE activates AMPK in the liver and muscles to improve glucose metabolism. NSE mediates its action by stimulating the adenosine
monophosphate-activated protein kinase (AMPK), an enzyme essential. It also reduces the enzymatic pathway involved in increasing fatty
acid production by the liver (ACC = acteyl-CoA carboxylase); thus, it increases the sensitivity to insulin. The activation of AMPK can inhibit
the pathway of gluconeogenesis in the liver. In the muscle, NSE activates AMPK towards allowing the increased synthesis and translocation
of Glut4 and consequently increases glucose transport in muscle. NSE also acts on the pancreas by increasing the secretion of insulin.

in vitro studies clearly demonstrate that NSE can enhance to increased synthesis of Glut4 [35], and this is also in
the proliferation of β cells and increase glucose stimulated accordance with our results (Figure 5).
insulin secretion [24]. Taken together, these actions can In conclusion, NSE greatly improves systemic glucose
explain the important increase in insulin levels observed in homeostasis and HDL-cholesterol in diabetic Meriones shawi
diabetic NSE-treated Meriones shawi (Figure 5). They further by acting through several mechanisms. Most importantly,
support the notion that N. sativa products can help maintain N. sativa increases circulating insulin and enhances the
pancreatic β-cell mass and hence mitigate the progression of sensitivity of peripheral tissues to the hormone. The latter
diabetes. Future studies will have to ascertain that this also effect can be attributed in part to an activation of the
occurs in the Merione shawi model. AMPK pathway in skeletal muscle and liver and to an
Our studies also attempted to examine certain key increased content of Glut4 in skeletal muscle (Figure 5). Such
intracellular components involved in glucose homeostasis. pleiotropic actions provide strong evidence in support of
Western blot analysis showed that NSE treatment in vivo the traditional use of N. sativa seeds for the treatment of
can significantly increase the total amount of Glut4 glucose diabetes. They further call for high-quality clinical studies
transporters in skeletal muscle, which play a major role in to determine the optimal conditions for complementary or
controlling hyperglycemia [31]. Moreover, Glut4 proteins alternative treatment in diabetic patients.
are known to be subject to transcriptional regulation that
allows for their increased synthesis [32], with a resultant Acknowledgments
contribution in reducing hyperglycemia. Further studies will
be necessary to determine the mechanisms underlying the These studies were supported by a Marion L. Munroe
action of NSE to increase skeletal muscle Glut4, but this Memorial grant from the Canadian Diabetes Association as
action is likely very relevant to the overall glucose-lowering well as by a grant from the Canadian Institutes of Health
activity of the plant (Figure 5). Research. A. B. Andaloussi received a travel studentship from
Finally, we assessed ACC, a key component of the the Agence universitaire de la Francophonie to carry out the
insulin-independent, metabolic sensing, AMPK pathway Meriones shawi experiments in Morocco. P. S. Haddad was
[33]. Indeed, our group recently reported on the stimulation a National Research Scientist of the Fonds de la recherche
of ACC and the AMPK pathway by NSE in both skeletal en santé du Québec from 2002 to 2007. The laboratory of
muscle and hepatocyte cell lines in vitro [25]. In the present Dr. John Thor Arnason is gratefully acknowledged for the
studies, we found that NSE treatment in vivo can increase the preparation of Nigella sativa seed extracts.
phosphorylation of ACC in liver and skeletal muscle tissues.
The phosphorylation of ACC reduces its activity and results
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