Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 77–84

Original paper

Targeting tumor necrosis factor alpha (TNF-a) in diabetic rats could approveavenues for an efficient strategy for diabetic therapy

Karolin K. Abdul-Aziz a,*, Muobarak J. Tuorkey b

a Zoology Department, Division of Physiology, Damanhour University, Egyptb Medical Biochemistry, Faculty of Science, Damanhour University, Egypt

A R T I C L E I N F O

Keywords:

Tumor necrosis factor a (TNF-a)

Protein kinase B (Akt)

Forkhead box O-1 (FOXO-1)

Myeloperoxidase (MPO)

Mucosa-associated lymphoid tissues

(MALT)

A B S T R A C T

Background: Several studies held belief that downregulation of TNF-a may be effective for preventing

diabetes and it’s complications. However, it is not known whether TNF-a downregulation in long-term

can generate any biological adverse.

Aim: The aim of the present study was to clarify what the impact is for such treatment with specific

antibody for TNF-a on the other biological activities after 4 weeks.

Methods: Using western blot, IHC, Elisa, biochemical assays and scanning electron microscope.

Results: Results show that TNF-a, FOXO-1, IL-6 and MPO, when expressed in diabetic rats, collectively

induce dramatic changes in diabetic rats. Since, TNF-a is involved in activation of transcription factor

FOXO1 along with oxidative stress mediated by neutrophils. On one hand, IL-6 mediates neutrophils

activation leading to an augmentation in stress mediators. And FOXO1 is activated in order to eliminate

these oxidative mediators, on the other hand. Data show also that the prominent defect in mucosal IgA

and IL-2 secretions may be the leading reasons for digestive atrophy. Finally, Akt-1 inhibits the cleavage

of caspase 3, so, it could prevent the incidence of apoptosis.

Conclusion: Findings of this study reveal how TNF-a can be mechanistically coupled to greater diabetic

complications potential.

� 2012 Diabetes India. Published by Elsevier Ltd. All rights reserved.

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1. Introduction

Diabetes type 1 is a multi-factorial disease characterized by anaggressive destruction of the insulin producing b-cells oftenmediated by multiple autoimmune reactions. Approximately440,000 cases of type 1 diabetes in childhood, more than 25%were in south-east Asian region and more than 20% from theEuropean region (International Diabetes Federation) [1]. Diabetestype 2 is a metabolic disorder that is primarily characterized byinsulin resistance accompanied by insulin secretion depressionthat in turn leads to hyperglycaemia. Therefore, the primaryrepercussion of diabetes is rise in blood glucose levels due to theimperilment in the cellular glucose consumption/uptake. The lowinsulin levels inhibit the transport of glucose across cellmembranes causing a high blood glucose level, which subsequent-ly leads to hyperglycaemia. The complications of diabetes areretinal damage, renal failure and elevation of ketone bodies. In thisregard, reactive oxygen species (ROS) could involve in insulinresistance [2]. The streptozotocin, a glucosamine-nitrosoureacompound obtained from Streptomyces has been extensively used

* Corresponding author.

E-mail address: k.kamel2002@yahoo.com (K.K. Abdul-Aziz).

1871-4021/$ – see front matter � 2012 Diabetes India. Published by Elsevier Ltd. All r

http://dx.doi.org/10.1016/j.dsx.2012.08.004

as an experimental tool to develop animal models to studydiabetes and associated complications. Since, it enters pancreatic bcells through glucose transporter 2 channels in the plasmamembrane and causes cellular toxicity and local immuneresponses that lead to hypoinsulinemia and hyperglycemia inanimals [3]. Examination of bones from STZ-induced diabetic micewas found to exhibit a significant decrease in bone volume fraction[4] low bone formation. The effect of diabetes on bone stiffness isless clear, with some reports of increased stiffness. Under normalphysiological conditions, bone undergoes dynamic microstructuralremodeling throughout life to accommodate mechanical stress andcalcium demand [5]. Bone remodeling is a coupled process of boneresorption and formation, and requires coordination of all threetypes of bone cells, namely osteocytes, osteoblasts and osteoclasts[6]. Under toxic insults and or mechanical stress, osteocytes act asmechanosensors to detect changes in the flow of bone fluid withinbone canaliculi, and respond by transmitting signals to theosteoblasts via their syncytial processes [6]. Numerous experi-mental, epidemiological, and clinical studies have established thatthe proinflammatory cytokines are key mediators in the process ofosteoclast differentiation and bone resorption [7–10]. Thesechanges in bone have also established in chronic inflammationsdue to increased production of proinflammatory cytokinesincluding TNF-a, IL-1, and IL-6. It has also been established that

ights reserved.

K.K. Abdul-Aziz, M.J. Tuorkey / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 77–8478

upregulated proinflammatory cytokines are primary mediators ofosteopenia or osteoporosis [11]. Since, the removal of cartilagecoupled with bone formation and remodeling is dependent on thecoordinated expression of cytokines that initiate and regulate thefracture healing process normal fracture repair process [12].

2. Materials and methods

2.1. Animals and experimental design

Thirty male albino rats weighing approximately 100–120 gwere used in this study. The experimental procedures wereapproved by the Committee of Animal Care and Use at DamanhourUniversity. Rats were arranged into three main groups; 10 ratsserve as control group and 20 rats were involved as experimentaldiabetic groups. Diabetes was induced by intraperitoneal injectionof a single dose of streptozotocin (60 mg/kg/BW in citrate bufferpH 4.6). Rats were considered to be diabetic when serum glucoselevels exceeded 250 mg/dl (Diagnostics, Indianapolis, IN, USA). Agroup of diabetic rats received an i.p. injection of anti-TNF-a(10 mg/kg/BW) in 1 ml of phosphate-buffered saline on days 21and 25. After one month of the last injection, both control andexperimental animals were sacrificed and blood, duodenum andfemur bones were collected and freezed.

2.2. Biochemical analysis

Plasma cytokines, IL-2, IL-4, IL-6 and TNF-a levels weredetermined by ELISA using the commercial kits according to themanufacturer’s instructions, as well as, Caspase-3 in duodenumtissue. Mucosal IgA, total proteins and plasma glucose level weredetermined by commercial kits. For determination of neutrophilactivity hypochlorous acid production by myeloperoxidase wasdetermined by measuring accumulation of taurine chloramine [13].Briefly, 10 mL of fresh heparinized blood samples or purifiedmyeloperoxidase (10 nmol/L) was incubated with 10 mmol/L taurinein 10 mmol/L sodium phosphate buffer (pH 7.4) plus 140 mmol/Lsodium chloride (PBS) at 21 8C for 5 min. The reaction was started byaddition of 30 mmol/L hydrogen peroxide and stopped with 20 mg/mlcatalase. The amount of taurine chloramine formed was assayed with5-thio-2-nitrobenzoic acid. The activity of MPO was measuredfollowing the decrease of absorbance at 412 nm. One unit is theamount of MPO that can produce 1.0 nmole of taurine chloramine(hypochlorous acid) at pH 6.5 at 25 8C during 30 min in the presenceof 100 mmol/L chloride and 100 mmol/L of hydrogen peroxide.

2.3. Bone analysis

2.3.1. Determination of ash content

Ash is the inorganic residue remaining after the water andorganic matter have been removed by heating in the presence ofoxidizing agents, which provides a measure of the total amount ofminerals within a femur bone of five replicates. Femur bone samplesof 1 g were used in the analysis of ash content. Bone samples weredefatted by solvent extraction; this facilitates the release of themoisture and prevents spattering. Dry ashing procedures use a hightemperature muffle furnace capable of maintaining temperatures ofbetween 500 and 600 8C. Water and other volatile materials arevaporized and organic substances are burned in the presence of theoxygen in air to CO2, H2O and N2. Most minerals are converted tooxides, sulfates, phosphates, chlorides or silicates. The ash contentcan be expressed on either a dry or a wet basis:

%Ashðdry basisÞ ¼ Mash

Mdry� 100

����

where MASH refers to the mass of the ashed sample, and Mdry andMash refer to the original masses of the dried and wet samples.

2.4. Determination of calcium and magnesium

Femur bones were separated critically dried at 100 8C for 24 hand ash was determined. A known weight of ash was dissolved in1 ml concentrated nitric acid and completely digested in hot plateat 60 8C with regular added bi-distilled water and complete thevolume to 5 ml in both control and experimental groups. Calciumand magnesium were measured by atomic absorption spectro-photometer [14].

2.5. X-ray analysis

Femur bones of both control and experimental groups weredried up to a critical point by a standard technique suitable forpreparing samples for electron microscopy. Five bones of eachgroup were chosen for the experiment. The dried samples wereexamined by a micro-CT desktop system, which based on thecombination of X-ray projection microscopy with a tomographicalreconstruction technique [15,16]. Accurate beam-hardening cor-rection was implemented. Gray values in the virtual cross-sectionswere calibrated as calcium mineral density in bone. From thesecross-sections, three-dimensional models were created. Mineralscontent were calculated directly from images and expressed aspercentage per volume and per weight.

2.6. Immunohistochemistry

Samples of fumer were decalcified in 20% EDTA and embeddedin paraffin wax as described previously [17]. Endogenousperoxidase activity was quenched by a H2O2/methanol wash,followed by hyaluronidase treatment. Samples were blocked withgoat serum in phosphate-buffered saline (PBS) for 1 h andincubated for 1 h with the primary antibody against phospho-FOXO1 (pSer256) (diluted 1:50). Afterwards, they were thenincubated for 1 h with the relevant secondary antibody, biotiny-lated goat anti-rat IgG (F6258). Color was developed by incubationwith DAB for 5 min; sides were then counterstained with H&E andmounted with Vecta-Mount.

2.7. Histological observations

Femur bones of both control and experimental groups wereincised immediately at the end of treatment, fixed in 10% formalsaline, followed by decalcification in 5% EDTA and dehydrated inascending grades of ethyl alcohol, cleared in xylol and mounted inmolten paraplast 58–62 8C. Five mm thick longitudinal histologicalsections were stained with hematoxylin and eosin and examinedunder bright field light microscope.

2.8. Duodenum alteration

2.8.1. Scanning electron microscope

About 1 mm slice of tissue from the middle portion of theduodenum, jejunum, ileum and cecum were fixed in 4%gluteraldyhyde. Tissue samples for SEM were processed asdescribed previously [18,19]. The lumen was cut open underthe dissection microscope to select the right orientationbefore mounting on the stud. Specimens were dried in acritical point drying apparatus (BALTEC-SPD 030) using liquidcarbon dioxide as the medium. Sputter-coated with gold(BALTEC-SCD 005 vacuum coater) at 100 ml, 7 mA for 3 minbefore being examined with a Jeol-SEM (JSM-6400, Japan) at8 kV.

Table 1Calcium, magnesium (mmol/100 mg) and ash weight (g) of femur of rat.

Ash (g) Mg (mmol/100 mg) Ca (mmol/100 mg) Subject

0.2800 � 0.005 0.2122 � 0.006 1.738 � 0.053 Control

0.3213 � 0.006*** 0.2656 � 0.005** 1.416 � 0.028* Diabetic

0.253 � 053a 0.1922 � 0.07 1.61 � 0.03 Anti-TNF-a

* p < 0.05.** p < 0.01.*** p < 0.001.

Table 2X-ray analysis of femur and percentages of phosphorus (P), calcium (Ca), copper

(Cu) and zinc in femur of rat.

Zn Cu Ca P Subject

2.9 � 0.421 1.1 � 0.385 78.8 � 6.543 17.32 � 0.2788 Control

2.6 � 0.327** 6.5 � 1.262* 12.9 � 3.123* 27.20 � 0.4011* Diabetic

2.33 � 0.861* 2.19 � 1.56 67.45 � 7.46 22.32 � 0.7 01 Anti-TNF-a

* p < 0.05.** p < 0.01.

K.K. Abdul-Aziz, M.J. Tuorkey / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 77–84 79

2.9. Western blot analysis

Twenty mg of duodenum tissue homogenate was added to lysisbuffer containing 20 mM Tris–HC1 (pH 7.5), 1% Triton X-100,137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodiumorthovanadate, 25 mM b-glycerophosphate, 2 mM sodium pyro-phosphate, 1 mM phenylmethylsulfonyl fluoride and 1 g/mlleupeptin. Equal amounts of aliquots of tissue homogenatecontaining 60 mg of protein were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pro-tein was then fractionated using a 10% separating gel. Thefractionated proteins were then transferred electrophoreticallyto nitrocellulose paper, and the proteins were immunoblotted withspecific antibody for phospho-PKB/AKT (pSer473) (diluted 1:1000)and incubated for 1 h and then at 4 8C overnight. After washingwith Tris-buffered saline Tween-20, the membranes were incu-bated with horseradish peroxidase (HRP)-conjugated secondaryantibody. The nitrocellulose papers were developed using 5-bromo-4-chloro-3-indolyl phosphate/4-nitroblue tetrazolium.Quantification of the bands was carried out using NIH ImageJsoftware. To ensure equal loading of the protein, b-actin was usedas an endogenous control.

2.9.1. Data analysis

All data collected were subjected to analysis of variance amongthe groups using one way ANOVA. Significant differences betweenmean values were considered at p � 0.05.

3. Results

3.1. Blood glucose alteration

Blood glucose level was recorded 178.00 � 59.842 mg/dl,573.26 � 119.398 mg/dl and 225.61 � 135.774 mg/dl in the control,diabetic and anti-TNF-a groups. Of note, the level of glycemia wassignificantly higher by factors of 3.22 and 2.54 compared with controland anti-TNF-a (Fig. 1).

3.2. Fumer bone alteration

To investigate the alteration in the femur, we have determinedthe most important minerals in bone based on biochemicalreaction. Data show that diabetic rats exhibited a high decrease ofcalcium and ash content, meanwhile, magnesium content wasmarkedly increased (Table 1). Accordingly, to verify our data, X-rayanalysis on femur was carried out. Following X-ray analysis, the

Fig. 1. Blood glucose level in blood plasma of rats.

percentages of calcium and zinc content were markedly decreased,however, percentages of phosphorus and copper were increased(Table 2) in diabetic group. However, no significant difference wasrecorded when control and anti-TNF-a groups were compared,except for Zn, which significantly decreased in anti-TNF-a group.Histological analysis of bone of control group showed that thefemoral epiphyseal region is formed of regularly arranged fourzones: zone of resting cartilage composed of small scatteredchondrocytes, zone of proliferation formed of regular arrangedcolumn of cartilage cells aligned near each other, zone ofhypertrophied cartilage cells and zone of calcified cartilage anddegeneration and death of chondrocytes (Fig. 2a). In experimen-tally diabetic rat, the femoral head revealed a considerablethinning of femoral epiphyseal cartilage associated with markeddegeneration of chondrocytes and loss of cartilage column. Finecollections of chondrocytes were sparsely distributed in theepiphyseal plate. Abnormal derangement of epiphyseal line wasdetected. The trabecular bone attained considerable thinning. Asignificant reduction in chondrocytes areas and numbers werenoted in diabetes group compared with the rest groups. Since,chondrocytes areas were 15.80 � 3.268 mm2, 8.98 � 1.427 mm2 and11.43 � 2.582 mm2 for control, diabetic and anti-TNF-a groups,respectively (Fig. 2 b). The number of chondrocytes was highlysignificant decrease into 66.77 � 7.679 mm2 compared with controland treated group, which were recorded 91.4544 � 22.275 mm2 and82.68 � 15.465 mm2, respectively. FOXO1 expression was onlydetected in chondrocytes nucleus in form of brown stain, however,its expression was completely absent in chondrocytes nucleus in eachof control and anti-TNF-a groups.

3.3. Duodenum alteration

In general, the duodenum is composed of four main distinctiveregions: mucosa, submucosa, muscularis and adventitia. Themucosa is arranged into finger like projections called villi withintervening short glands called crypts. The peripheral surface ofthe villi is lined by a simple columnar epithelium. The goblet cellsare regularly arranged in between the epithelial cells. Thesubmucosa is enclosed by loose collagenous tissues and infiltratedby blood vessels. The muscularis is composed of dispersed smoothmuscles. The adventitia represents the outermost cell layers and isformed of collagenous tissues outlined by serosa of simplesquamous epithelium. Scanning electron micrographs of duode-num of rats in Fig. 3 showing that: in control group, normaldistribution for mucus layer on the epithelium (A), well recognizefinger-like shape (B), tall, spatulate villi with horizontally-arranged

Fig. 2. Immunohistochemistry of fumer paraffin embedded sections reveal Expression of FOXO1. Magnification �40. Scale bar: 10 mm. Arrows are marked apoptotic cells,

trabecular bone (TB), cartilage column (CC), matrix (M). Quantification of immunohistochemistry staining using Image J software. For morphometric analysis chondrocytes

units (defined as �4 mm in length), number of chondrocytes was quantified as described.

K.K. Abdul-Aziz, M.J. Tuorkey / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 77–8480

surface cleft (C), with will recognized goblet cells (D). The villi heightreached to 220 mm with broad width of 110 mm in control group (C).In diabetic, degeneration of mucosal covering epithelium (A1) andjagged tips with protuberances were observed (B1). Dome-shapedcells and atrophied degenerative phases of their peripheral borders(C1) (an abrasion on the villus surface). Massive reduction of glandularopening of goblet cells (D1). The villi height reached to 195 mm withbroad width of 85 mm manifesting more reduction comparing withcontrol and anti-TNF-a treated groups (C1). In anti-TNF-a treatedgroup, mucus probably coating the degenerated cells formed strandsover the villi (Fig. 3A2). Villi still have broad finger-like shape (C1) witha slightly compressed laterally and recesses on the surface (B1). Gobletcells with their openings can be seen between the epithelial cells (D1).The villi height reached to 218 mm with broad width of 97 mm(Fig. 3C1). Histological analysis of duodenum (Fig. 4a) revealed denseleukocytes infiltration associated with villus core separation weredetected, submucosal edema, reduction of cryps size sloughing ofepithelial cells and massive reduction of goblet cells. Fainly alcian bluestaining of goblet cells manifested reduction of their numbers withinthe villi. Since, the percentage ratio of goblet cells number (Fig. 4b) wassignificantly reduced in diabetic to 7.78 � 2.336 when compared withcontrol (11.91 � 5.007) and anti-TNF-a (10.71 � 3.016) groups (Fig. 5).

3.4. Cytokines profile

Data reflect a prominent decrease (P < 0.001) in the level of IL-2in diabetic group compared with control and anti-TNF-a groups.Since, the level of IL-2 was recorded 246.091 � 17.249 Pg/gm,295.770 � 14.660 Pg/gm and 281.936 � 15.577 Pg/gm in diabetic,control and anti-TNF-a groups, respectively. In contrast, the level of

IL-6 was significantly (P < 0.01) elevated in diabetic group(69.06 � 3.27 Pg/gm) as compared with control (50.34 � 4.82 Pg/gm)and the anti-TNF-a diabetic treated group (54.34 � 4.82 Pg/gm). Onthe other hand, the level of TNF-a was prominently decline in the anti-TNF-a diabetic treated group (82.451 � 11.888 Pg/gm) by factors1.6�2 compared with control (131.618 � 12.147 Pg/gm) and by factors�2 compared with diabetic group (163.895 � 14.135 Pg/gm). Theirwas a significant increase (P < 0.01) in the level of TNF-a in diabeticgroup compared with control group.

3.5. Akt-1 phosphorlyation

As a result of blocking of TNF-a, phosphorylation of AKT wasincreased by factors of 1.8 and 2.02 compared with control anddiabetic groups, respectively (Fig. 6).

3.6. Mucosal immunoglobulin A

In diabetic group, the level of mucosal IgA was highlysignificantly decline (p < 0.001) compared with control andanti-TNF-a groups. Since, data recorded 147.775 � 2.466 mg/gm,130.109 � 1.533 mg/gm and 144.541 � 2.792 for control, diabeticand anti-TNF-a groups, respectively. Their was also a significantdifference (p < 0.05) was noted when compared the anti-TNF-agroup and control group (Fig. 7).

3.7. Caspase-3 activity

The activity of Caspase-3 was significantly increased in diabeticgroup (431.63 � 27.13 U/mg) compared with control and the

Fig. 3. Scanning electron micrographs of duodenum of rats showing that: in control group, normal distribution for mucus layer on the epithelium (A), well recognize finger-

like shape (B), tall, spatulate villi with horizontally-arranged surface cleft (C), with will recognized goblet cells (D). In diabetic, degeneration of mucosal covering epithelium

(A1), jagged tips with protuberances were observed (B1). Dome-shaped cells and atrophied degenerative phases of their peripheral borders (C1) (an abrasion on the villus

surface) and massive reduction of glandular opening of goblet cells (D1). In anti-TNF-a treated group, mucus probably coating the degenerated cells formed strands over the

villi (A2). Villi still have broad finger-like shape (C1) with a slightly compressed laterally and recesses on the surface (B1). Goblet cells with their openings can be seen between

the epithelial cells (D1).

Fig. 4. Alcian blue staining of duodenum of control (A), diabetic (B) and anti-TNF-a (C). Diabetic rats have a progressive decrease of the number of goblet cells (blue) along the

small intestine. The number of goblet cells was counted in the duodenum (n = 5 rats).

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Fig. 5. Biochemical changes of IL-2, IL-6 and TNF-a expressed as Pg/gm.

K.K. Abdul-Aziz, M.J. Tuorkey / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 77–8482

anti-TNF-a groups, since, they were recorded 367.25 � 23.57 U/mg,385.25 � 23.57 U/mg, respectively (Fig. 8).

3.8. Neutrophils activity

Myeloperoxiase (MPO) is the most abundant protein inneutrophils (also found in monocytes). Meloperoxidase can alsouse hydrogen peroxide resulting from the reduction of superoxideradicals to oxidize chloride to hypochlorous acid (HPO). It isimportant to clarify that the increased basal neutrophil MPOactivity is not related to soluble plasma MPO due to intracellularMPO activity. In diabetic group, data show that the activity ofmyeloperoxidase was significantly increased by factors 1.43 and2.25 compared with control and anti-TNF-a groups, respectively.Since, MPO was recorded 21.42 � 3.68 U/ml, 48.22 � 4.03 U/ml and33.58 � 3.42 U/ml for control, diabetic and anti-TNF-a groups,respectively (Fig. 9).

4. Discussion

IgA-producing plasma cells comprise �20% of total plasma cellsof peripheral lymphoid tissues, in human and mouse, whereasmore than 80% of plasma cells produce IgA in mucosa-associatedlymphoid tissues [20]. Secretory IgA is constitutively released intothe lumen via the transport system of the intestinal epithelia [21];it prevents invading pathogens from binding to mucosal epithelialcells and neutralizes their toxins [22,23]. In this study, a significantreduction in the mucosal IgA was recorded in diabetic group when

Fig. 6. Total proteins were extracted and subjected to Western blotting using antibody di

1), or as a loading control beta actin.

compared whether with control or treated groups. Of note, IgA isthe most abundant immunoglobulin in the body and has a centralrole in mucosal immunity [24,25]. That could explain the dramaticalteration and atrophied changes in the duodenum of diabetic rats.Since, in response to antigenic stimulation by the intestinalcommensal bacteria, IgA release is induced from IgA-producingplasma cells [26]. As a result of anti-TNF-a injection, the level ofbasal mucosal immunoglobulin A was elevated by factor 1.09. Thiselevation in IgA may provide a stringent cellular protection formucosal and epithelial cells from the destructive effect of differentinflammatory and oxidized mediators. In this regard, a recentstudy by Hiroyuki Tezuka et al. (2007) has been addressed one ofthe most biologically important and longstanding questions inimmunology, which was why this ‘biased’ IgA synthesis takes placein the mucosa-associated lymphoid tissues (MALT), but not otherlymphoid organs [20]. They showed that IgA class-switchrecombination (CSR) is impaired in inducible-nitric-oxide(iNOS)-synthase-deficient mice [20]. Since, iNOS regulates the T-cell-dependent IgA CSR through expression of transforminggrowth factor-b receptor, and the T-cell-independent IgA CSRthrough production of a proliferation-inducing ligand and a B-cell-activating factor of the tumor necrosis factor (TNF) family.

On the other hand, it has been established that IL-2 plays apivotal role in programming T cells for activation-induced celldeath. IL-2 played an additional role in immune regulation,exogenous IL-2 distraction could reverse the excessive death ofisolated CD25+ T cells and restored their proliferative activity [27].Surprisingly, delivery of IL-2 a few days after birth resulted in lowefficiency of disease improvement [28], therefore, IL-2 had to beprovided at very early stages of CD25+ T dysfunction. In this study,the level of IL-2 was significantly decreased in diabetic groupcompared with other groups. As a result of targeting TNF-a theirstill was a significant decrease in IL-2 levels compared with controlgroup, however, this decrease confer a minor effect compared withdiabetic group.

Since, oxidative stress has been widely emerged for thepathogenesis of several diseases; we here in this study tried toinvestigate the activity of the major promoter for the reactivespecies production in order to understand consequence of events.Data show that the activity of myeloperoxidase (MPO) in wholeblood was significantly higher in diabetic group compared withcontrol and treated groups. That could put a finger on the crucialrole of hyper neutrophils activity in diabetic complications. It isimportant to clarify that the increased basal neutrophil MPOactivity is not related to soluble plasma MPO due to intracellularMPO activity [29]. In this context, it is very important to mention afact that in order to eliminate functionless cells, IL-6 is the centralinducer for neutrophil activation under stress conditions.

rected against Akt-1 protein kinase. Protein levels of phosphorylated Akt-1 (p-AKT-

Fig. 7. Immunoglobulin A in mucus layer of rats duodenum.Fig. 9. Myeloperoxidase (MPO) activity, one unit of MPO expressed as the amount of

MPO that can produce 1.0 nmole of taurine chloramine (hypochlorous acid).

K.K. Abdul-Aziz, M.J. Tuorkey / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 77–84 83

Therefore, the proinflammatory IL-6 could predict more preciselythe severity and duration of inflammation, particularly in its acutephase than TNF-a [30]. As a result of the increased levels ofreactive oxygen species in diabetic group, a prominent expressionfor the transcription factor FOXO1 has noted in the nucleus ofchondrocytes, since, oxidative stress is the main mediator forFOXO1 upregulation. In general, FOXO1 is activated in order toeliminate oxidative stress hazard effects [31,32]. Furthermore,during periods of oxidative stress, FoxO transcription factors canlead to apoptosis [33]. Thus, the elevation of FOXO1 expressioncould explain the augmented levels of caspase 3 in diabetic group.In this concern, the increased levels of TNF-a may also beresponsible for the increased cleaved caspase 3 levels in thediabetic group. Of note, there was a prominent reduction inchondrocytes areas and numbers along with prominent apoptoticchanges in diabetic group, thus, TNF-a up-regulation couldinvolve in such events. Data show that TNF-a up-regulationenhances susceptibility of minerals resorption in cartilage andbone. Additionally, upregulation of FOXO1 by TNF-a has beeninvolved in pro-apoptotic alteration. In this regard, it has beenproposed that conditions with prolonged or high levels of FOXO1activation may be deleterious by inducing apoptosis [34,35].

On the other hand, it has been concluded that Akt mediates thephysiological responses of insulin on glucose uptake [36] andglycogen synthesis [37]. In this study protein kinase B (Akt)phosphorylation was significantly decreased in diabetic groupcompared with control and anti-TNF-a treated groups. In thecontrary, as a result of down-regulation of TNF-a by the specificantibody phosphorylation of Akt-1 was induced in treated group,thus in turn, a significant decrease in the level of the executor

Fig. 8. Caspase-3 activity in duodenum wall of rats expressed as U/mg.

capase-3 was noted. Since, Akt typically inhibits the cleavage ofcaspase 3, and thus, it could prevent the incidence of apoptosis[38]. That could emerge as a reason for apoptotic events in diabeticgroup. Additionally, it is very important to state that there is areverse relation between the activation of Akt-1 and the presenceor absence of FoxO1 in the nucleus [39]. On phosphorylation byAkt-1, Foxo1 is transported out of the nucleus due to phosphor-ylation. Therefore, FOXO1 expression has noted in chondrocytesnucleuses of the diabetic rats, due to TNF-a upregulation andreactive oxygen species produced by neutrophils.

In conclusion, data of this study confirmed that the elevatedlevels of TNF-a upregulate FOXO1 expression in chondrocytescells, which in turn, accelerate cartilage resorption, decreasecartilage areas and numbers. Therefore, over expression of TNF-acould involve in activation of transcription factor FOXO1 alongwith oxidative stress induced by neutrophils, which activated bythe proinflammatory cytokine 6 (IL-6). Impairment in interleukin 2(IL-2) and mucosal immunoglobulin A secretions in diabeticpatients may be the main factors for the digestive tract atrophy andorigins for inflammatory bowel disease.

Conflict of interest statement

None.

Acknowledgments

We would like to thank Prof Dr. Ibrahim El-Shorbagy forinsightful discussions on bone developmental biology. We wouldalso like to thank Dr. Ghad Tabl for sympathetic and keenassistance in western blot technique, as well as Mohamad Al-Gazaly and Noha Nazeeh for technical assistance. We also expressour gratitude to Mr. Mohamad Younis for assistance with animalcare and handling.This work was financially supported by aresearch grant from Faculty of Science, Damanhour University(DFS-122 BIO-D) to Biology department.

References

[1] Roglic G, Unwin N, Bennett PH, Mathers C, Tuomilehto J, Nag S, et al. Theburden of mortality attributable to diabetes: realistic estimates for the year2000. Diabetes Care 2005;28(9):2130–5.

[2] Houstis N, Rosen ED, Lander ES. Reactive oxygen species have a causal role inmultiple forms of insulin resistance. Nature 2006;440(7086):944–8.

[3] Motyl K, McCabe LR. Streptozotocin, type I diabetes severity and bone.Biological Procedures Online 2009;11:296–315.

[4] Botolin S, Faugere MC, Malluche H, Orth M, Meyer R, McCabe LR. Increasedbone adiposity and peroxisomal proliferator-activated receptor-gamma2 ex-pression in type I diabetic mice. Endocrinology 2005;146(8):3622–31.

K.K. Abdul-Aziz, M.J. Tuorkey / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 77–8484

[5] Wongdee K, Charoenphandhu N. Osteoporosis in diabetes mellitus: possiblecellular and molecular mechanisms. World Journal of Diabetes 2011;2(3):41–8.

[6] Teitelbaum SL. Bone resorption by osteoclasts. Science 2000;289(5484):1504–8.

[7] Mundy GR. Osteoporosis and inflammation. Nutrition Reviews 2007;65(12 (Pt2)):S147–51.

[8] Pfeilschifter J, Koditz R, Pfohl M, Schatz H. Changes in proinflammatorycytokine activity after menopause. Endocrine Reviews 2002;23(1):90–119.

[9] Khosla S. Minireview: the OPG/RANKL/RANK system. Endocrinology2001;142(12):5050–5.

[10] Ammann P, Rizzoli R, Bonjour JP, Bourrin S, Meyer JM, Vassalli P, et al.Transgenic mice expressing soluble tumor necrosis factor-receptor are pro-tected against bone loss caused by estrogen deficiency. Journal of ClinicalInvestigation 1997;99(7):1699–703.

[11] Cao JJ. Effects of obesity on bone metabolism. Journal of Orthopaedic Surgeryand Research 2011;6:30.

[12] Ai-Aql ZS, Alagl AS, Graves DT, Gerstenfeld LC, Einhorn TA. Molecular mecha-nisms controlling bone formation during fracture healing and distractionosteogenesis. Journal of Dental Research 2008;87(2):107–18.

[13] Kettle AJ, Winterbourn CC. Assays for the chlorination activity of myeloper-oxidase. Methods in Enzymology 1994;233:502–12.

[14] Trudeau DL, Freier EF. Determination of calcium in urine and serum by atomicabsorption spectrophotometry (AAS). Clinical Chemistry 1967;13(2):101–14.

[15] Sasov A, Van DD. Desktop X-ray microscopy and microtomography. Journal ofMicroscopy 1998;191(2):151–8.

[16] Howell PG, Boyde A. Volumes from which calcium and phosphorus X-raysarise in electron probe emission microanalysis of bone: Monte Carlo simula-tion. Calcified Tissue International 2003;72(6):745–9.

[17] Alblowi J, Kayal RA, Siqueira M, McKenzie E, Krothapalli N, McLean J, et al. Highlevels of tumor necrosis factor-alpha contribute to accelerated loss of cartilagein diabetic fracture healing. American Journal of Pathology 2009;175(4):1574–85.

[18] Maneewan B, Yamauchi K. Recovery of duodenal villi and cells in chickens refedprotein, carbohydrate and fat. British Poultry Science 2005;46(4):415–23.

[19] Yamauchi K, Isshiki Y. Scanning electron microscopic observations on theintestinal villi in growing White Leghorn and broiler chickens from 1 to 30days of age. British Poultry Science 1991;32(1):67–78.

[20] Tezuka H, Abe Y, Iwata M, Takeuchi H, Ishikawa H, Matsushita M, et al.Regulation of IgA production by naturally occurring TNF/iNOS-producingdendritic cells. Nature 2007;448(7156):929–33.

[21] Mostov KE. Transepithelial transport of immunoglobulins. Annual Review ofImmunology 1994;12:63–84.

[22] Matsunaga T, Rahman A. What brought the adaptive immune system tovertebrates? – The jaw hypothesis and the seahorse. Immunological Reviews1998;166:177–86.

[23] Suzuki K, Meek B, Doi Y, Muramatsu M, Chiba T, Honjo T, et al. Aberrantexpansion of segmented filamentous bacteria in IgA-deficient gut. Proceedings

of the National Academy of Sciences of the United States of America2004;101(7):1981–6.

[24] Brandtzaeg P, Baekkevold ES, Farstad IN, Jahnsen FL, Johansen FE, Nilsen EM,et al. Regional specialization in the mucosal immune system: what happens inthe microcompartments? Immunology Today 1999;20(3):141–51.

[25] van EM, Damen CA, van Spriel AB, Vidarsson G, van GE, van de Winkel JG. IgAand the IgA Fc receptor. Trends in Immunology 2001;22(4):205–11.

[26] Macpherson AJ, Uhr T. Induction of protective IgA by intestinal dendritic cellscarrying commensal bacteria. Science 2004;303(5664):1662–5.

[27] Kaminitz A, Askenasy EM, Yaniv I, Stein J, Askenasy N. Apoptosis of purifiedCD4+ T cell subsets is dominated by cytokine deprivation and absence of othercells in new onset diabetic NOD mice. PLoS One 2010;5(12):e15684.

[28] Chentoufi AA, Gaudreau S, Nguyen A, Sabha M, Amrani A, Elghazali G. Type Idiabetes-associated tolerogenic properties of interleukin-2. Clinical and De-velopmental Immunology 2011 2011;289343.

[29] Capeillere-Blandin C, Gausson V, Nguyen AT, scamps-Latscha B, Drueke T,Witko-Sarsat V. Respective role of uraemic toxins and myeloperoxidase in theuraemic state. Nephrology Dialysis Transplantation 2006;21(6):1555–63.

[30] Sathyanarayan G, Garg PK, Prasad H, Tandon RK. Elevated level of interleukin-6predicts organ failure and severe disease in patients with acute pancreatitis.Journal of Gastroenterology and Hepatology 2007;22(4):550–4.

[31] Gibson G. Active role of chondrocyte apoptosis in endochondral ossification.Microscopy Research and Technique 1998;43(2):191–204.

[32] Kayal RA, Siqueira M, Alblowi J, McLean J, Krothapalli N, Faibish D, et al. TNF-alpha mediates diabetes-enhanced chondrocyte apoptosis during fracturehealing and stimulates chondrocyte apoptosis through FOXO1. Journal ofBone and Mineral Research 2010;25(7):1604–15.

[33] Taylor PC. Anti-TNF therapy for rheumatoid arthritis and other inflammatorydiseases. Molecular Biotechnology 2001;19(2):153–68.

[34] Rokudai S, Fujita N, Kitahara O, Nakamura Y, Tsuruo T. Involvement of FKHR-dependent TRADD expression in chemotherapeutic drug-induced apoptosis.Molecular and Cellular Biology 2002;22(24):8695–708.

[35] Gilley J, Coffer PJ, Ham J. FOXO transcription factors directly activate bim geneexpression and promote apoptosis in sympathetic neurons. Journal of CellBiology 2003;162(4):613–22.

[36] Hajduch E, Litherland GJ, Hundal HS. Protein kinase B (PKB/Akt) – a keyregulator of glucose transport? FEBS Letters 2001;492(3):199–203.

[37] Srivastava AK, Pandey SK. Potential mechanism(s) involved in the regulation ofglycogen synthesis by insulin. Molecular and Cellular Biochemistry1998;182(1–2):135–41.

[38] Panjala SR, Jiang Y, Kern TS, Thomas SA, Steinle JJ. Increased tumor necrosisfactor-alpha, cleaved caspase 3 levels and insulin receptor substrate-1 phos-phorylation in the beta-adrenergic receptor knockout mouse. Molecular Vi-sion 2011;17:1822–8.

[39] Haegebarth A, Bie W, Yang R, Crawford SE, Vasioukhin V, Fuchs E, et al. Proteintyrosine kinase 6 negatively regulates growth and promotes enterocytedifferentiation in the small intestine. Molecular and Cellular Biology2006;26(13):4949–57.