Alexaner Gosslau,Emmanuel Zahariah,Shiming Li,Chi-Tang Ho

a Department of Science(Biology),City University of New York,BMCC,New York,NY 10007,United States

b Department of Chemistry and Chemical Biology,Rutgers University,Piscataway,NJ 08854-8087,United States

c Oncopath Genomics,Monmouth Junction,NJ 08852,United States

d Department of Food Science,Rutgers University,New Brunswick,NJ 08901-8520,United States

Keywords:

ABSTRACT

1.Introduction

The incidence of Type 2 diabetes(T2D)has dramatically increased in recent years and represents one of the most escalating global health concerns[1–3].Currently at 425 million worldwide,the International Diabetes Federation projects 629 million people will develop T2D by 2045 with a high proportion of the diabetic population still remaining undiagnosed(www.idf.org).Chronic inflammation is recognized as a key pathologic link between obesity and T2D in hypertrophic adipose tissue.Dysfunctional lipid metabolism gives rise to increased circulating free fatty acids(FFAs)leading to hyperlipidemia,lipid peroxidation and the accumulation of necrotic,apoptotic and autophagic adipocytes[3–7].These events of dyslipidemia lead to an infiltration of pro-inflammatory immune cells responsible for progression of T2D[2–5,8–10].Accumulation of free radicals released by immunocompetent cells,or derived from conditions of hyperglycemia and dyslipidemia,causes a disruption of the insulin signaling cascade.More reactive radicals formed by high glucose in a vicious cycle,expedite an impairment of the insulin receptor,causing a further disconnection of signaling pathways to glucose transporters,thus leading to chronic hyperglycemia and insulin resistance[3,4,11–13].A dramatic rise in T2D is expected with an aging population and epidemic obesity due to the connection with chronic inflammation[2,4,14].

Two different types macrophages contribute to inflammation in adipose tissue.Alternative M1 type macrophages predominate in lean adipose tissue and have mainly anti-inflammatory functions through IL-4,IL-10 and IL-13.M2 classical macrophages induce inflammation through secretion of pro-inflammatory cytokines and chemokines[3–5,9,14].The infiltration with M2 macrophages in adipose tissue derived from obese individuals is visible as crownlike structures[5,14].The release of TNF-α,IL-1β,IL-6 and ICAM-1 stimulate the inflammatory cascade in a positive feedback manner via NFκB and AP-1 signaling,thus generating free radicals such as reactive nitrogen species(RNS)and reactive oxygen species(ROS)[2,4,5,9,12–17].JNK and NFκB signaling lead to increased serine phosphorylation of insulin receptor substrates(e.g.,IRS1 and IRS2)which,in turn,inhibit the tyrosine kinase activity of the insulin receptor[18,19].An impairment of PI-3 K/AKT insulin signaling pathway causes a decrease of translocation and insertion of GLUT-4 leading to chronic hyperglycemia[20,21].The formation of advanced glycated endproducts(AGE)by reactive carbonyl species(RCS;e.g.,methylglyoxal)and glycated proteins(e.g.,HbA1c)will ultimately lead to cell,tissue and organ damage,causing nephropathy,cardiovascular disease,retinopathy,neuropathy or different cancers[3,9,11,12,16,22,23].

Therapies for diabetes mostly involve control of hyperglycemia or insulin resistance.Metformin is considered to be the first-line anti-hyperglycemic drug treatment for T2D,by mechanisms of increased cellular insulin sensitivity and suppression of hepatic glucose production[24,25].Anti-diabetic drugs often fail to control the inflammatory processes leading to T2D and may have side effects.Natural extracts effective against T2D by exerting modest effects at multiple steps in the complex inflammatory cascade with potentially lesser side effects may be advantageous for long-term therapy[7,26,27].Peels of Citrus genus contain high amounts of flavonoids which exhibit strong anti-inflammatory effects[17,28–36].Previously,we showed significant anti-inflammatory effects of a flavonoid-enriched orange peel extract(OPE)comparable with ibuprofen[17,36].In obese rodent models,positive effects of Citrus peel were observed against T2D.Lower levels of fasted blood glucose and improved glucose tolerance had been shown[37–39].In addition,anti-dyslipidemic effects of citrus peel extracts were demonstrated by a decrease in cholesterol,triglycerides and LDL[37–39].An inhibition of inflammatory pathways is indicated by increased levels of anti-inflammatory adiponectin and IL-10,whereas levels of pro-inflammatory markers IL-6,MCP-1,IFN-γ and TNF-α were decreased[39].In several of these T2D obese models,citrus peel extracts significantly reduced weight gain[37–40].

There is a growing body of evidence pointing to the benefits of natural anti-inflammatory extracts in T2D which led us to test the effects of OPE in ZDF rats.The ZDF rat model reflects the relationship between obesity,chronic inflammation and T2D.Here we show that lipophilic OPE showed significant effects against inflammation in adipose tissue,ambivalent effects against hyperglycemia,and hyperlipidemic effects.Our results suggest a potential therapeutic application of OPE affecting inflammatory and glycemic pathways.Furthermore,a combination with other antidiabetic citrus peel or natural extracts might be promising to address the complexity of different pathways leading to T2D.

2.Materials and methods

2.1.Materials and chemicals

For RNA isolation from whole blood and epididymal adipose tissue,Trizol reagent and RNeasyTMTotal RNA Kit or RNeasyTMLipid Mini Kit(Qiagen,Chatsworth,CA)was used.Oligo-dT,dNTPs,SuperscriptTMII reverse transcriptase were purchased from Invitrogen,Life Technologies(Grand Island,NY).TaqMan qPCR probes,primers and master mix were from Applied Biosystems,Life Technologies(Grand Island,NY).Other chemicals were purchased from Sigma(St.Louis,MO).

2.2.Preparation and analysis of the orange peel extract

Different batches of sweet orange peel extract(OPE,from coldpressed orange peel oil)were purchased previously from Florida Flavors Company(Lakeland,FL).The OPE mixture(10 g)was then dissolved in a mixture of methylene chloride(2 mL)and hexanes(2 mL)and loaded onto a 120 g pre-conditioned silica gel flash column.The isocratic solvent was 10%ethyl acetate and 90%hexanes and kept eluting in 30 min.Then,another isocratic solvent system with 85%of ethyl acetate and 15%of hexanes was introduced and kept eluting while collecting eluents as one fraction for another 30 min.At the end of the procedure,OPE(8 g)without essential oils was obtained.

2.2.1.Chemical characterization of the OPE extract

The HPLC was equipped with a reversedphaseC16amidecolumn(Ascentis RP-Amide,3 μm,150 mm×4.6 mm ID),from Supelco(Bellefonte,PA).Gradient elution was used with a mobile phase composed of water(solvent A)and acetonitrile(ACN,solvent B).The optimized condition is as follows:a 20 min gradient was started with 40%of B,linearly increased to 55%of B in 10 min,then linearly increased to 70% in 15 min,and finally ramped to 80% in 20 min.Flow rate was 1.0 mL/min and the column temperature was maintained at 35°C.Detection wavelength was 326 nm and the injection volume was 10 μL.The orange peel extract(OPE)is particularly enriched with polymethoxyflavones(PMFs)[36].

2.3.Zucker diabetic fatty(ZDF)rat model for T2D

Zucker diabetic fatty(ZDF)rats,based on a missense mutation in the leptin receptor gene[41]and control animals were studied at Charles River Laboratories(CRL).40 six week old male ZDF were used.At 10 weeks of age,fed glucose(via glucometer)was assessed and 40 animals with blood glucose levels≥250 mg/dL were selected along with 8 age matched lean controls.Rats were singly housed on a normal light cycle in the animal facility and received a control diet 5008(LabDiets,changed weekly)for the duration the study.All protocols were approved by the IACUC(Piedmont research center).

2.3.1.Treatment

For experiments,the 40 ZDF rats were randomized and combined with the lean control group into six different cohorts by HbA1c levels:group 1(Lean ZDF control treated with vehicle(0.5%HPMC+0.2%Tween));group 2(ZDF control treated with vehicle);group 3(ZDF treated with metformin(250 mg/kg));group 4(ZDF treated with ibuprofen(100 mg/kg);group 5(ZDF treated with OPE(75 mg/kg));group 6(ZDF treated with OPE(150 mg/kg)).The OPE concentrations were chosen based on earlier studies[17].Oral gavage doses were formulated weekly,and released from the pharmacy in daily aliquots for dosing.Animals were gavaged once daily for 30 days at 10 mL/kg.Rats were weighted twice a week and food intake was recorded weekly.Body mass index(BMI)was determined after animals were weighed using animal length(tip of the nose to the tip of the tail).Fed(at 8 h,prior to test article dosing)and 5 h fasted(at 13 h)blood glucose was checked at Baseline,Day 8,15,and 22,and 28.On study day 29 an oral glucose tolerance test was conducted on overnight fasted animals.Food was returned to all animals following the final time point.

2.3.2.Sampling

On study day 30,blood was collected at CRL.HbA1c was measured at 8 h on a drop of tail whole blood and then animals were fasted.At 11 h,animals were dosed per normal and euthanized at 13 h by CO2asphyxiation.Blood was withdrawn by cardiac puncture and 1 mL of whole blood placed into cryo vials for RNA extraction.Remaining blood was centrifuged(at 2200 x g for 10 min at 22°C)and serum(500μL)pipetted into 96 well plates on dry ice for analysis of the following:insulin,adiponectin,clinical chemistry panel with lipid parameters,and the inflammatory panel.Liver,epididymal fat,kidney,heart and spleen were collected and a representative piece was snap frozen.100 mg of tissue was transferred into 1.5 mL cold RNA later tubes and stored overnight at 4°C before being moved to-20°C.

Table 1 Effects of OPE on weight and food intake.

2.4.HbA1c analysis

Glycated hemoglobin(HbA1c)levels in venous whole blood were determined at CRL using the HbA1c Now test kit(Bayer,Whippany,NJ).HbA1c(mmol)was measured in treatment groups by immunoassay with anti-HbA1c using tail nick whole blood.

2.5.Oral glucose tolerance test

On study day 29 an oral glucose tolerance test was conducted on overnight fasted animals at CRL.All animals had been dosed per normal daily routine at 8 h.One hour later animals were gavaged with glucose at 2 g/kg.Whole blood glucose sampling occurred at the following times(min)relative to glucose dose:0,15,30,60,90 and 120 min and were determined using a veterinary glucometer(Alpha Trak,Abbott Laboratories,Abbott Park,IL).

2.6.Clinical chemistry analysis

Plasma analysis of insulin,adiponection,cytokines and a full clinical chemistry panel with lipid parameters was performed at CRL using commercially available ELISA and colorimetric kits.

2.7.TaqMan qPCR analysis

For gene expression analysis by TaqMan qPCR,we employed five inflammatory surrogate genes(COX-2,TNF-α,ICAM-1,IL-1β,and IL-6),previously selected and validated in cell-based,animal and clinical studies by whole genome Affymetrix and custom-made Oligo microarrays[17].RNA was isolated from whole blood samples with Trizol reagent,followed by chloroform and isopropanol extraction.Total RNA was precipitated using the RNeasyTM(Qiagen,Chatsworth,CA)for whole blood or RNeasyTMLipid Mini Kit for adipose tissue.Total RNA was reverse transcribed using standard protocols and reagents from Invitrogen,Life Technologies(Grand Island,NY).TaqMan qPCR was run on a Roche 480 Lightcycler(Roche Life Science,Indianapolis,IN)for 50 cycles with concentrations ranging from 0.01 to 100 ng for the standard curve.Gene expression of COX-2(Rn01483828_m1),TNF-α(Rn01525859_g1),ICAM-1(Rn00564227_m1),IL-1β(Rn00580432m1),IL-6(Rn01410330_m1),and GAPDH(Rn01775763_g1)were analyzed using probes,primers and master mix from Applied Biosystems(Life Technologies).After normalization to GAPDH,gene expression was expressed either as delta CT mean values+/-standard deviation or as delta-delta ct values to show the comparison with ZDF vehicle controls.

2.8.Statistics

Results were expressed as mean values+standard deviation for the different treatment groups(n=8 for each group).Statistical comparisons of data were performed using the student’s t-test.Significant differences from the control group with P＜0.05,0.01 or 0.001 were indicated as*,**,and***,respectively.

3.Results

3.1.Effects of OPE on body weight and food intake

Food intake,body weight and BMI were significantly lower in lean control rats compared to the ZDF control group(Table 1).All treatment groups showed a decline in body weight between day 25 and day 29 due to the overnight fasting on day 28.Chronic treatment with low and high concentrations of OPE did not cause any significant changes in body weight or BMI,respectively.Furthermore,the overall assessment by clinical chemistry(total proteins,albumin,globulin,gamma-glutamyl transpeptidase,creatinine,Ca,P,Na,K and Cl)and organ morphology revealed that treatment with low and high concentrations of OPE was well tolerated and did not cause any toxicity or damaging effects(data not shown).

Fig.1.Effects of OPE and metformin on glucose-related parameters.The effects of chronic treatment by OPE in low(l;magenta square)or high(h;brown circle)concentrations and metformin(MET;blue triangle)were compared with the ZDF vehicle control group(CONT;black square)and lean controls(LEAN;light blue rhombus).A)Animals were fasted for 5 h and blood glucose levels were analyzed on day 1,8,15,22 and 29 and expressed as mg/dL.B)Normal fed blood glucose levels were analyzed and expressed as mg/dL.C)On day 29 an oral glucose tolerance test(OGGT)was performed.All groups were treated with glucose(2 g/kg).After 15,30,60,90 and 120 min blood glucose levels were determined as mg/dL.D)Insulin levels were analyzed on day 30 and expressed as ng/mL.Mean values+standard deviation for the different treatment groups.*,**,and***indicate significant differences from the ZDF vehicle control group with P＜0.05,0.01 or 0.001,respectively.

3.2.Effects of OPE on glucose homeostasis

In the next set of experiments we analyzed the effects of OPE on glucose-related parameters.Fig.1A shows a significant decrease in fasted blood glucose levels after 8 days of metformin treatment in ZDF rats.High concentrations of OPE(150 mg/kg)also induced a significant decrease in fasted glucose on day 22(P＜0.05).The overnight fasting on day 28,however,induced a decline of blood glucose levels to around 250 mg/dL on day 29 in all treatment groups.Lean control animals did not show major changes in glucose levels throughout the study.Fed glucose levels showed significant higher levels on day 22 and 28 in response to metformin(Fig.1B).Also ZDF rats receiving high concentrations of OPE showed elevated levels of fed glucose starting on day 15(P＜0.01)with lesser significance on day 22 and 29 compared to ZDF rats(P＜0.05).

An oral glucose tolerance test(OGTT)was performed on day 29 of the study(Fig.1C).Metformin,as expected induced a significant decline in blood glucose in a time-dependent manner as compared to the ZDF vehicle control starting after 15 min(P＜0.01).High concentrations of OPE caused a significant increase in blood glucose 120 min after the glucose challenge(P＜0.01).In lean controls we did observe a similar pattern of glucose clearance of significant smaller amounts of blood glucose.

Analysis of plasma insulin levels on day 30 of the study revealed significant lower levels of insulin in lean controls as compared to ZDF rat treatment groups(Fig.1D).Metformin treated ZDF rats showed significantly(P＜0.05)higher levels of insulin release(1.54 ng/mL)when compared to ZDF controls.Also OPE induced an increase of insulin release although to a lesser degree as compared to metformin in a non-significant manner.We also quantified glycated hemoglobin(HbA1c)in our treatment groups on day 1 and day 30(data not shown).The lean control group showed significant lower levels of HbA1c on day 1 as well as after 30 days as compared to ZDF vehicle controls(P＜0.001).However,both concentrations of OPE but also metformin did not significantly affect HbA1c levels after 30 days of treatment as compared to the ZDF controls.

3.3.Effects of OPE on fat metabolisms

Adiponectin,a hormone produced and secreted exclusively by adipocytes has gained attention due to its anti-diabetic and antiinflammatory effects[6,14].In accordance,significantly higher adiponectin levels were determined in plasma derived from the lean control group(11.6 ug/mL)as compared to ZDF controls(6.7ug/mL).No major effects were observed in metformin and OPE treated animals after 30 days(Fig.2A).For triglycerides(TGs),a significant reduction(P＜0.001)in the levels were observed in the lean control animals(around 70 mg/dL)when compared to the ZDF control animals(around 430 mg/dL)but no major changes in response to metformin or OPE(Fig.2B).Free fatty acids(FFA)were reduced in plasma derived from lean control rats(P＜0.01)as compared to the ZDF rats(Fig.2C).OPE treatment induced a dosedependent significant increase in FFAs.No significant changes in FFAs were observed in metformin treated animals.Fasting cholesterol and HDL,but not LDL,were significantly reduced in the lean control group compared with ZDF control rats(Fig.2D–F).No significant differences in cholesterol,HDL and LDL were observed for metformin treated ZDF rats.However,ZDF rats treated with high concentrations of OPE showed significant higher levels of cholesterol and LDL(P＜0.01),whereas the slight increase of HDL was shown in the non-significant range.ent induced a dose-dependent significant increase in FFAs.No significant changes in FFAs were observed in metformin treated animals.Fasting cholesterol and HDL,but not LDL,were significantly reduced in the lean control group compared with ZDF control rats(Fig.2D–F).No significant differences in cholesterol,HDL and LDL were observed for metformin treated ZDF rats.However,ZDF rats treated with high concentrations of OPE showed significant higher levels of cholesterol and LDL(P＜0.01),whereas the slight increase of HDL was shown in the non-significant range.

Fig.2.Effects of OPE and metformin on levels of lipid related mediators.The effects of chronic treatment by OPE in low(l;magenta)or high(h;brown)concentrations and metformin(MET;blue)were compared with the ZDF vehicle control group(CONT;black)after 30 days.Lean controls(LEAN)are indicated in light blue.A)Levels of adiponectin were analyzed and expressed as ug/mL.B)Levels of triglycerides were analyzed and expressed as mg/dL.C)Levels of free fatty acids(FFA)were analyzed and expressed as mg/dL.D)Levels of cholesterol were analyzed and expressed as mg/dL.E)Levels of low density lipoproteins(LDL)were analyzed and expressed as mg/dL.F)Levels of high density lipoproteins(HDL)were analyzed and expressed as mg/dL.Mean values+standard deviation for the different treatment groups.*,**,and***indicate significant differences from the ZDF vehicle control group with P＜0.05,0.01 or 0.001,respectively.

3.4.Effects of OPE on inflammation in fat tissue and whole blood

Previous studies have suggested a link between systemic inflammation so we next analyzed the expression of inflammatory genes in blood and epididymal adipose.A panel of six cytokines in whole blood was quantified by ELISA analysis as a measure for systemic inflammation(Fig.3).IL-4,IL-10 and IL-13 are involved in the reduction of the inflammatory response through inhibition of NFκB signaling and cytokine release from macrophages.IL-6 and IFN-γ possess both pro-inflammatory and anti-inflammatory properties,whereas TNF-α is a pro-inflammatory cytokine[3,9,14].IL-4 was significantly increased in lean controls(P＜0.01)and Ibuprofen treated ZDF rats(P＜0.05)as compared to ZDF controls(Fig.3A).Treatment with OPE induced a dose-dependent increase of IL-4 in a significant manner(P＜0.05).For IL-6,no significant changes were observed throughout all groups compared to ZDF controls(Fig.3B).A similar pattern was observed for IL-10 where no significant changes were observed for lean animals and the different treatment groups compared to ZDF controls(Fig.3C).IL-13 levels increased in response to ibuprofen treatment in ZDF rats(P＜0.05),whereas the increase in the lean control group and the two OPE groups was non-significant(Fig.3D).For IFN-γ only marginal,non-significant increases were observed for the different groups.For TNF-α we did not observe a major regulation in lean controls and in response to ibuprofen.OPE treatment induced even higher levels of TNF-α although also in a non-significant manner(Fig.3F).

In the next set of experiments we quantified the expression of a subset of inflammatory genes in whole blood(Fig.4A)and epididymal fat tissue(Fig.4B):cyclooxygenase-2(COX-2),intracellular adhesion molecule-1(ICAM-1),interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)by TaqMan qPCR analysis.Gene expression levels were normalized to GAPDH(Delta ct values+/-SD indicated as¨I¨)or normalized to the ZDF controls(Delta delta ct values indicated as¨II¨).The expression of COX-2 in blood derived from lean control rats was similar to that of ZDF control animals(Fig.4A,I).The OPE group did not majorly affect COX-2 expression,whereas ibuprofen treatment induced a significant down-regulation of COX-2(P＜0.05),as demonstrated by higher delta ct values.Similarly,there were no differences for ICAM-1 between lean controls,ZDF animals treated with OPE and ZDF controls.However,ZDF rats treated with ibuprofen showed a drastic down-regulation of ICAM-1(P＜0.001).Also,IL-1β levels were significant reduced in response to ibuprofen as compared to ZDF controls(P＜0.05),whereas no significant changes were observed for OPE or lean controls.IL-6 was significantly up-regulated in lean control rat blood(P＜0.05)but not in OPE or ibuprofen treated animals.Finally,a down-regulation of TNF-α was observed in lean controls(P＜0.05)and to a higher degree by ibuprofen(P＜0.001).No major changes in TNF-α expression were observed in the OPE group.

The expression of these genes was then analyzed in epididymal fat tissue(Fig.4B).COX-2 expression was significantly down regulated in the ibuprofen treated animals(P＜0.05)but not in fat tissue from the lean control group.ZDF rats treated with OPE induced a significant up-regulation COX-2 to even higher levels(P＜0.01)compared to ibuprofen.For ICAM-1 a similar pattern of gene regulation was observed in whole blood(A)and epididymal fat tissue(B)of lean controls and ibuprofen treated animals.Ibuprofen induced a downregulation of ICAM-1 although to a lesser level(P＜0.05)as compared to whole blood(P＜0.001).In contrast to our whole blood analysis,OPE induced a prominent decrease in ICAM-1 expression to lower levels as compared to ibuprofen(P＜0.05).No differences throughout the treatment groups were observed for IL-1β expression in fat tissue.The regulation of IL-6 was different in fat tissue as compared to blood.Ibuprofen treatment significantly upregulated IL-6(P＜0.01),whereas no major changes were observed between lean controls,OPE-treated animals and the ZDF control group.As observed for whole blood,TNF-α was down-regulated in lean control animals as compared to the ZDF controls but to a larger degree(P＜0.001).OPE treated ZDF rats showed a down-regulation of TNF-α expression levels comparable to the ibuprofen group(both P＜0.05).

Fig.3.Effects of OPE and ibuprofen on levels of inflammatory cytokines.The effects of chronic treatment by OPE in low(l;magenta)or high(h;brown)concentrations,and ibuprofen(IBU;blue)were compared with the ZDF vehicle control group(CONT;black)after 30 days in whole blood by ELISA analysis.Lean controls(LEAN)are indicated in light blue.Levels of A)interleukin-4(IL-4),B)interleukin-6(IL-6),C)interleukin-10(IL-10),D)interleukin-13(IL-13),E)interferon-γ(IFN-γ)and F)tumor necrosis factor-α(TNF-α)were analyzed and expressed as pg/mL.Mean values+standard deviation for the different treatment groups.*,**,and***indicate significant differences from the ZDF vehicle control group with P＜0.05,0.01 or 0.001,respectively.

4.Discussion

In our study,we investigated the impact of a flavonoidsenriched orange peel extract(OPE)on the inflammatory response in obesity and pathogenesis using the ZDF model for T2D.Previously,we showed strong anti-inflammatory effects as validated in a human cell-based model for inflammation and in vivo mouse carrageenan-induced mouse paw edema model[36].ZDF rats have significantly higher body weight,BMI and food intake with higher plasma levels of glucose,as compared to lean control rats.In addition,ZDF animals have significantly higher HbA1c levels,triglycerides,free fatty acids,cholesterol,HDL,and slightly increased levels of LDL.In epididymal adipose tissue and plasma,the inflammatory cytokine TNF-α was upregulated,confirming a central role in the adipocyte inflammatory cascade[3,9,14,22].There have been controversial reports on the role of IL-6 signaling in obesity-related insulin resistance[3,7,13,14].Earlier studies showed elevated levels of IL-6 in blood of obese patients with T2D[42,43].This contrasts our observation of an upregulation of IL-6 in whole blood of lean controls indicating an anti-inflammatory role of IL-6 in the ZDF model.However,the increased adiponectin in lean controls is in accordance with studies in patients with T2D,showing a negative correlation of plasma adiponectin with visceral fat[6,14].It is well documented that a more favorable glycemic,lipidemic as well as inflammatory profile in a normal weight population can have a positive impact on human health.This is in accordance with studies showing that caloric restriction can induce an extension of life span in mammals[44].In several T2D studies,a reduction of weight gain by citrus peel extracts had been demonstrated[37–40].Although we did not observe a change of body weight or BMI,we noted an increase in levels of FFAs by OPE.The differential effects might be due to a different molecular composition of distinct citrus peel extracts.

Fig.4.Effects of OPE and ibuprofen on the expression of inflammatory genes.The effects of chronic treatment by OPE(brown)and IBU(blue)were compared with the ZDF vehicle control group(CONT;black)after 30 days in whole blood(A)or epididymal adipose tissue(B).Lean controls(LEAN)are indicated in light blue.After RNA isolation and reverse transcription,expression of either COX-2,ICAM-1,IL-1β,IL-6 and TNF-α was analyzed by TaqMan qPCR and expressed either according to the delta CT method(I)using GAPDH as internal control or the delta-delta CT method(II)compared to ZDF vehicle controls as described in¨Materials and Methods¨.Mean values+standard deviation for the different treatment groups.*,**,and***indicate significant differences from the ZDF vehicle control group with P＜0.05,0.01 or 0.001,respectively.

Metformin is a widely used T2D drug which significantly decreased fasted blood glucose levels after 8 days and improved glucose tolerance in our study.Anti-hyperglycemic effects of metformin correlated to a significant insulin release.Interestingly,some studies indicate that insulin may exert an anti-inflammatory response,independent of its effects on glycemia by mechanisms of NO release,and inhibition of NFκB signaling thus decreasing ICAM-1 and MCP-1 expression[45,46].Also OPE induced a significant attenuation of fasted blood glucose after 22 days and dose-dependent increase in insulin although in a non-significant manner.OPE also induced an increase of fed glucose although to a lesser degree as compared to metformin.However,OPE did not improve glucose tolerance and significant higher glucose levels were observed.Our study did not reveal effects against dyslipidemia in contrast to studies with other citrus peel extracts which showed a decrease in cholesterol,triglycerides and LDL[37–39,47].On the contrary,OPE treatment caused an increase of free fatty acids in a dose-responsive manner as well as elevated levels of cholesterol and LDL.The different effects on fat metabolism in our study might be based on a difference in the chemical composition of OPE as compared to other citrus peel extracts.Systemic inflammation is well accepted as causal in T2D development supported by clinical studies[3,4,9,10].The analysis of inflammatory mediators revealed a significant down-regulation of COX-2,ICAM-1,and TNF-α in epididymal adipose in OPE to a higher degree as compared to ibuprofen.The decrease of TNF-α is significant due to its central role in the adipocyte inflammatory cascade[3,9,14,22].ICAM-1 is a major adhesion molecule governing monocytes or other leukocytes to the site of inflammation[15].Therefore,the decrease of ICAM-1 by OPE is significant to lower chronic inflammation in adipose tissue.Studies on the impact of COX-2 on diabetes are limited and show evidence of COX-2 mediated inflammation[48–50].In our study,we did not find significant differences between COX-2 expression in lean and ZDF controls.However,the decline of COX-2 by OPE in adipose tissue,as well as by ibuprofen in whole blood and epididymal adipose tissue may indicate a contribution to T2D.IL-6 in obesity-related T2D symptoms has been reported to have pro-inflammatory and anti-inflammatory properties[3,5,7,14].The upregulation of IL-6 in lean controls in whole blood and ibuprofen in adipose tissue,suggest an anti-inflammatory function in ZDF rats.On the other hand,no major changes were observed in the OPE group in both tissues.In whole blood no inflammatory cytokines or mediators were down-regulated but IL-4 was upregulated in a dose-responsive manner.IL-4 plays an important role as anti-inflammatory mediators in the inflammatory cascade[4,5,7,9].

Our study showed strong anti-inflammatory effects of lipophilic OPE in adipose tissue.However,the effects against hyperglycemia revealed mixed results as indicated by a decrease of fasted blood glucose but a worsened glucose tolerance.OPE treatment also caused an increase of free fatty acids as well as elevated levels of cholesterol and LDL.Although considered as an initial assessment,our data suggest a potential therapeutic application of OPE to reduce inflammation in hypertrophic adipose tissue.A combination with citrus peel or other extracts with hypoglycemic and dyslipidemic effects might be a promising strategy against T2D in a population with epidemic obesity rates.Further studies are required to clarify the role of OPE in the management of type 2 diabetes in more predictable long-term and clinical settings.

Acknowledgements

This research was supported by the National Institute of Health,USA(R43/44 AT007889).