Plant Archives Volume 20 No. 2, 2020 pp. 6170-6174
e-ISSN:2581-6063 (online), ISSN:0972-5210
THE INSULIN RESISTANCE AND ITS RELATION TO PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTOR-GAMMA (PPAR)
POLYMORPHISM IN DIABETES MELLITUS TYPE II IN EGYPT
Mahmoud El-Rehany1, Walaa Hozayen2 and Sara Nabil Shokrany3
1
Biochemistry department, Faculty of Pharmacy, Deraya University, Egypt.
Biochemistry department, Faculty of Science, Beni-Suef University, Egypt.
3
Medical biochemistry department, Faculty of Postgraduates studies for advanced science,
Beni-Suef University Egypt.
2
Abstract
Pathogenesis of type 2 diabetes mellitus (T2DM) and development of insulin resistance are described by multi-stimuli
factors. Peroxisome proliferator-activated receptor-2(PPAR-gamma2) polymorphism might play a vital role in type 2 diabetes
mellitus and insulin resistance. The adipose tissue-released cytokines as interleukin-1 (IL-1) and Tumor Necrosis FactorAlpha (TNF-) may be contributory factors. Homeostatic model assessment (HOMA IR) is an approximating equation for
insulin resistance from fasting glucose and insulin concentrations divided by a constant. HOMA-IR has been observed to
have a linear link with the glucose clamp and minimum model estimations of insulin sensitivity/resistance in several studies
of distinct people. The goal of the study is to determine the relation between PPAR-2, TNF-, IL-1 and HOMA-IR with
T2DM in Egypt.
Key Words: PPAR-, TNF-, T2DM, IL-1, HOMA-IR , Polymorphism
Introduction
Type 2 diabetes mellitus (T2DM) is a series of
metabolic disorders elucidated by high blood glucose
levels, which results from deficiency in insulin excretion
or action or both leading to complications (Saxena M, et
al., 2009).Diabetes mellitus is predicted to reach nearly
5% of the world’s population (about 366 million) in 2030
in the proportion of people >65 years of age (Wild, S., et
al., 2004).
Peroxisome proliferator-activated receptors (PPARs)
are ligand-activated transcription factors that are portion
of the super family contains receptors for steroid
hormones, thyroid hormones, retinoic acid and fat-soluble
vitamin A and D. The main role of PPARs is to adjust
glucose, energy balance, fatty acid and lipoprotein
metabolism, cell proliferation and differentiation,
inflammation and atherosclerosis (Grygiel-Górniak B,
2014). PPAR was the first gene reproducibly related
to T2DM. The relation between the substitution of alanine
by proline at codon 12 of PPAR2 (Ala12 allele) and the
risk for T2DM has been vastly studied since (Yen CJ, et
al., 1997), first notified this polymorphism.
A large and different family of small, low molecular
weight cell signaling proteins acting as intermediate
complex interaction is called “cytokines”, which involve
interleukins and interferons (Banerjee M., Saxena M.,
2012). The adipose tissue-released cytokines as
interleukin-1 (IL-1) and Tumor Necrosis Factor-Alpha
(TNF alpha) may be contributory factors (Wieser V, et
al., 2013). The pro-inflammatory IL-1 family members;
IL-1 and IL1 were among the first identified cytokines
(Dinarello CA , 2011). IL-1alpha looks to play a more
important role than IL-1beta in the development of
atherosclerosis in mice (Freigang, S., et al., 2013).
However, the role of IL-1alpha in patients with type 2
remains to be investigated. IL-1, a key inflammatory
mediator during T2D, stimulates insulin resistance, impairs
-cells function, and causes apoptosis (Karstoft K,
Pedersen BK, 2016).
Through all pro-inflammatory biomarkers, TNF-
first identified to be included in the pathogenesis of insulin
resistance, and glucose related abnormalities that linked
to T2DM (Imai Y, et al., 2013). TNF- has a pivotal role
in the development of insulin resistance as it decreases
Insulin resistance and its relation to peroxisome proliferator-activated receptor-gamma (PPAR) polymorphism
6171
the expression of glucose transporter type 4 (GLUT4)
which is an insulin-regulated glucose transporter and
located mainly in adipocytes, skeletal and cardiac muscles
(Olson, A.L., 2012) .High level of TNF-á in circulation is
related to the development of insulin resistance and
diabetes (Swaroop J.J., et al., 2012).
glucose test. Fasting blood glucose and post-prandial blood
glucose were examined enzymatically using kit provided
by (Spinreact, Spain). Fasting insulin was measured by
commercially kit ( Chemux Bioscience, Inc). After
centrifugation, whole blood was stored at -20 for DNA
extraction.
HOMA IR was first reported in 1985 by (Matthews,
et al., 1985). It is a method used to quantify insulin
resistance and beta cell function from fasting glucose
and insulin (or C-peptide) concentrations. It was
calculated by the form: serum insulin (µIU/ml) × FPG
(mg/dl)/405 (Bergman, et al., 1985).
Genomic DNA extraction and genotyping
In this study, we are going to assess the correlation
between PPAR2 (Ala12 allele), TNF-, IL-1 and
HOMA-IR with T2DM.
Materials and Methods
Study population
This study was done on 110 patients with type 2
diabetes mellitus aged 28-87 years compared with 30
healthy controls aged 20-55 years.
Biochemical analysis
Blood samples was collected from the patients and
healthy control after a 10-h fasting on sodium fluoride
tubes, and plasma was separated by centrifugation at
room temperature for fasting blood glucose and fasting
insulin. After 2-h of meal, peripheral blood was collected
from the patients and healthy control for post-prandial
Fig. 1: Electrophoretic pattern of PCR products. Lane (1- 9)
the PCR products at 270 bp, lane (10) 50 bp ladder.
Table 1: Biochemical characteristics of study subjects.
Control
30
91.83 ± 2.47
119.9 ±3.951
5.193±0.1979
29.65 ±1.022
1.163±0.0422
Diabetic female
post- menopause
47
148.9±9.098
264.4±13.73
14.81 ±1.341
31.94±0.736
5.447±0.638
Diabetic female
pre- menopause
30
168.2±10.16
259.2±14.24
15.06 ± 1.549
31.92±0.894
6.547±0.902
DNA was extracted from whole blood samples
according to the method of (Medrano, et al., 1990). The
final preparation of genomic DNA used for PCR
technique was free of RNA and protein and had an A 260
\ A280 ratio more than 1.7 in all samples.
A single PCR product was produced using a
commercial Kit (MyTaq™ Red Mix) and visualized on
the agarose gel after exposure to UV light. PCR was
performed through 35 cycles by the following steps:
denaturation at 94°C for 30 sec; annealing at 60°C for
30 sec; and extension at 72°C for 30 sec. A single product
of 270 bp was produced as shown in Fig. 1. The PCR
product was digested with restriction enzyme BstU-I at
37æ%c for 15 minutes, then applied to a 2.5% agarose
gel and stained with ethidium bromide. Two different
patterns were observed, a non-cut (non-polymorphic) for
wild (Pro/Pro) variant, cutted (polymorphic) for
heterogeneous type (Pro/Ala ) as shown in Fig. 2.
Determination of TNF- and IL-1 level by using
Western blotting method (Harlow, E.D. and Lane, D,
1988) .
Fig. 2: Electrophoretic pattern of PCR product digest. Lanes
(1- 4) wild pro/pro shows one band at 270 bp, lanes (57) shows heterozygous pattern with 2 bands at 270,
227 bp, lane (8) 50 bp ladder.
Diabetic male
Characteristic
33
158 ± 9.59
260.9±17.01
11.5 ± 1.39
30.18±1.016
4.509±0.596
n
Fasting blood sugar(mg/dl)
Post prandial blood sugar(mg/dl)
Fasting insulin (µIU/ml)
BMI
HOMA-IR
6172
Mahmoud El-Rehany et al.
Statistical analysis
Data analysis was all done using software GraphPad
Prism7. Quantitative inputs were given by mean and
standard deviation, while qualitative data were given by
frequency distribution. Chi Square used to examine the
significant difference for proportion and calculation of
Odds ratio, and one way ANOVA test for multiple
comparison. The probability of < 0.05 was used as a cut
off point for all significant tests.
Results
Biochemical variables
The biochemical parameters of the T2DM patients
(male, female pre-menopause, female post-menopause)
and controls are summarized in table 1.
Genotypes and allele frequencies
The genotype distribution and the allele frequencies
between the control and T2DM patients are shown in
table 2. The frequencies of GG, GC, and CC genotypes
in control group were 80%, 16.67%, 3.33% while in T2DM
patients males were 84.85%, 12.12%, 3.03% and in T2DM
patients female pre-menopause were 96.67%, 3.33%, 0%
where in T2DM patients female post-menopause were
89.36%, 10.64%, 0%.
Determination of TNF- and IL-1 using Western
Blotting shows significant between T2DM and healthy
control (P<0.0001) as shown in table 3.
Discussion
Type 2 diabetes mellitus (T2DM) is a general health
problem in the world with a high diffusion which is the
most noticeable disease in developing countries (Morita,
et al., 2005). Gene association studies have identified
several common variants implicated in T2DM. One of it
is Peroxisome Proliferator Activated Receptor 2
(PPAR2). A Pro12Ala polymorphism at extreme amino
terminus of PPAR-2 gene has been studied but its effect
on obesity and insulin sensitivity is unclear (Allan F.
Moore, Jose C. Florez, 2008). In several additional studies,
the Ala12 allele was related to lower BMI, improved
Table 3: Correlation of TNF-alpha and IL-1 in patients with
type2 diabetes mellitus and healthy volunteers.
P value
control
<0.0001* 1.014 ± 0.015
<0.0001* 1.082 ± 0.072
characteristic
TNF-alpha
IL-1
* Statistically highly significant.
insulin sensitivity, and reduced risk of type 2 diabetes
(Deeb, et al., 1998). Although most studies have shown
a statistically significant Type2 Diabetes reduction given
by Ala variant (Zeggini, et al., 2005; Ghoussaini, et al.,
2005), some others have not (Badii, et al., 2008;
Bouassida, et al., 2005) suggesting variability in the
contribution of this variant to the risk of T2DM.
The present study aimed to study the Pro12Ala
polymorphism in PPAR2 gene in Egyptian population.
Various biochemical parameters were analyzed in
controls and Type 2 diabetics. The association of this
polymorphism with T2DM was studied. The relationship
of this polymorphism with insulin and other biochemical
parameters were also studied.
Association of Pro12Ala and Type 2 Diabetes
mellitus
On genotype analysis, Pro/Pro homozygotes were
24 (80%) in controls and 99 (90%) in cases that include
males, females pre-menopause and females postmenopause. The number of Pro/Ala heterozygotes were
5 (16.67%) in controls and 10 (9.09%) in cases. The
number of Ala/Ala homozygotes were 1 (3.33%) in
controls and 1 (0.91%) in cases that was a male. These
results did not detect any statistically significant between
the Pro12Ala SNP and T2DM. This result corroborates
the findings of study done on South Indian population
from Chennai (Radha, et al., 2006). Another study on
South Indian population reported no association of
Pro12Ala SNP with metabolic syndrome (Vimaleswaran,
et al., 2007). Also, a study was done on Palestinians was
unable to explain a significant association of Pro12Ala
variant and T2DM (Ereqat, et al., 2009). The finding of
the present study differs from a study done on Caucasians,
Table 2: Genotypes distribution of PPARã2 (Ala12 allele)
polymorphisms in healthy control and T2DM patients.
Total
30
33
30
47
30
110
T2DM
6.024 ± 0.086
6.562 ± 0.4506
Ala/Ala (CC) Pro/Ala (GC) Pro/Pro (GG)
Group
1 (3.33%)
5 (16.67%)
24 (80%)
Control
1 (3.03%)
4 (12.12%)
28 (84.85%)
Dia. male
- (0%)
1 (3.33%)
29 (96.67%)
Dia.F. pre
- (0%)
5 (10.64%)
42 (89.36%) Dia.f. post
Control
Control
Control
1 Cases 1
5 Cases 10
24 Cases 99
Total no.
(0.91%)
(9.09%)
(90%)
where an association of this polymorphism was found
with the insulin sensitivity (Altshuler, et al., 2000).
The difference in reports of various studies may
suggest that the effect of genetic variation may be
restricted to particular ethnic groups. This may also
be due to the influence of other genetic variants in the
candidate gene or the interaction of certain yet
uncharacterized genetic factors with environmental
factors.
Association of TNF- and IL-1 with Type 2
Insulin resistance and its relation to peroxisome proliferator-activated receptor-gamma (PPAR) polymorphism
Diabetes mellitus
In 1993, tumor necrosis factor (TNF) was known as
a pro-inflammatory yield of adipose tissue that is produced
from models of diabetes and obesity, providing proof for
a functional relation between obesity and inflammation
(Hotamisligil, et al., 1993). TNF- interferes with the
signals of the activated insulin receptor, promoting insulin
resistance (Ouchi, et al., 2011) . TNF- was related to
homeostasis model assessment (HOMA-IR) in some
studies (Abbatecola, et al., 2004; Löfgren, et al., 2000),
but not in others (Koistinen, et al., 2000; Bruun, et al.,
2003). Our study confirms the correlation between TNF and HOMA-IR as it shows high levels of TNF- and
HOMA-IR in T2DM compared with control. Our results
are in agreement with some studies (Abbatecola, et al.,
2004; De Rekeneire, et al., 2006) but not with others
(Bruun, et al., 2003; Bastard, et al., 2002).
High plasma IL-1 levels were linked to
hyperglycemia and insulin resistance in T2DM patients
(Donath, M.Y., Shoelson, S.E., 2011). Hyperglycemia is
recognized to stimulate the production and release of IL1â in various cell types (Maedler, et al., 2017) and IL-1
may result in islet -cells dysfunction, reduce insulin
secretion and thus raise the risk of diabetes (Maedler, et
al., 2017; Herder, et al., 2015). Our results show high
levels of IL-1 and HOMA-IR in T2DM compared with
control.
In summary, our study shows non-significant
association between the Pro12Ala SNP and T2DM, but
TNF-, IL-1 and HOMA-IR have a significant
association with T2DM.
References
Abbatecola, A.M., L. Ferrucci, R. Grella, et al., (n.d.).
6173
polymorphism of the PPAR-2 gene and type 2 diabetes
mellitus in the Qatari consanguineous population. Acta
Diabetologica, 45(1): 15-21.
Banerjee, M. and M. Saxena (2012). Interleukin-1 (IL-1) family
of cytokines: Role in Type 2 Diabetes. Clin. Chim. Acta,
413(15-16): 1163-1170.
Bastard, J.P., M. Maachi, J.T. van Nhieu, C. Jardel, E. Bruckert,
A. Grimaldi, J.J. Robert, J. Capeau and B. Hainque (2002).
Adipose tissue IL-6 content correlates with resistance to
insulin activation of glucose uptake both in vivo and in
vitro. The Journal of Clinical Endocrinology and
Metabolism, 87(5): 2084-2089.
Bergman, R.N., D.T. Finegood and M. Ader (1985). Assessment
of Insulin Sensitivity in Vivo. Endocrine Reviews, 6(1):
45–86.
Bouassida, K.Z., L. Chouchane, K. Jellouli, S. Cherif, S. Haddad,
S. Gabbouj and J. Danguir (2005). The peroxisome
proliferator activated receptorö2 (PPARö2) Pro12Ala
variant: lack of association with type 2 diabetes in obese
and non obese Tunisian patients. Diabetes and
Metabolism, 31(2): 119-123.
Bruun, J.M., C. Verdich, S. Toubro, A. Astrup and B. Richelsen
(2003). Association between measures of insulin
sensitivity and circulating levels of interleukin-8,
interleukin-6 and tumor necrosis factor-alpha. Effect of
weight loss in obese men. European journal of
endocrinology, 148(5): 535-542.
De Rekeneire, N., R. Peila, J. Ding, L.H. Colbert, M. Visser, R.I.
Shorr, S.B. Kritchevsky, L.H. Kuller, E.S. Strotmeyer, A.V.
Schwartz and B. Vellas (2006). Diabetes, hyperglycemia,
and inflammation in older individuals: the health, aging
and body composition study. Diabetes care, 29(8): 19021908.
Deeb, S.S., L. Fajas, M. Nemoto, J. Pihlajamäki, L. Mykkänen, J.
Kuusisto, M. Laakso, W. Fujimoto and J. Auwerx (1998). A
Pro12Ala substitution in PPAR2 associated with
decreased receptor activity, lower body mass index and
improved insulin sensitivity. Nature genetics, 20(3): 284287.
Abbatecola, A.M., L. Ferrucci, R. Grella, S. Bandinelli, M. Bonafè,
M. Barbieri, A.M. Corsi, F. Lauretani, C. Franceschi and G.
Paolisso (2004). Diverse effect of inflammatory markers on
insulin resistance and insulin resistance syndrome in the
elderly. Journal of the American Geriatrics Society, 52(3):
399-404.
Dinarello, C.A. (2011). Interleukin-1 in the pathogenesis and
treatment of inflammatory diseases. blood, 117(14): 37203732.
Allan F. Moore and Jose C. Florez (2008). Genetic Susceptibility
to Type 2 Diabetes and Implications for Antidiabetic
Therapy. Annual Review of Medicine, 59(1): 95-111.
Donath, M.Y. and S.E. Shoelson (2011). Type 2 diabetes as an
inflammatory disease. Nature Reviews Immunology, 11(2):
98-107.
Altshuler, D., J.N. Hirschhorn, M. Klannemark, C.M. Lindgren,
M.C. Vohl, J. Nemesh, C.R. Lane, S.F. Schaffner, S. Bolk, C.
Brewer and T. Tuomi (2000). The common PPAR Pro12Ala
polymorphism is associated with decreased risk of type 2
diabetes. Nature genetics, 26(1): 76-80.
Ereqat, S., A. Nasereddin, K. Azmi, Z. Abdeen and R. Amin
(2009). Impact of the Pro12Ala polymorphism of the PPARGamma 2 gene on metabolic and clinical characteristics in
the Palestinian type 2 diabetic patients. PPAR research,
2009.
Badii, R., A. Bener, M. Zirie, A. Al-Rikabi, M. Simsek, A.O. AlHamaq, M. Ghoussaini, P. Froguel and N.J. Wareham
(2008). Lack of association between the Pro 12 Ala
Freigang, S., F. Ampenberger, A. Weiss, T.D. Kanneganti, Y.
Iwakura, M. Hersberger and M. Kopf (2013). Fatty acidinduced mitochondrial uncoupling elicits inflammasome
independent IL-1alpha and sterile vascular inflammation
6174
Mahmoud El-Rehany et al.
in atherosclerosis. Nature Immunology, 14: 1045–1053.
from nucleated red blood cells. Biotechniques, 8(1):.
Ghoussaini, M., D. Meyre, S. Lobbens, G. Charpentier, K.
Clément, M.A. Charles, M. Tauber, J. Weill and P. Froguel
(2005). Implication of the Pro12Ala polymorphism of the
PPAR-gamma 2gene in type 2 diabetes and obesity in the
French population. BMC medical genetics, 6(1): 1-11.
Morita, T., S. Tabata, M. Mineshita, T. Mizoue, M.A. Moore
and S. Kono (2005). The metabolic syndrome is associated
with increased risk of colorectal adenoma development:
the Self-Defense Forces health study. Asian Pacific
journal of cancer prevention, 6(4): 485–489.
Grygiel-Górniak, B. (2014). Peroxisome proliferator-activated
receptors and their ligands: nutritional and clinical
implications -a review. Nutr. J., 13: 17.
Olson, A.L. (2012). Regulation of GLUT4 and insulin-dependent
glucose flux. ISRN molecular biology, 2012.
Harlow, E.D. and D. Lane (1988). A laboratory manual. New
York: Cold Spring Harbor Laboratory, 579.
Herder, C., E. Dalmas, M. Böni-Schnetzler and M.Y. Donath
(2015). The IL-1 pathway in type 2 diabetes and
cardiovascular complications. Trends in Endocrinology
and Metabolism, 26(10): 551-563.
Hotamisligil, G.S., N.S. Shargill and B.M. Spiegelman (1993).
Adipose expression of tumor necrosis factor-alpha: direct
role in obesity-linked insulin resistance. Science,
259(5091): 87-91.
Imai, Y., A.D. Dobrian, J.R. Weaver, M.J. Butcher, B.K. Cole,
E.V. Galkina and M.A. Morris (2013). Interaction between
cytokines and inflammatory cells in islet dysfunction,
insulin resistance and vascular disease. Diabetes Obesity
and Metabolism, 15(13): 117-129.
Karstoft, K. and B.K. Pedersen (2016). Exercise and type 2
diabetes: focus on metabolism and. Immunology and Cell
Biology, 94: 146-150.
Koistinen, H., J.P. Bastard, E. Dusserre, P. Ebeling, N. Zegari, F.
Andreelli, C. Jardel, M. Donner, L. Meyer, P. Moulin and B.
Hainque (2000). Subcutaneous adipose tissue expression
of tumour necrosis factor-alpha is not associated with
whole body insulin resistance in obese nondiabetic or in
type-2 diabetic subjects. European journal of clinical
investigation, 30(4): 302-310.
Löfgren, P., V. van Harmelen, S. Reynisdottir, E. Näslund, M.
Rydén, S. Rössner and P. Arner (2000). Secretion of tumor
necrosis factor-alpha shows a strong relationship to
insulin-stimulated glucose transport in human adipose
tissue. Diabetes, 49(5): 688-692.
Maedler, K., P. Sergeev, F. Ris, J. Oberholzer, H.I. Joller-Jemelka,
G.A. Spinas, N. Kaiser, P.A. Halban and M.Y. Donath (2017).
Glucose-induced cell production of IL-1 contributes to
glucotoxicity in human pancreatic islets. The Journal of
Clinical Investigation, 127(4): 1589-1589.
Matthews, D.R., J.P. Hosker, A.S. Rudenski, B.A. Naylor, D.F.
Treacher and R.C. Turner (1985). Homeostasis model
assessment: insulin resistance and -cell function from
fasting plasma glucose and insulin concentrations in man.
Diabetologia, 28: 412-419.
Medrano, J.F., E. Aasen and L. Sharrow (1990). DNA extraction
Ouchi, N., J.L. Parker, J.J. Lugus and K. Walsh (2011).
Adipokines in inflammation and metabolic disease. Nature
reviews immunology, 11(2): 85.
Radha, V., K.S. Vimaleswaran, H.N.S. Babu, N. Abate, M.
Chandalia, P. Satija, S.M. Grundy, S. Ghosh, P.P. Majumder,
R. Deepa and S.M. Rao (2006). Role of Genetic
Polymorphism Peroxisome Proliferator–Activated
Receptor-2 Pro12Ala on Ethnic Susceptibility to Diabetes
in South-Asian and Caucasian Subjects: Evidence for
heterogeneity. Diabetes care, 29(5): 1046-1051.
Saxena, M., C.G. Agrawal, S. Gautam, H.K. Bid and M. Banerjee
(2009). Overt Diabetic Complications in obese Type 2
Diabetes Mellitus Patients from North India. Arch. Appl
Sci. Res., 1: 57-66.
Swaroop, J.J., D. Rajarajeswari and J. Naidu (2012). Association
of TNF- with insulin resistance in type 2. Indian Journal
of Medical Research, 135(1): 127-130.
Vimaleswaran, K.S., V. Radha, R. Deepa and V. Mohan (2007).
Absence of association of metabolic syndrome with
PPARGC1A, PPARG and UCP1 gene polymorphisms in
Asian Indians. Metabolic syndrome and related
disorders, 5(2): 153-162.
Wieser, V., A.R. Moschen and H. Tilg (2013). Inflammation,
Cytokines and Insulin Resistance:. Archivum
Immunologiae et Therapiae Experimentalis, 61(2): 119125.
Wild, S., G. Roglic, A. Green, R. Sicree and H. King (2004).
Global prevalence of diabetes: estimates for the year 2000
and projections for 2030. Diabetes care, 27(5): 1047-1053.
Yen, C.J., B.A. Beamer, C. Negri, K. Silver, K.A. Brown, D.P.
Yarnall, D.K. Burns, J. Roth and A.R. Shuldiner (1997).
Molecular scanning of the human peroxisome proliferator
activated receptor gamma (hPPAR gamma) gene in diabetic
Caucasians: identification of a Pro12Ala PPAR gamma 2
missense mutation. Biochem. Biophys Res. Commun, 241:
270-274.
Zeggini, E., J.R.C. Parkinson, S. Halford, K.R. Owen, M. Walker,
G.A. Hitman, J.C. Levy, M.J. Sampson, T.M. Frayling, A.T.
Hattersley and M.I. McCarthy (2005). Examining the
relationships between the Pro12Ala variant in PPARG and
Type 2 diabetes related traits in UK samples. Diabetic
medicine, 22(12): 1696-1700.
Mahmoud El Rehany