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Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inflammation
INTIMP-03895; No of Pages 6 International Immunopharmacology xxx (2015) xxx–xxx
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Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inflammation Mahmoud Abdel-Latif Zoology Department, Faculty of Science, Beni-Suef University, 62511 Salah Salem Street, Beni-Suef, Egypt
a r t i c l e
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Article history: Received 20 April 2015 Received in revised form 17 September 2015 Accepted 23 September 2015 Available online xxxx Keywords: Diethylcarbamazine citrate Insulin Adipose tissue Inflammation
a b s t r a c t Diethylcarbamazine citrate (DEC) had been known as anti-inflammatory drug but its effect on obesity-induced insulin resistance as a result of released inflammatory mediators from adipose tissue (AT) was not known. White male albino mice were fed with high fat diet (HFD) for 18 weeks to induce obesity. DEC at different three doses (12, 50 and 200 mg/kg) was orally administered twice a week starting at week 6. Body, liver and adipose tissue weights were taken, while glucose tolerance, insulin resistance, blood triglycerides and levels of adipokines (leptin, TNF-α, IL-6 and MCP-1) were tested. The activity of cyclooxygenase (COX) in the liver tissue homogenate was also tested. In addition, NF-κBp65 localization in liver cell cytoplasmic and nuclear fractions was detected using Western blotting. The only effective anti-inflammatory dose was 50 mg/kg to reduce (p b 0.05) the high levels of glucose, insulin and triglycerides in serum. DEC was not anti-obesity drug because the weights of body, liver and adipose tissues were not changed. Hyperleptinemia was decreased (p b 0.001) and associated with a reduction in serum levels of TNF-α, IL-6 and MCP-1 (p b 0.001). In addition, the activity of COX in DEC treatment decreased significantly (p b 0.01), while NF-κBp65 localization in nuclear extracts was obviously inhibited in 50 mg/kg treated group. It could be concluded that DEC was the only effective dose against mouse insulin resistance but not lipid accumulation. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Obesity is a growing public health problem and its prevalence has reached epidemic proportions in recent decades [1]. Obesity and associated metabolic syndrome are accompanied by heightened levels of proinflammatory mediators, not only systemically but also locally in metabolically critical tissues such as AT, liver and skeletal muscle [2]. A plethora of studies has shown that an increase in proinflammatory mediators such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and macrophage chemotactic protein (MCP)-1 and a decrease in released adiponectin were evident [3–5]. These dysregulations of adipokine secretory patterns from AT result in adipocyte lipolysis and hepatic steatosis, eventually leading to systemic leptin- and insulin resistance [6]. Major changes of immune cell populations – especially of macrophages – within adipose tissue play a key role in propagating obesity-induced adipose tissue inflammation [7,8]. Diethylcarbamazine (DEC) interferes with arachidonic acid (AA) metabolism for the clearance of microfilariae in Wuchereria bancrofti infected individuals. It inhibits prostaglandins for the clearance of blood microfilariae [9]. DEC has been known as an anti-inflammatory drug because of its success to reduce the experimentally induced inflammation in lung and liver [10,11]. These reduced inflammatory responses could
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be attributed to the inactivation of NF-κB and thus suppressing the induction of NF-κB-dependent genes. DEC has also been known as an inhibitor for both COX and lipoxygenase (LO) pathways [12]. The drug was found to reduce hepatic inflammation and injury through reduction of inflammatory mediators and elevation of IL-10 [13]. Because of the anti-inflammatory effects of DEC, it was hypothesized that the drug can prevent obesity-linked adipose tissue inflammation and associated insulin resistance. This study aimed to test the effect of DEC at different doses on the levels of released adipokines and insulin resistance. The results could indicate that 50 mg/kg dose was the most effective. 2. Materials and methods 2.1. Animals and experimental protocol Five-week-old male outbred Swiss mice (n = 60) at 10 g body weight were obtained from the National Institute of Ophthalmology, Giza, Egypt, housed five in a cage in a standard experimental animal laboratory and maintained under specific pathogen free conditions [14]. Animals were kept under controlled conditions of light (12 h light– dark cycle with lights on at 6 am) and temperature (24 ± 1 °C) without any stressful causes. All mice received water and food ad libitum. The mice were offered normal pellet diet (NPD) for a 1-week adaptation period and were then divided randomly into five groups consisting of 12
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Please cite this article as: M. Abdel-Latif, Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inf..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.021
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mice each. One group was offered NPD while the other 4 groups were offered high fat diet (HFD) containing 58% fat, 25% protein and 17% carbohydrate, as a percentage of total kcal [15]. Feeding on NPD or HFD continued for 18 weeks [16] where the body weight was monitored at two weeks intervals. After six weeks of dietary manipulation, the 4 HFD-fed groups were treated at gavage with 0, 12, 50 and 200 mg of DEC in normal saline/kg body weight twice a week for a period of 12 weeks. At the end of 18 weeks period, the animals were euthanized after blood collection by decapitation. Blood was collected from the orbital sinus into tubes, kept on ice for 10 min, centrifuged at 3000 ×g for 20 min, and serum samples were collected and stored at − 80 °C for subsequent analyses. Epididymal (gonadal), perirenal fat pads and liver were dissected out, weighed and directly stored at −80 °C for subsequent analyses. Liver tissue was homogenized in 0.1 M Tris–HCL, pH 7.8 containing 1 mM EDTA, centrifuged 3000 ×g for 20 min at 4 °C and supernatants were taken and stored at −80 °C. This study was approved by the Research Ethics Committee of Beni-Suef University. Experimental procedures are in accordance with Principles of Laboratory Animal Care formulated by the National Institutes of Health (National Institutes of Health Publication number 96-23, revised 1996).
2.5. Preparation of cytoplasmic and nuclear extracts Cytoplasmic and Nuclear extracts were prepared according to the previously described method [19]. Livers were homogenized in a Potter-Elvehjem homogenizer (Braun, Melsungen, Germany) with a loose-fitting Teflon pestle at 1000 rpm with eight up and-down strokes in ice-cold homogenization medium (250 mM sucrose, 5 mM MgCl2, 10 mM Tris–HCl, pH 7.4), called buffer A. After filtration, the homogenate was centrifuged at 600 ×g for 10 min at 4 °C in a Beckman TJ-6 centrifuge (Beckman Instruments; Munich, Germany). To prepare the cytoplasmic extract the obtained supernatant was centrifuged at 100.000 × g at 4 °C for 30 min in a Beckman L5-65B ultracentrifuge (swinging-bucket rotor). The pellet obtained by the first centrifugation was resuspended in buffer A and again centrifuged at 600 ×g at 4 °C for 10 min. This pellet of crude nuclei obtained after the second centrifugation was resuspended in 9 volumes of buffer B (2.2 M sucrose, 1 mM MgCl2, 10 mM Tris–HCl, pH 7.4), homogenized, and centrifuged at 70.000 ×g at 4 °C for 80 min in a Beckman L5-65B ultracentrifuge. The purified nuclei were resuspended in buffer A. 2.6. Western blotting analysis
2.2. Oral glucose tolerance test (OGTT) This was performed at the end of 18 weeks in the same set of mice. Following a 6-h period of feed deprivation, 5 μL of tail blood was used to measure the blood glucose levels using the One Touch Ultra glucometer. Next, 2 g/kg body weight of 20% D-glucose was injected orally. Serial blood glucose measures were taken at 30, 60, and 120 min after the injection.
2.3. Serum insulin, adipokines and triglycerides Commercially available ELISA kits were used to measure serum insulin (Raybiotech, Norcross, USA), leptin, TNF-α, IL-6 and MCP-1 (R & D systems, Minneapolis, USA). Homeostasis model assessment for insulin resistance (HOMA-IR) values was estimated as follows: HOMA-IR = [fasting insulin (μU/mL) × fasting glucose (mmol/L)]/22.5 [17]. The serum triglycerides were measured by an enzymatic colorimetric method using commercial kit (Spinreact, Girona, Spain). All assays were performed according to the manufacturers' protocols.
2.4. Cyclooxygenase activity assay The method had been performed in a clear plastic 96-well plate [18]. Briefly, stock solutions from N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD; Sigma) in dimethyl sulfoxide (DMSO) at 20 mM, porcine hematin (Sigma) in 1 M NaOH (40 mM) and AA (Sigma) in 98% ethanol (1 mM). The reaction mixture contained final concentrations of TMPD at 100 μM, hematin at 1 μM and AA at 100 μM in Tris/HCl buffer (100 mM, pH 8). To 0.2 ml of reaction mixture/well, 10 μL of liver tissue homogenate was added and absorbance was read at 590 nm after 5 min. The absorbance in the absence of AA was subtracted from the absorbance in the presence of AA and the net enzyme activity (expressed as the change in OD at 590 nm, ΔA590) is then calculated. One unit is defined as the amount of enzyme that will catalyze the oxidation of 1.0 nmol of TMPD per minute at 25 °C. COX activity in units was calculated as follows: COX activity = Δ A590 ÷ 10 min ÷ 0.00826\mu M−1 ÷ 0.01# mL × 1000 ÷ 2$ = nmol/min/mL (U/mL). Δ A590 is the difference in absorbance between wells containing complete mixture and control wells without AA. $ Two molecules of TMPD are required for the reduction of PGG2 to PGH2. # 0.01 mL is the enzyme sample volume.
The protein quantity of cytoplasmic and nuclear extracts was performed using Bicinchoninic acid kit (Sigma). Twenty micrograms protein for detection of NF-ĸBbp65 was separated in SDS-10% polyacrylamide gels and transferred to nitrocellulose membranes (0.45 μm; Heidelberg; Serva Electrophoresis GmbH, Germany) by electroblotting. Membranes were washed in PBS/Tween buffer (PBS containing 0.3% Tween-20) and incubated for 1 h at room temperature (RT) in blocking buffer containing 5% non-fat milk in PBS/Tween-20, followed by washing and incubation with the anti-mouse NF-κBp65 (1:1000; AbD Serotec, Puchheim, Germany) in the same buffer overnight at RT. The immunocomplexes were detected by using horseradish peroxidase-labeled rabbit anti-mouse antibody (1:5000; KPL, Maryland, USA). After 2 h of incubation at room RT, bands were developed by adding substrate (50 mg 3,3′-Diaminobenzidine tetrahydrochloride and 100 μL H2O2 in 100 ml PBS). The intensity of immunoreactive bands was quantified by densitometry using NIH-Image software (version 1.59; National Institutes of Health, Bethesda, MD). 2.7. Statistical analysis SPSS (version 20) statistical program (SPSS Inc., Chicago, IL) was used to carry out a one-way analysis of variance (ANOVA) on our data. When significant differences by ANOVA were detected, analysis of differences between the means of the treated and control groups were performed by using Dunnett's t test. 3. Results 3.1. Body, adipose tissues and liver weights The body weight in HFD was significantly higher (p b 0.001) than NPD while the treated group with 50 mg DEC/Kg did not show any significant changes in comparison to HFD (Fig. 1). Similarly, adipose tissues (epididymal and perirenal) and liver weights in HFD were significantly (p b 0.01 and p b 0.05, respectively) higher than NPD but no significant reduction appeared in treated group (Fig. 2). 3.2. Effect of DEC on serum glucose, triglycerides and insulin OGTT showed that the hyperlipidic diet promoted an increase at all time points when compared to control (HFD versus NFD). The AUC (area under the curve) analysis increased significantly (p b 0.01) in HFD compared with NFD (Fig. 3). The dose 50 mg/kg of DEC was only effective to reduce the glucose levels at all the time points. The AUC
Please cite this article as: M. Abdel-Latif, Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inf..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.021
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Fig. 1. Evolution of the average gain in body weight (gm) of mice for twelve weeks of feeding on HFD or NPD. The gavage with DEC (50 mg/kg) indicated no change in weight. Data are the mean ± SEM, n = 12. HFD versus NPD showed statistical significance (p b 0.001) at all time points.
analysis indicated a significant (p b 0.05) decrease after 50 mg/kg treatment (HFD + 50 mg versus HFD). Similarly, the same dose was the most effective to significantly (p b 0.05) reduce the level of triglycerides (Fig. 4). In consistence with the results of glucose and triglyceride levels, Insulin and HOMA-IR score were significantly (p b 0.05 and p b 0.01, respectively) higher in HFD compared to NFD (Fig. 5). The dose 50 mg/kg of DEC was the only effective for a significant reduction in both of insulin level and insulin resistance (p b 0.05 and p b 0.01, respectively). The treatment with DEC could reverse the increased glucose, triglycerides and insulin resistance.
3.3. Effect of DEC on serum leptin, proinflammatory cytokines and MCP-1 DEC at different three doses was tested in these assays. Serum leptin in the HFD group was significantly (p b 0.001) higher than that in NFD group. The 50 mg/kg group showed reduced (p b 0.001) leptin compared with that in the HFD group (Fig. 6a). Serum levels of TNF-α and IL-6 increased significantly (p b 0.001) in the HFD group compared to those in NFD group (Fig. 6b, c). However, treatment with DEC at the same dose significantly (p b 0.001) decreased these levels compared with those in the HFD group. Similarly, Serum MCP-1 levels in NFD and HFD groups were significantly (p b 0.001) different (Fig. 6d). Treatment with DEC tended to decrease MCP-1 levels significantly (p b 0.001) compared to those in the HFD group. Doses at 12 and 200 mg/kg could have anti-inflammatory effect but the reduction level was not statistically significant compared to HFD group.
Fig. 3. OGTT (a) and AUC (b) after 18 weeks of feeding on HFD or NPD. DEC administrations at 12, 50 and 200 mg/kg indicated the most effective dose at 50 mg/kg. The OGTT reductions in NPD or HFD + 50 mg/kg were significant (p b 0.05) at all-time points (0, 30, 60 and 120 min) compared to HFD. Data are the mean ± SEM, n = 12. a, p b 0.01; b, p b 0.05.
3.4. Effect of DEC on COX activity in liver tissue Using the colorimetric method, the COX activity in liver tissues of NPD, HFD and HFD treated with different three doses of DEC was determined (Fig. 7). COX activity in HFD was higher than NPD (p b 0.05) while its activity was strongly reduced after DEC treatment at dose 50 mg/kg compared to HFD (p b 0.01). Doses at 12 and 200 mg/kg could also reduce COX activity but their effects were not statistically significant. 3.5. Effect of DEC on translocated NF-κBp65 to the liver cell nucleus Using Western blotting (Fig. 8), NF-κBp65 was strongly translocated to nuclear fraction of HFD compared to NDP group. DEC at different three doses could inhibit this translocated or activated NF-κBp65, however DEC at dose 50 mg/kg had the most observable cytoplasmic localization. Quantitative detection for the immunoreactive band density revealed nuclear abundance in HFD compared to NPD (p b 0.001) and very low level in nuclear fraction of 50 mg/kg group compared to HFD (p b 0.001). 4. Discussion In this study, the beneficial effect of chronic intake of DEC at three different doses (12, 50 and 200 mg/kg) on the development of obesity
Fig. 2. Effect of DEC administrations at 50 mg/kg on the weight (gm) of adipose (perirenal &gonadal) and liver tissues in obese mice fed on HFD. No significant reduction (p N 0.05) in weights after treatment appeared while the weights in HFD versus NPD were significant. *(p b 0.05) and **(p b 0.01).
Fig. 4. Effect of DEC administrations at 0, 12, 50 and 200 mg/kg on the level of blood triglycerides (mg/dl). The dashed line represents the level of triglycerides in NPD. The most effective dose was 50 mg/kg (p b 0.05). Data are the mean ± SEM, n = 12.
Please cite this article as: M. Abdel-Latif, Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inf..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.021
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Fig. 7. Effect of DEC (50 mg/kg) on the activity of COX in liver tissue. Activity in HFD versus NPD (a, p b 0.05) or DEC versus HFD (b, p b 0.01) was statistically determined. Data are the mean ± SEM, n = 12.
Fig. 5. Effect of DEC administrations at 12, 50 and 200 mg/kg on the level of blood insulin (a) and calculated insulin resistance (b). Significant reductions in insulin and insulin resistance compared to HFD were observed only at 50 mg/kg. Data are the mean ± SEM, n = 12. In blood insulin level, a, p b 0.05; b, p b 0.05, while in insulin resistance, a, p b 0.01; b, p b 0.01.
and insulin resistance as well as the modulation of inflammatory markers in mice fed on high fat diet was investigated. Chronic consumption of high fat diet for 18 weeks induced obesity, hyperinsulinemia, and hyperglycemia [20]. Treatment with DEC started at week 6 and continued twice a week where 50 mg/kg was the most effective dose for reduction of hyperinsulinemia and hyperglycemia. This dose was also
the most effective for reduction of inflammatory markers released from inflammed adipose tissue. The same dose was formerly found to depress the induced inflammatory responses in liver and lung [10,13]. Thus, the anti-inflammatory action of DEC was limited to the application of 50 mg/kg. In this study, the later dose had been also effective against type 2 diabetes which occurs due to insulin resistance as a result of inflammatory reactions. The increased inflammatory responses and translocation of NF-κB to the nucleus interferes with the signaling pathways of insulin in the hepatocytes [21]. Nevertheless, 12 mg/kg was found preventive to experimentally induced pulmonary eosinophilic inflammation in BALB/c mice [22]. This kind of experimentally induced inflammation might be more sensitive to DEC treatment than the currently studied inflammation model. Thus, the current study was different in mouse strain, schedule of DEC administration, HFD-induced
Fig. 6. Effect of DEC (50 mg/kg) on the blood levels of leptin (a), TNF-α (b), IL-6 (c) and MCP-1 (d). Levels between NPD and HFD or HFD with DEC were compared. Data are the mean ± SEM, n = 12. a, p b 0.001; b, p b 0.001.
Please cite this article as: M. Abdel-Latif, Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inf..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.021
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Fig. 8. Immunolocalization of NF-kBp65 in cytoplasmic and nuclear fractions of liver cells in NPD (lane 1), HFD (lane 2), HFD +12 (lane 3), 50 (lane 4) and 200 mg/kg (lane 5) mice groups. Quantitative detection of NF-kBp65 was done using Western blotting (a) and density of immunoreactive bands (b). Densities between NPD and HFD or HFD with DEC were compared. Data are the mean ± SEM, n = 6. a, b, c and d represent the degree of statistical significance (p b 0.001).
obesity and kind of assayed cytokines. DEC dose recommended by world health organization in mass drug treatments of filariasis was single annual 6 mg/kg [23]. This was a successful dose for the disease removal and could be equivalent to 12 or 50 mg/kg doses in experimental animals. Although the application of 50 mg/kg for 12 days seemed enough for induction of a successful antiinflammatory effect [12], different treatment schedule with three different doses was applied in this study to test the possible effects against obesity-related insulin resistance. The mice were obese and the weights of adipose tissues and liver were comparable in weight to those fed on NPD. The antiinflammatory dose of DEC could not decrease the body, adipose tissues or liver weights. Although DEC could reduce leptin resistance which causes high adiposity and food intake [24], its effect on the weights of body, adipose tissues and liver was not significant. It can be suggested that DEC treatment used in this study was only reversed and not preventive as the treatment started at week 6 after the start of high fat diet feeding [25]. The reduced hyperleptinemia was related to the reduced adipose tissue inflammation which causes decreased leptin signaling in the brain hypothalamus [26]. It can be assumed that the decreased leptin resistance after DEC treatment was not enough to affect adiposity. Indeed, DEC effect on fatty acid oxidation should be clarified because DEC had been known as an inhibitor for AA metabolism or oxidation [12]. In the current study, DEC at different three doses could reduce the activity of COX in liver tissue homogenates. However, the dose 50 mg/kg was the most effective. The method used in this study to assay COX was found sensitive to the known COX inhibitors like indomethacin [27]. This evidence could emphasize the inhibitory action of the drug on AA metabolism in the liver cell. The oxidation of fatty acids plays a great role in inhibition of lipid accumulation and thus the insulin resistance [28]. DEC could reduce the glucose intolerance and insulin resistance but could not effectively inhibit the lipid accumulation. However, DEC was able to reduce the level of triglycerides in serum indicating an inhibition for lipid metabolism disorder [29]. The reduced blood triglycerides had been found also with the most preventive compounds against insulin resistance and glucose intolerance [30,31].
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Because of the central role of insulin signaling in suppressing lipolysis, insulin resistance in adipose tissue is associated with increased release of free fatty acids, which in turn fuels increased hepatic generation of triglycerides. The decreased TNF-α and IL-6 by 50 mg/kg DEC contributed to the potent anti-inflammatory action which was correlated with the reduced insulin resistance. TNF-α and IL-6 modulate insulin signaling, leading to impaired insulin action on insulin receptor substrate-1 -associated phosphatidylinositol 3-kinase and translocation of glucose transporter subtype 4 [32]. MCP-1 plays an important role in macrophage infiltration into adipose tissue, which then induces inflammation and insulin resistance [5]. The reduced level of serum MCP-1 by DEC could largely contribute to the anti-inflammatory action a result of reduced macrophage infiltration. Furthermore, inhibition of NF-κBp65 translocation into the nucleus was obviously observed at dose 50 mg/kg. However, other doses could show also inhibitive effect. This finding was also observed in previous studies on DEC antiinflammatory action [10,11]. This confirms the inhibitive action of DEC on intracellular signaling pathways which interfere with insulin-receptor signaling. The ability of DEC to reduce murine lung inflammation was found largely dependent on 5-lipooxygenase inhibition and leukotriene products [12,33]. Furthermore, leukotrienes have been found very essential as inflammatory mediators in mediating HFD induced insulin resistance [34]. Adipocytes in obesity were found as the major source of leukotrienes [35]. Hence, leukotriene inhibitors could reduce the levels of TNF-α, IL-6 and MCP-1. The inhibition of leukotriene production was linked to the reduced COX activity since DEC has been known as an inhibitor for AA metabolism which leads to both 5-LO and COX pathways [33]. DEC at higher dose (200 mg/kg) could not show a dose dependent effect on inflammatory responses in comparison to 50 mg/kg. It could be explained as the higher dose was able to activate phagocytes (neutrophils and macrophages). This effect was similarly observed on increased respiratory burst by higher dose (500 mg/kg) in comparison to a lower dose (50 mg/kg) in BALB/c mice [36]. Hence, the increased activation of phagocytic cells at higher doses could play a role against the mode of action of DEC as anti-inflammatory drug and thus as inhibitor of insulin resistance. Indeed, lower and higher doses of DEC should be tested as inhibitors for leukotrienes release. In conclusion, DEC at a dose of 50 mg/kg could have a protective effect against insulin resistance in mice fed high fat diet. This alleviation of insulin resistance could occur by reducing serum proinflammatory cytokine levels and hence inhibition of activated cellular signaling represented by translocated NF-κBp65 into nucleus. Conflicts of interest There are no conflicts of interest. Acknowledgments This work was supported by the Advanced Science Research Institute (ASRI) of Beni-Suef University, Beni-Suef city, Egypt. The authors had no commercial interest in the study. References [1] Y. Wang, M.A. Beydoun, L. Liang, B. Caballero, S.K. Kumanyika, Will all Americans become overweight or obese? Estimating the progression and cost of the US obesity epidemic, Obesity 16 (2008) 2323–2330. [2] J.M. Olefsky, C.K. Glass, Macrophages, inflammation, and insulin resistance, Annu. Rev. Physiol. 72 (2010) 219–246. [3] G.S. Hotamisligil, N.S. Shargill, B.M. Spiegelman, Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance, Science 259 (1993) 87–91. [4] J.P. Bastard, C. Jardel, E. Bruckert, P. Blondy, J. Capeau, M. Laville, Elevated levels of interleukin 6 are reduced in serum and subcutaneous adipose tissue of obese women after weight loss, J. Clin. Endocrinol. Metab. 85 (2000) 3338–3342.
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Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inflammation Mahmoud Abdel-Latif Zoology Department, Faculty of Science, Beni-Suef University, 62511 Salah Salem Street, Beni-Suef, Egypt
a r t i c l e
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Article history: Received 20 April 2015 Received in revised form 17 September 2015 Accepted 23 September 2015 Available online xxxx Keywords: Diethylcarbamazine citrate Insulin Adipose tissue Inflammation
a b s t r a c t Diethylcarbamazine citrate (DEC) had been known as anti-inflammatory drug but its effect on obesity-induced insulin resistance as a result of released inflammatory mediators from adipose tissue (AT) was not known. White male albino mice were fed with high fat diet (HFD) for 18 weeks to induce obesity. DEC at different three doses (12, 50 and 200 mg/kg) was orally administered twice a week starting at week 6. Body, liver and adipose tissue weights were taken, while glucose tolerance, insulin resistance, blood triglycerides and levels of adipokines (leptin, TNF-α, IL-6 and MCP-1) were tested. The activity of cyclooxygenase (COX) in the liver tissue homogenate was also tested. In addition, NF-κBp65 localization in liver cell cytoplasmic and nuclear fractions was detected using Western blotting. The only effective anti-inflammatory dose was 50 mg/kg to reduce (p b 0.05) the high levels of glucose, insulin and triglycerides in serum. DEC was not anti-obesity drug because the weights of body, liver and adipose tissues were not changed. Hyperleptinemia was decreased (p b 0.001) and associated with a reduction in serum levels of TNF-α, IL-6 and MCP-1 (p b 0.001). In addition, the activity of COX in DEC treatment decreased significantly (p b 0.01), while NF-κBp65 localization in nuclear extracts was obviously inhibited in 50 mg/kg treated group. It could be concluded that DEC was the only effective dose against mouse insulin resistance but not lipid accumulation. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Obesity is a growing public health problem and its prevalence has reached epidemic proportions in recent decades [1]. Obesity and associated metabolic syndrome are accompanied by heightened levels of proinflammatory mediators, not only systemically but also locally in metabolically critical tissues such as AT, liver and skeletal muscle [2]. A plethora of studies has shown that an increase in proinflammatory mediators such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and macrophage chemotactic protein (MCP)-1 and a decrease in released adiponectin were evident [3–5]. These dysregulations of adipokine secretory patterns from AT result in adipocyte lipolysis and hepatic steatosis, eventually leading to systemic leptin- and insulin resistance [6]. Major changes of immune cell populations – especially of macrophages – within adipose tissue play a key role in propagating obesity-induced adipose tissue inflammation [7,8]. Diethylcarbamazine (DEC) interferes with arachidonic acid (AA) metabolism for the clearance of microfilariae in Wuchereria bancrofti infected individuals. It inhibits prostaglandins for the clearance of blood microfilariae [9]. DEC has been known as an anti-inflammatory drug because of its success to reduce the experimentally induced inflammation in lung and liver [10,11]. These reduced inflammatory responses could
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be attributed to the inactivation of NF-κB and thus suppressing the induction of NF-κB-dependent genes. DEC has also been known as an inhibitor for both COX and lipoxygenase (LO) pathways [12]. The drug was found to reduce hepatic inflammation and injury through reduction of inflammatory mediators and elevation of IL-10 [13]. Because of the anti-inflammatory effects of DEC, it was hypothesized that the drug can prevent obesity-linked adipose tissue inflammation and associated insulin resistance. This study aimed to test the effect of DEC at different doses on the levels of released adipokines and insulin resistance. The results could indicate that 50 mg/kg dose was the most effective. 2. Materials and methods 2.1. Animals and experimental protocol Five-week-old male outbred Swiss mice (n = 60) at 10 g body weight were obtained from the National Institute of Ophthalmology, Giza, Egypt, housed five in a cage in a standard experimental animal laboratory and maintained under specific pathogen free conditions [14]. Animals were kept under controlled conditions of light (12 h light– dark cycle with lights on at 6 am) and temperature (24 ± 1 °C) without any stressful causes. All mice received water and food ad libitum. The mice were offered normal pellet diet (NPD) for a 1-week adaptation period and were then divided randomly into five groups consisting of 12
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Please cite this article as: M. Abdel-Latif, Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inf..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.021
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mice each. One group was offered NPD while the other 4 groups were offered high fat diet (HFD) containing 58% fat, 25% protein and 17% carbohydrate, as a percentage of total kcal [15]. Feeding on NPD or HFD continued for 18 weeks [16] where the body weight was monitored at two weeks intervals. After six weeks of dietary manipulation, the 4 HFD-fed groups were treated at gavage with 0, 12, 50 and 200 mg of DEC in normal saline/kg body weight twice a week for a period of 12 weeks. At the end of 18 weeks period, the animals were euthanized after blood collection by decapitation. Blood was collected from the orbital sinus into tubes, kept on ice for 10 min, centrifuged at 3000 ×g for 20 min, and serum samples were collected and stored at − 80 °C for subsequent analyses. Epididymal (gonadal), perirenal fat pads and liver were dissected out, weighed and directly stored at −80 °C for subsequent analyses. Liver tissue was homogenized in 0.1 M Tris–HCL, pH 7.8 containing 1 mM EDTA, centrifuged 3000 ×g for 20 min at 4 °C and supernatants were taken and stored at −80 °C. This study was approved by the Research Ethics Committee of Beni-Suef University. Experimental procedures are in accordance with Principles of Laboratory Animal Care formulated by the National Institutes of Health (National Institutes of Health Publication number 96-23, revised 1996).
2.5. Preparation of cytoplasmic and nuclear extracts Cytoplasmic and Nuclear extracts were prepared according to the previously described method [19]. Livers were homogenized in a Potter-Elvehjem homogenizer (Braun, Melsungen, Germany) with a loose-fitting Teflon pestle at 1000 rpm with eight up and-down strokes in ice-cold homogenization medium (250 mM sucrose, 5 mM MgCl2, 10 mM Tris–HCl, pH 7.4), called buffer A. After filtration, the homogenate was centrifuged at 600 ×g for 10 min at 4 °C in a Beckman TJ-6 centrifuge (Beckman Instruments; Munich, Germany). To prepare the cytoplasmic extract the obtained supernatant was centrifuged at 100.000 × g at 4 °C for 30 min in a Beckman L5-65B ultracentrifuge (swinging-bucket rotor). The pellet obtained by the first centrifugation was resuspended in buffer A and again centrifuged at 600 ×g at 4 °C for 10 min. This pellet of crude nuclei obtained after the second centrifugation was resuspended in 9 volumes of buffer B (2.2 M sucrose, 1 mM MgCl2, 10 mM Tris–HCl, pH 7.4), homogenized, and centrifuged at 70.000 ×g at 4 °C for 80 min in a Beckman L5-65B ultracentrifuge. The purified nuclei were resuspended in buffer A. 2.6. Western blotting analysis
2.2. Oral glucose tolerance test (OGTT) This was performed at the end of 18 weeks in the same set of mice. Following a 6-h period of feed deprivation, 5 μL of tail blood was used to measure the blood glucose levels using the One Touch Ultra glucometer. Next, 2 g/kg body weight of 20% D-glucose was injected orally. Serial blood glucose measures were taken at 30, 60, and 120 min after the injection.
2.3. Serum insulin, adipokines and triglycerides Commercially available ELISA kits were used to measure serum insulin (Raybiotech, Norcross, USA), leptin, TNF-α, IL-6 and MCP-1 (R & D systems, Minneapolis, USA). Homeostasis model assessment for insulin resistance (HOMA-IR) values was estimated as follows: HOMA-IR = [fasting insulin (μU/mL) × fasting glucose (mmol/L)]/22.5 [17]. The serum triglycerides were measured by an enzymatic colorimetric method using commercial kit (Spinreact, Girona, Spain). All assays were performed according to the manufacturers' protocols.
2.4. Cyclooxygenase activity assay The method had been performed in a clear plastic 96-well plate [18]. Briefly, stock solutions from N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD; Sigma) in dimethyl sulfoxide (DMSO) at 20 mM, porcine hematin (Sigma) in 1 M NaOH (40 mM) and AA (Sigma) in 98% ethanol (1 mM). The reaction mixture contained final concentrations of TMPD at 100 μM, hematin at 1 μM and AA at 100 μM in Tris/HCl buffer (100 mM, pH 8). To 0.2 ml of reaction mixture/well, 10 μL of liver tissue homogenate was added and absorbance was read at 590 nm after 5 min. The absorbance in the absence of AA was subtracted from the absorbance in the presence of AA and the net enzyme activity (expressed as the change in OD at 590 nm, ΔA590) is then calculated. One unit is defined as the amount of enzyme that will catalyze the oxidation of 1.0 nmol of TMPD per minute at 25 °C. COX activity in units was calculated as follows: COX activity = Δ A590 ÷ 10 min ÷ 0.00826\mu M−1 ÷ 0.01# mL × 1000 ÷ 2$ = nmol/min/mL (U/mL). Δ A590 is the difference in absorbance between wells containing complete mixture and control wells without AA. $ Two molecules of TMPD are required for the reduction of PGG2 to PGH2. # 0.01 mL is the enzyme sample volume.
The protein quantity of cytoplasmic and nuclear extracts was performed using Bicinchoninic acid kit (Sigma). Twenty micrograms protein for detection of NF-ĸBbp65 was separated in SDS-10% polyacrylamide gels and transferred to nitrocellulose membranes (0.45 μm; Heidelberg; Serva Electrophoresis GmbH, Germany) by electroblotting. Membranes were washed in PBS/Tween buffer (PBS containing 0.3% Tween-20) and incubated for 1 h at room temperature (RT) in blocking buffer containing 5% non-fat milk in PBS/Tween-20, followed by washing and incubation with the anti-mouse NF-κBp65 (1:1000; AbD Serotec, Puchheim, Germany) in the same buffer overnight at RT. The immunocomplexes were detected by using horseradish peroxidase-labeled rabbit anti-mouse antibody (1:5000; KPL, Maryland, USA). After 2 h of incubation at room RT, bands were developed by adding substrate (50 mg 3,3′-Diaminobenzidine tetrahydrochloride and 100 μL H2O2 in 100 ml PBS). The intensity of immunoreactive bands was quantified by densitometry using NIH-Image software (version 1.59; National Institutes of Health, Bethesda, MD). 2.7. Statistical analysis SPSS (version 20) statistical program (SPSS Inc., Chicago, IL) was used to carry out a one-way analysis of variance (ANOVA) on our data. When significant differences by ANOVA were detected, analysis of differences between the means of the treated and control groups were performed by using Dunnett's t test. 3. Results 3.1. Body, adipose tissues and liver weights The body weight in HFD was significantly higher (p b 0.001) than NPD while the treated group with 50 mg DEC/Kg did not show any significant changes in comparison to HFD (Fig. 1). Similarly, adipose tissues (epididymal and perirenal) and liver weights in HFD were significantly (p b 0.01 and p b 0.05, respectively) higher than NPD but no significant reduction appeared in treated group (Fig. 2). 3.2. Effect of DEC on serum glucose, triglycerides and insulin OGTT showed that the hyperlipidic diet promoted an increase at all time points when compared to control (HFD versus NFD). The AUC (area under the curve) analysis increased significantly (p b 0.01) in HFD compared with NFD (Fig. 3). The dose 50 mg/kg of DEC was only effective to reduce the glucose levels at all the time points. The AUC
Please cite this article as: M. Abdel-Latif, Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inf..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.021
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Fig. 1. Evolution of the average gain in body weight (gm) of mice for twelve weeks of feeding on HFD or NPD. The gavage with DEC (50 mg/kg) indicated no change in weight. Data are the mean ± SEM, n = 12. HFD versus NPD showed statistical significance (p b 0.001) at all time points.
analysis indicated a significant (p b 0.05) decrease after 50 mg/kg treatment (HFD + 50 mg versus HFD). Similarly, the same dose was the most effective to significantly (p b 0.05) reduce the level of triglycerides (Fig. 4). In consistence with the results of glucose and triglyceride levels, Insulin and HOMA-IR score were significantly (p b 0.05 and p b 0.01, respectively) higher in HFD compared to NFD (Fig. 5). The dose 50 mg/kg of DEC was the only effective for a significant reduction in both of insulin level and insulin resistance (p b 0.05 and p b 0.01, respectively). The treatment with DEC could reverse the increased glucose, triglycerides and insulin resistance.
3.3. Effect of DEC on serum leptin, proinflammatory cytokines and MCP-1 DEC at different three doses was tested in these assays. Serum leptin in the HFD group was significantly (p b 0.001) higher than that in NFD group. The 50 mg/kg group showed reduced (p b 0.001) leptin compared with that in the HFD group (Fig. 6a). Serum levels of TNF-α and IL-6 increased significantly (p b 0.001) in the HFD group compared to those in NFD group (Fig. 6b, c). However, treatment with DEC at the same dose significantly (p b 0.001) decreased these levels compared with those in the HFD group. Similarly, Serum MCP-1 levels in NFD and HFD groups were significantly (p b 0.001) different (Fig. 6d). Treatment with DEC tended to decrease MCP-1 levels significantly (p b 0.001) compared to those in the HFD group. Doses at 12 and 200 mg/kg could have anti-inflammatory effect but the reduction level was not statistically significant compared to HFD group.
Fig. 3. OGTT (a) and AUC (b) after 18 weeks of feeding on HFD or NPD. DEC administrations at 12, 50 and 200 mg/kg indicated the most effective dose at 50 mg/kg. The OGTT reductions in NPD or HFD + 50 mg/kg were significant (p b 0.05) at all-time points (0, 30, 60 and 120 min) compared to HFD. Data are the mean ± SEM, n = 12. a, p b 0.01; b, p b 0.05.
3.4. Effect of DEC on COX activity in liver tissue Using the colorimetric method, the COX activity in liver tissues of NPD, HFD and HFD treated with different three doses of DEC was determined (Fig. 7). COX activity in HFD was higher than NPD (p b 0.05) while its activity was strongly reduced after DEC treatment at dose 50 mg/kg compared to HFD (p b 0.01). Doses at 12 and 200 mg/kg could also reduce COX activity but their effects were not statistically significant. 3.5. Effect of DEC on translocated NF-κBp65 to the liver cell nucleus Using Western blotting (Fig. 8), NF-κBp65 was strongly translocated to nuclear fraction of HFD compared to NDP group. DEC at different three doses could inhibit this translocated or activated NF-κBp65, however DEC at dose 50 mg/kg had the most observable cytoplasmic localization. Quantitative detection for the immunoreactive band density revealed nuclear abundance in HFD compared to NPD (p b 0.001) and very low level in nuclear fraction of 50 mg/kg group compared to HFD (p b 0.001). 4. Discussion In this study, the beneficial effect of chronic intake of DEC at three different doses (12, 50 and 200 mg/kg) on the development of obesity
Fig. 2. Effect of DEC administrations at 50 mg/kg on the weight (gm) of adipose (perirenal &gonadal) and liver tissues in obese mice fed on HFD. No significant reduction (p N 0.05) in weights after treatment appeared while the weights in HFD versus NPD were significant. *(p b 0.05) and **(p b 0.01).
Fig. 4. Effect of DEC administrations at 0, 12, 50 and 200 mg/kg on the level of blood triglycerides (mg/dl). The dashed line represents the level of triglycerides in NPD. The most effective dose was 50 mg/kg (p b 0.05). Data are the mean ± SEM, n = 12.
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Fig. 7. Effect of DEC (50 mg/kg) on the activity of COX in liver tissue. Activity in HFD versus NPD (a, p b 0.05) or DEC versus HFD (b, p b 0.01) was statistically determined. Data are the mean ± SEM, n = 12.
Fig. 5. Effect of DEC administrations at 12, 50 and 200 mg/kg on the level of blood insulin (a) and calculated insulin resistance (b). Significant reductions in insulin and insulin resistance compared to HFD were observed only at 50 mg/kg. Data are the mean ± SEM, n = 12. In blood insulin level, a, p b 0.05; b, p b 0.05, while in insulin resistance, a, p b 0.01; b, p b 0.01.
and insulin resistance as well as the modulation of inflammatory markers in mice fed on high fat diet was investigated. Chronic consumption of high fat diet for 18 weeks induced obesity, hyperinsulinemia, and hyperglycemia [20]. Treatment with DEC started at week 6 and continued twice a week where 50 mg/kg was the most effective dose for reduction of hyperinsulinemia and hyperglycemia. This dose was also
the most effective for reduction of inflammatory markers released from inflammed adipose tissue. The same dose was formerly found to depress the induced inflammatory responses in liver and lung [10,13]. Thus, the anti-inflammatory action of DEC was limited to the application of 50 mg/kg. In this study, the later dose had been also effective against type 2 diabetes which occurs due to insulin resistance as a result of inflammatory reactions. The increased inflammatory responses and translocation of NF-κB to the nucleus interferes with the signaling pathways of insulin in the hepatocytes [21]. Nevertheless, 12 mg/kg was found preventive to experimentally induced pulmonary eosinophilic inflammation in BALB/c mice [22]. This kind of experimentally induced inflammation might be more sensitive to DEC treatment than the currently studied inflammation model. Thus, the current study was different in mouse strain, schedule of DEC administration, HFD-induced
Fig. 6. Effect of DEC (50 mg/kg) on the blood levels of leptin (a), TNF-α (b), IL-6 (c) and MCP-1 (d). Levels between NPD and HFD or HFD with DEC were compared. Data are the mean ± SEM, n = 12. a, p b 0.001; b, p b 0.001.
Please cite this article as: M. Abdel-Latif, Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inf..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.021
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Fig. 8. Immunolocalization of NF-kBp65 in cytoplasmic and nuclear fractions of liver cells in NPD (lane 1), HFD (lane 2), HFD +12 (lane 3), 50 (lane 4) and 200 mg/kg (lane 5) mice groups. Quantitative detection of NF-kBp65 was done using Western blotting (a) and density of immunoreactive bands (b). Densities between NPD and HFD or HFD with DEC were compared. Data are the mean ± SEM, n = 6. a, b, c and d represent the degree of statistical significance (p b 0.001).
obesity and kind of assayed cytokines. DEC dose recommended by world health organization in mass drug treatments of filariasis was single annual 6 mg/kg [23]. This was a successful dose for the disease removal and could be equivalent to 12 or 50 mg/kg doses in experimental animals. Although the application of 50 mg/kg for 12 days seemed enough for induction of a successful antiinflammatory effect [12], different treatment schedule with three different doses was applied in this study to test the possible effects against obesity-related insulin resistance. The mice were obese and the weights of adipose tissues and liver were comparable in weight to those fed on NPD. The antiinflammatory dose of DEC could not decrease the body, adipose tissues or liver weights. Although DEC could reduce leptin resistance which causes high adiposity and food intake [24], its effect on the weights of body, adipose tissues and liver was not significant. It can be suggested that DEC treatment used in this study was only reversed and not preventive as the treatment started at week 6 after the start of high fat diet feeding [25]. The reduced hyperleptinemia was related to the reduced adipose tissue inflammation which causes decreased leptin signaling in the brain hypothalamus [26]. It can be assumed that the decreased leptin resistance after DEC treatment was not enough to affect adiposity. Indeed, DEC effect on fatty acid oxidation should be clarified because DEC had been known as an inhibitor for AA metabolism or oxidation [12]. In the current study, DEC at different three doses could reduce the activity of COX in liver tissue homogenates. However, the dose 50 mg/kg was the most effective. The method used in this study to assay COX was found sensitive to the known COX inhibitors like indomethacin [27]. This evidence could emphasize the inhibitory action of the drug on AA metabolism in the liver cell. The oxidation of fatty acids plays a great role in inhibition of lipid accumulation and thus the insulin resistance [28]. DEC could reduce the glucose intolerance and insulin resistance but could not effectively inhibit the lipid accumulation. However, DEC was able to reduce the level of triglycerides in serum indicating an inhibition for lipid metabolism disorder [29]. The reduced blood triglycerides had been found also with the most preventive compounds against insulin resistance and glucose intolerance [30,31].
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Because of the central role of insulin signaling in suppressing lipolysis, insulin resistance in adipose tissue is associated with increased release of free fatty acids, which in turn fuels increased hepatic generation of triglycerides. The decreased TNF-α and IL-6 by 50 mg/kg DEC contributed to the potent anti-inflammatory action which was correlated with the reduced insulin resistance. TNF-α and IL-6 modulate insulin signaling, leading to impaired insulin action on insulin receptor substrate-1 -associated phosphatidylinositol 3-kinase and translocation of glucose transporter subtype 4 [32]. MCP-1 plays an important role in macrophage infiltration into adipose tissue, which then induces inflammation and insulin resistance [5]. The reduced level of serum MCP-1 by DEC could largely contribute to the anti-inflammatory action a result of reduced macrophage infiltration. Furthermore, inhibition of NF-κBp65 translocation into the nucleus was obviously observed at dose 50 mg/kg. However, other doses could show also inhibitive effect. This finding was also observed in previous studies on DEC antiinflammatory action [10,11]. This confirms the inhibitive action of DEC on intracellular signaling pathways which interfere with insulin-receptor signaling. The ability of DEC to reduce murine lung inflammation was found largely dependent on 5-lipooxygenase inhibition and leukotriene products [12,33]. Furthermore, leukotrienes have been found very essential as inflammatory mediators in mediating HFD induced insulin resistance [34]. Adipocytes in obesity were found as the major source of leukotrienes [35]. Hence, leukotriene inhibitors could reduce the levels of TNF-α, IL-6 and MCP-1. The inhibition of leukotriene production was linked to the reduced COX activity since DEC has been known as an inhibitor for AA metabolism which leads to both 5-LO and COX pathways [33]. DEC at higher dose (200 mg/kg) could not show a dose dependent effect on inflammatory responses in comparison to 50 mg/kg. It could be explained as the higher dose was able to activate phagocytes (neutrophils and macrophages). This effect was similarly observed on increased respiratory burst by higher dose (500 mg/kg) in comparison to a lower dose (50 mg/kg) in BALB/c mice [36]. Hence, the increased activation of phagocytic cells at higher doses could play a role against the mode of action of DEC as anti-inflammatory drug and thus as inhibitor of insulin resistance. Indeed, lower and higher doses of DEC should be tested as inhibitors for leukotrienes release. In conclusion, DEC at a dose of 50 mg/kg could have a protective effect against insulin resistance in mice fed high fat diet. This alleviation of insulin resistance could occur by reducing serum proinflammatory cytokine levels and hence inhibition of activated cellular signaling represented by translocated NF-κBp65 into nucleus. Conflicts of interest There are no conflicts of interest. Acknowledgments This work was supported by the Advanced Science Research Institute (ASRI) of Beni-Suef University, Beni-Suef city, Egypt. The authors had no commercial interest in the study. References [1] Y. Wang, M.A. Beydoun, L. Liang, B. Caballero, S.K. Kumanyika, Will all Americans become overweight or obese? Estimating the progression and cost of the US obesity epidemic, Obesity 16 (2008) 2323–2330. [2] J.M. Olefsky, C.K. Glass, Macrophages, inflammation, and insulin resistance, Annu. Rev. Physiol. 72 (2010) 219–246. [3] G.S. Hotamisligil, N.S. Shargill, B.M. Spiegelman, Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance, Science 259 (1993) 87–91. [4] J.P. Bastard, C. Jardel, E. Bruckert, P. Blondy, J. Capeau, M. Laville, Elevated levels of interleukin 6 are reduced in serum and subcutaneous adipose tissue of obese women after weight loss, J. Clin. Endocrinol. Metab. 85 (2000) 3338–3342.
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Please cite this article as: M. Abdel-Latif, Diethylcarbamazine citrate ameliorates insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inf..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.021