Cucurbita ficifolia Bouché (Cucurbitaceae) andD-chiro-inositol modulate the redox state and inflammationin 3T3-L1 adipocytesÁngeles Fortis-Barreraa,b,c, Francisco Javier Alarcón-Aguilarb, Tania Banderas-Dorantesb,Margarita Díaz-Floresc, Rubén Román-Ramosb, Miguel Cruzc and Rebeca García-Macedoc
aPosgrade in Experimental Biology, Division of Health and Biological Sciences. Metropolitan Autonomous University of Iztapalapa, bDepartment ofHealth Sciences, Division of Health and Biological Sciences, Metropolitan Autonomous University of Iztapalapa, and cUnit of Medical Investigationin Biochemistry, Hospital of Specialties, CMNSXXI, Mexican Institute of Social Security, Doctores, Mexico DF, Mexico
CorrespondenceRebeca García-Macedo, Hospital ofSpecialties, CMNSXXI, Mexican Institute ofSocial Security, 330 S. Cuauhtémoc, Col.Doctores, Mexico DF 06720, México.E-mail: [email protected]
Received December 22, 2012Accepted June 18, 2013
doi: 10.1111/jphp.12119
Abstract
Objectives Cucurbita ficifolia (characterised by its D chiro inositol (DCI)content) and of synthetic DCI on the redox state, mRNA expression and secre-tions of proinflammatory cytokines. Additionally, we evaluated the insulin-mimetic action of both treatments by assessing protein kinase B (PKB) activationin 3T3-L1 adipocytes.Methods Adipocytes were treated with C. ficifolia and synthetic DCI. Theredox state was determined by spectrophotometry as changes in the reducedglutathione/oxidised glutathione (GSH/GSSG) ratio, glutathione peroxidase andglutathione reductase activities; H2O2 levels were measured by flow cytometry. ThemRNA expression and the protein level of cytokines were determinate by real-time reverse transcription polymerase chain reaction and enzyme-linked immu-nosorbent assay, respectively. The activation of PKB activation was detected byWestern blot.Key findings C. ficifolia extract and synthetic DCI reduced oxidative stress bydecreased H2O2 levels, increased glutathione peroxidase activity and changes inthe GSH/GSSG ratio. Furthermore, DCI decreased the mRNA expression andsecretion of tumour necrosis factor-α, interleukin 6 (IL-6) and resistin, whileC. ficifolia reduced protein levels of resistin and increased IL-6 levels. Only DCIdemonstrated insulin-mimetic action.Conclusions The antioxidant and anti-inflammatory effects of C. ficifolia extractcan be explained in part by its DCI content, which modulates the GSH/GSSG ratioand contributes to a reduced proinflammatory state. C. ficifolia and DCI treat-ments may reduce the disturbances caused by oxidative stress. Additionally, DCImay improve insulin sensitivity through its insulin-mimetic effects.
Introduction
In Mexico, traditional medicine uses different plants for thetreatment of type 2 diabetes (T2D), including Cucurbitaficifolia Bouché (Cucurbitaceae). The fruit juice preparedfrom this plant has a hypoglycaemic effect in animals withexperimental diabetes[1,2] and in T2D patients.[3] We recentlydemonstrated that the aqueous C. ficifolia extract has anti-oxidant and anti-inflammatory effects, in addition to aglucose-lowering effect, in streptozotocin-induced diabeticmice.[4,5] Apparently, the hypoglycaemic effect is due to theextract’s content of D-chiro-inositol (DCI),[6] which is part
of the structure of inositol phosphoglycans. These com-pounds are mediators of the action of insulin[7] and are acti-vators of the glycogen synthase and pyruvate dehydrogenaseenzymes.[8] Diabetic db/db mice, diabetic Rhesus monkeysand T2D patients showed a linear correlation betweendecreased urinary DCI excretion and the degree of insulinresistance.[7–9] Previously, it was reported that DCI decreasesthe level of oxidative stress in endothelial cells,[10] and DCIseems to prevent the inflammatory neuronal damagesobserved in rats treated with streptozotocin.[11]
Oxidative stress and a proinflammatory state are involvedin the development of obesity, T2D and their vascularcomplications.[12–14] In this context, an increase in the levelof reactive oxygen species modifies the redox state ofadipose tissue because of the decrease in the activity ofantioxidant enzymes, such as glutathione peroxidase(GPX).[12,15] In the glutathione redox cycle, the function ofGPX is to scavenge and inactivate hydrogen peroxide(H2O2) with water formation and inactivate lipid peroxideswith lipid hydroxyls formation, through the oxidation ofreduced glutathione (GSH) to produce oxidised glutathione(GSSG). GSSG is converted into GSH by glutathione reduc-tase (GR).[12] GSH is a prominent intracellular antioxidantthat regulates physiological homeostasis and participates inthe maintenance of the intracellular redox state,[16] whichcan be determined by the GSH/GSSG ratio.[5] Alterations inthe redox state of adipose tissue activate inflammatorypathways involving nuclear factor κB (NF-κB), which issensitive to redox changes and regulates the transcriptionof proinflammatory cytokines such as tumour necrosisfactor-α (TNF-α), interleukin 6 (IL-6) and resistin.[16,17]
These cytokines and reactive oxygen species, such as H2O2,produce changes in the phosphorylation of proteinsinvolved in insulin signal transduction, for example, insulinreceptor substrate 1 and protein kinase B (PKB).[18–20] Thesechanges are the cause of insulin resistance in obese individ-uals and are important factors for the development ofT2D.[21]
The crucial role of the control of oxidative stress andinflammation in adipose tissue associated with obesity andT2D has driven efforts to develop new therapeutic alterna-tives to control these alterations. In this study, we investi-gated and compared the effects of the aqueous C. ficifoliaextract (used on the basis its DCI content) and syntheticDCI on parameters of oxidative stress and inflammation.Additionally, we assessed the activation of PKB in 3T3-L1adipocytes. Our results showed that DCI may be an impor-tant component that contributes to the antioxidant activityof the extract, suggesting that the use of C. ficifolia and syn-thetic DCI may prevent or delay the disturbances caused byoxidative stress. This research provides novel evidence ofthe pharmacological anti-inflammatory effects of DCI. Thiscompound may improve insulin sensitivity because of itsinsulin-mimetic effects; therefore, this compound may bebeneficial in the treatment of obesity and T2D.
Materials and Methods
Plant material and preparation of theaqueous C. ficifolia extract
The botanical identification of C. ficifolia plant was per-formed by the Medicinal Plant Herbarium of the MexicanInstitute of Social Security (specimen voucher num. 11119)
in Mexico City. To prepare the extract, the endocarp ofmature fruit, free of seeds, was dried at room temperatureand ground using a 2-mm mesh in an electric mill (ThomasWiley Laboratory Mill Model 4, Ramsey, MN, USA). Thismaterial (400 g) was macerated in water for 72 h and every24 h; the aqueous phase was removed and freeze-dried. Theyield was 35% (w/v). The product was dissolved in culturemedium and filtered (0.22 μm) immediately before its use.
Quantitative analysis of the D-chiro-inositolcontent in the aqueous C. ficifolia extract
The DCI content in the aqueous C. ficifolia extract wasdetermined by high-performance liquid chromatography(HPLC) with a Waters 2697 separation module, a refractiveindex detector (Waters, Milford, MA, USA) and aLiChrospher NH2 column (5 μm, 4 × 250 mm, 100 A°). Theelution system was an isocratic mixture of acetonitrile andmethanol (8 : 2). Aliquots of 20 μl of the DCI (Sigma-Aldrich, St Louis, MO, USA) standard (1, 3 and 6 mg/ml)and the extract (2 mg/ml (w/v)) were injected into the sameHPLC system.
Cell culture
3T3-L1 murine fibroblasts were grown in Dulbecco’s modi-fied Eagle’s medium (DMEM, Gibco, Grand Island, NY,USA) supplemented with 25 mm glucose, 1 mm pyruvate,2 mm glutamine, 0.01 mm nonessential amino acids,20 μg/ml gentamicin and 10% fetal bovine serum (FBS), at37°C in an atmosphere of 5% CO2. The growth mediumwas changed every 2 days until confluence was reached. At 2days postconfluence (day 0), the fibroblasts were differenti-ated into adipocytes with methylisobutylxanthine (500 μm),dexamethasone (0.5 μm) and insulin (1.2 μm) in DMEMplus 10% FBS. Two days later, the medium was replacedonly with 1.2 μm insulin for 2 more days. The adipocyteswere subjected to different treatments on day 8 of differen-tiation. On this day, over 95% of the cells expressed a char-acteristic adipocyte phenotype.
Cell viability
Adipocytes in medium DMEM with 25 mm glucose wereincubated for 24 and 48 h with extract at different concen-trations, based on its content of DCI (0.125, 0.25, 0.50, 0.75and 1 mm), in the following assays the concentration of theextract refers to this compound. In another set of experi-ments, synthetic DCI was added at the same concentrations.The controls (untreated cells) were exposed in DMEM with5 and 25 mm glucose for 24 or 48 h. The cell viability wasquantified using the CellTiter 96 AQueous One SolutionCell Proliferation Assay kit (Promega, Madison, WI, USA).
Ángeles Fortis-Barrera et al.C. f. and DCI redox state, inflammation
Adipocytes in DMEM supplemented with 25 mm glucosewere treated for 24 h with C. ficifolia extract at differentconcentrations (0.0125, 0.025, 0.05, 0.125 and 0.25 mm) orwith synthetic DCI at the same concentrations. A positivecontrol treated with 20 mm N-acetyl-L-cysteine (NAC,Boehringer Mannheim, Ingelheim, Germany) was included.In other experiments, adipocytes were treated withC. ficifolia extract or with synthetic DCI at 0.125 mm for48 h. Controls were incubated with 5 and 25 mm glucose inDMEM for 24 or 48 h. At the end of the incubations, thecells were washed, and detached with 0.025 mm trypsin and0.5 mm ethylenediaminetetraacetic acid (EDTA), recoveredby centrifugation at 129g for 10 min at 4°C and suspendedin PBS. An aliquot of each of the samples was mixed with1-methyl-2-vinylpyridinium trifluoromethanesulfonate topreserve GSSG. Both peptides were quantified using theGSH/GSSG Ratio Assay Kit (Calbiochem, Darmstadt,Germany).
Measurement of intracellular hydrogenperoxide concentration
In the following experiments, adipocytes were treatedwith C. ficifolia extract or synthetic DCI at 0.125 mmin DMEM with 25 mm glucose for 24 and 48 h. Theadipocytes were recovered as mentioned above and sus-pended in PBS. The samples were incubated with 10 μm2′,7′-dichlorofluorescein diacetate for 30 min at 37°C andwere analysed using a FACScan (Becton Dickinson,Immunocytometry Systems, San Jose, CA, USA) to measurethe intracellular H2O2 concentration using laser excitationand emission wavelengths of 488 and 530 nm, respectively.
Glutathione peroxidase- and glutathionereductase-specific activity
After the treatments the adipocytes were homogenised incold buffer (50 mm Tris, pH 7.4, 5 mm EDTA and 1 mmdithiothreitol) and centrifuged at 10 000g for 15 min at4°C. The supernatant was used for the quantification ofthe enzymatic activity of GPX and GR by nicotinamideadenine dinucleotide phosphate (NADP+) production,using the methods of Lawrence and Burk[22] and Beutler,[23]
respectively. Proteins were quantified by the method ofBradford.[24]
RNA extraction and quantitativereal-time reverse transcription polymerasechain reaction
Total RNA was isolated with TriPure isolation reagent(Roche, Indianapolis, IN, USA). Single-strand complemen-
tary cDNA was synthesised with the ImProm II ReverseTranscription System Kit (Promega) following the manufa-cturer’s instructions. The cDNA was amplified with SYBRGreen Master Mix (Roche) and using the following primers:IL-6 (F5′-TTCCATCCAGTTGCCTTCTT-3′ R5′ CAGAATTGCCATTGCACAAC-3′), TNF-α (F5′-CCTCCCTGTCATCAGTTCTA-3′ R5′-ACTTGGTGGTTTGCTACGAC-3′),resistin (F5′-GTACCCACGGGATGAAGAACCG-3′ R5′-GCAGAGCCACAGGAGCAG-3′), adiponectin (F5′-AAGGACAAGGCCGTTCTCT-3′ R5′-TATGGGTAGTTGCAGTCAGTTGG-3′) and housekeeping gene 36B4 (F5′-AAGCGCGTCCTGGCATTGTCT-3′ R5′-CCGCAGGGGCAGCAGTGGT-3′). The genes were amplified and measured in aLightCycler 2.0 Real-Time PCR detection system (RocheMolecular Biochemicals, Indianapolis, IN, USA), the rela-tive changes in the expression levels of mRNA were calcu-lated with the formula 2−ΔΔCt.
Quantification of adipokines byenzyme-linked immunosorbent assay
The adipocytes were treated for 24 h with different concen-trations of C. ficifolia extract or of synthetic DCI, or with5 μm rosiglitazone as a positive control. In another set ofexperiments, treatments were administered at 0.125 mm for48 h. After the incubation period, the culture medium wasrecovered, and the adipokine levels were measured usingenzyme-linked immunosorbent assay kits for TNF-α(Abcam, Cambridge, MA, USA), adiponectin (R & DSystems, Minneapolis, MN, USA), IL-6 and resistin(Cusabio, Hubei Province, China).
Protein kinase B activation and Westernblotting analyses
The adipocytes were treated with C. ficifolia extract or syn-thetic DCI at 0.125 mm for 24 h in DMEM with 25 mmglucose. After the incubation period, the cells were washedwith Krebs–Ringer phosphate (KRP) buffer plus 0.5% fattyacid-free albumin. Then, the adipocytes were incubated inKRP containing 10 mm glucose and 1 μm insulin for15 min. In other experiments, the adipocytes were incu-bated with these two treatments in KRP for 30 min, withoutinsulin stimulation.
At the end of the incubations, the adipocytes were lysedwith lysis buffer.[25] After centrifugation at 15 000g for20 min at 4°C, the aqueous phase was recovered. The pro-teins were quantified by the method of Bradford[24] andwere analysed by Western blotting after 10% sodiumdodecyl sulphate-polyacrilamide gel electrophoresis andtransfer to a polyvinylidene difluoride membrane. Themembrane was blocked with 5% albumin for 1 h and incu-bated overnight at 4°C with mouse primary anti-PKB,anti-phospho-PKB (ser-473) and anti-β-actin antibodies
Ángeles Fortis-Barrera et al. C. f. and DCI redox state, inflammation
coupled with peroxidase (Santa Cruz Biotechnology, Inc.,Santa Cruz, CA, USA). After being washed, the membraneswere incubated for 2 h with a secondary antirabbit antibody(Santa Cruz Biotechnology, Inc.), and the proteins weredetected by enhanced chemiluminescence (ECL Amersham,Buckinghamshire, UK).
Statistical analysis
The differences between the groups were determined bymeans of Student’s t-test and by analysis of variance test,supplemented by a Tukey–Kramer test. Statistical analysiswas performed with NCSS 2007 software (Kaysville, UT,USA). A P-value <0.05 was accepted as statisticallysignificant.
Results
Quantification of DCI in C. ficifolia extract
Figure 1 shows the chromatograms for the DCI standardand for the aqueous C. ficifolia extract. DCI was identified
in the extract by its retention time (9.12 min), and its con-centration was calculated as 3.3 mg per gram of extract.
Effects of C. ficifolia and DCI oncell viability
The administration of C. ficifolia extract at concentrationsof 0.125 and 0.25 mm (based on the content of DCI) in25 mm glucose did not affect the viability of adipocytesafter 24 and 48 h of incubation. However, concentrations of0.5 and 0.75 mm reduced the viability of adipocytes bymore than 90%, and 1 mm extract reduced the viability ofadipocytes by 100% (Table 1). DCI did not alter cell viabil-ity at any concentration (data not shown).
Effects of C. ficifolia extract and DCI onoxidative stress
Adipocytes incubated with 25 mm glucose showed signifi-cant reductions in their GSH and GSSG concentrations (of25% and 40%, respectively), and the GSH/GSSG ratioincreased (by 40%) after 24 h compared with the GSH/
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Figure 1 Identification of the D-chiro-inositol (DCI) in the aqueous extract of Cucurbita ficifolia. The high-performance liquid chromatography(HPLC) chromatograms show (a) the synthetic DCI and (b) its identification in the extract, identified by the retention time (9.12 min).
Table 1 Cell viability at different concentrations of Cucurbita ficifolia (characterised by its D-chiro-inositol (DCI) content) in 3T3-L1 adipocytesexposed to 25 mM glucose
Concentration 0.125 mM 0.25 mM 0.5 mM 0.75 mM 1 mM
Cells were treated with varying doses of Cucurbita ficifolia extract with respect to the DCI content (0.125–1 mM). Data are represented as % of cellviability. The reported values are the means ± standard error of the mean (SEM) (n = 4). *Indicates statistically significant differences betweentreated and control untreated cells P < 0.05 (analysis of variance (ANOVA)).
Ángeles Fortis-Barrera et al.C. f. and DCI redox state, inflammation
GSSG ratio of adipocytes exposed to 5 mm glucose(Figure 2). Adipocytes incubated with 25 mm glucose for24 h and with varying concentrations of C. ficifolia extractfrom 0.0125 mm to 0.125 mm exhibited increased GSH andGSSG concentrations (Figure 2a and 2b), which produced adecreased GSH/GSSG ratio (Figure 2c). While the adminis-tration of 0.05 mm to 0.25 mm DCI led to similar changesin these parameters leading to levels similar to thoseobserved in the positive control (NAC) (Figure 2d, 2e and2f), C. ficifolia extract administered at 0.25 mm did notinduce any change in GSH concentration but did increaseGSSG (by 100%), thereby diminishing the GSH/GSSG ratio(by 55%). Considering these results, we decided to useC. ficifolia extract and DCI at a concentration of 0.125 mmfor a 48 h treatment.
The differences in GSH and GSSG concentrations and inthe GSH/GSSG ratio of adipocytes incubated with 5 and25 mm glucose for 48 h were similar those observed after 24 hof incubation (Figure 3). After treatment for 48 h, both theGSH and the GSSG concentrations increased by approxi-mately 30% (Figure 3a and 3b), and the ratio of GSH/GSSGdecreased by approximately 15% (Figure 3c) relative to theuntreated control (25 mm glucose). These results were similarto those observed after treatment for 24 h.
In adipocytes exposed to 25 mm glucose, the increase inthe GSH/GSSG ratio after 24 and 48 h coincided with adecrease in GPX activity (by approximately 24%) and anincrease in the level of H2O2 (by approximately 25%) atboth time points, without significant changes in the activityof GR. In all cases, the levels of these parameters measuredin cells incubated with 5 mm glucose were used for com-parison (Figure 4). The administration of C. ficifolia extractand of DCI increased GPX activity (by 41% and 60%,respectively) after 24 h of incubation (Figure 4a). This effectwas maintained after 48 h (Figure 4d), and a significantincrease in GR activity was observed after treatment withDCI (Figure 4e). Both treatments also decreased theamounts of intracellular H2O2 after 24 h (by approximately40%) and 48 h (by approximately 31%) (Figure 4c and 4f).
Effects of C. ficifolia extract and DCI on theexpression of adipokines
Adipocytes incubated with 25 mm glucose showed signifi-cant increases in the mRNA and protein expression levels ofTNF-α, IL-6 and resistin compared with the expressionlevels of these adipokines in adipocytes incubated in 5 mmglucose (Figures 5, 6 and 7). Under these conditions, TNF-αmRNA expression increased by 46% and 35% after 24 and48 h of incubation, respectively, concurrent with an increaseof 24% in the protein levels of this adipokine at both timepoints (Figure 5). A 24-h treatment with different concen-trations of C. ficifolia extract did not affect the protein
TNF-α level (Figure 5a), DCI at concentrations of 0.125and 0.25 mm decreased the level of this cytokine (byapproximately 20%) (Figure 5b). In TNF-α and in the fol-lowing cytokines quantification, the results of the treat-ments were compared to an untreated control (25 mmglucose).
The increases in TNF-α mRNA expression induced by25 mm glucose incubation diminished after treatment with0.125 mm C. ficifolia extract and with DCI for 24 h (by 30%and 43%, respectively) and for 48 h (by 45 and 51%, respec-tively) (Figures 5c, e). Levels of TNF-α protein decreased byapproximately 25% after both incubation periods, but onlywhen DCI was given (Figure 5d and 5f).
In adipocytes incubated with 25 mm glucose, both themRNA expression and the protein levels of IL-6 increasedby approximately 25% after 24 h of incubation. After48 h of incubation, mRNA expression and protein levelsincreased by 45% and 52%, respectively, when comparedwith the levels found in adipocytes exposed to 5 mmglucose (Figure 6). The two treatments studied exhibitedopposing effects on IL-6 levels after 24 h of incubation.C. ficifolia extract (0.125 and 0.25 mm) increased IL-6protein levels (Figure 6a) while 0.25 mm DCI decreasedIL-6 protein levels by approximately 40%. The effect of DCIon IL-6 protein levels was similar to that observed aftertreatment with rosiglitazone (Figure 6b).
Incubation of adipocytes with 0.125 mm C. ficifoliaextract increased IL-6 mRNA expression after 24 and 48 hof incubation (by 31% and 40%, respectively) (Figure 6cand 6e). These increases in mRNA expression are consistentwith an augmented level of protein (Figure 6a, 6d and 6f).In contrast, 0.125 mm DCI decreased IL-6 mRNA expres-sion after 24 h (by 31%) (Figure 6c) and after 48 h (by 41%)(Figures 6e), without affecting protein concentration(Figure 6d and 6f).
In adipocytes exposed to 25 mm glucose for 24 h, resistinmRNA expression and secretion increased by approximately50% and 25%, respectively, compared with adipocytes incu-bated with 5 mm glucose. Similar results were found after48 h of incubation (Figure 7). In adipocytes treated withC. ficifolia extract with DCI concentrations ranging from0.05 to 0.25 mm and incubated for 24 h, resistin secretionwas reduced to approximately 20%. This decrement wassimilar to that obtained with rosiglitazone (Figure 7a and7b). Furthermore, both treatments with 0.125 mm concen-trations reduced resistin mRNA expression by approxi-mately 30% and 40% after 24 and 48 h, respectively(Figure 7c and 7e). Resistin protein levels decreased byapproximately 20% after both incubation durations(Figure 7d and 7f).
C. ficifolia extract and DCI did not induce any significantchanges in mRNA expression and protein levels ofadiponectin (data not shown).
Ángeles Fortis-Barrera et al. C. f. and DCI redox state, inflammation
Figure 2 Effects of Cucurbita ficifolia (C. ficifolia) extract and D-chiro-inositol (DCI) on the redox state of 3T3-L1 adipocytes after 24 h of treat-ment. Adipocytes in Dulbecco’s modified Eagle’s medium (DMEM) with 25 mM glucose were treated with varying doses of C. ficifolia with respectto the DCI content in the extract (0.0125, 0.025, 0.05, 0.125 and 0.25 mM) or with synthetic DCI at the same doses. N-acetyl-L-cysteine (NAC,20 mM) was included as a positive control, as well as untreated cells incubated in DMEM with 5 and 25 mM glucose. After 24 h of incubation, thefollowing compound concentrations and ratio were determined: (a, d) reduced glutathione (GSH), (b, e) oxidised glutathione (GSS) (c, f) and theGSH/GSSG ratio. The reported values are the means ± standard error of the mean (SEM) (n = 4). ØIndicates statistically significant differencesbetween cells exposed to 25 mM glucose and 5 mM glucose, P < 0.05 (Student’s t-test). *Indicates statistically significant differences betweentreated and untreated cells, both cultured with 25 mM glucose, P < 0.05 (analysis of variance (ANOVA)).
Ángeles Fortis-Barrera et al.C. f. and DCI redox state, inflammation
The activation of PKB, determined based on the level ofphosphorylation of ser-473, which is insulin dependent,was assessed in the adipocytes incubated with 5 or 25 mmglucose in the presence of 1 μm insulin. There were no dif-ferences between the two glucose concentrations (data notshown). After incubation of the adipocytes with 25 mmglucose and C. ficifolia or DCI for 24 h, the activation ofPKB tended to increase, although this change was notsignificant. PKB was activated significantly only by DCI(30 min) in the absence of insulin stimulation (Figure 8).
Discussion
In obesity, the metabolic deregulation of adipocytes maylead to the development of insulin resistance and T2D.[21,26]
In 3T3-L1 adipocytes, exposure to a high glucose concen-tration (25 mm) results in oxidative stress and an inflamma-tory state associated with insulin resistance, effects thatare not observed in adipocytes exposed to low glucose(5 mm).[27] In this study, an altered redox state was observed,as evidenced by the increase in the GSH/GSSG ratio as aconsequence of the decrease in GPX activity, which led toan increase in the concentration of H2O2 in adipocytesincubated with 25 mm glucose as compared with adipocytesexposed to 5 mm glucose.
H2O2, free fatty acids and TNF-α are factors that decreasethe GPX activity.[12,28] Therefore, our results suggest that theantioxidant effects of C. ficifolia extract and DCI restore the
normal redox state of adipocytes because of a decrease inthe intracellular H2O2. This effect may be responsible for theincrease in GPX activity reflected by the GSH and GSSGlevels and the consequent reduction in the GSH/GSSGratio. This ratio became similar to that observed inadipocytes incubated with NAC (a positive control) and inadipocytes incubated with 5 mm glucose. In another study,DCI was shown to reduce levels of superoxide anion (O2
−), amolecule that can be transformed to H2O2.[10] Therefore, theDCI contained in C. ficifolia extract and synthetic DCIcould prevent the formation of H2O2 by decreasing intracel-lular levels of O2
−.C. ficifolia extract and DCI both exhibited similar effects
on oxidative stress, although the extract increased GSH andGSSG concentrations from 0.0125 mm to 0.125 mm whilethe modifications induced by DCI were from 0.05 to0.25 mm. This finding indicates that the extract containsother antioxidant compounds in addition to DCI, including(±)-catechin, L-ascorbic acid, p-coumaric acid and gallicacid (data not shown). We did not rule out the possibilitythat C. ficifolia contains other compounds that contributeto its antioxidant effect. The loss of the extract’s antioxidanteffect at 0.25 mm as a result of the increase in GSSG con-centration and the decrease in the GSH/GSSG ratio may bedue to pro-oxidant activity. Some antioxidants, such asascorbic acid, lead to the formation of reactive oxygenspecies under certain conditions.[29]
Adipocytes incubated with 25 mm glucose exhibitedinflammatory states, as shown by increases in the mRNAexpression and secretion levels of TNF-α, IL-6 and resistin.
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Figure 3 Effects of Cucurbita ficifolia (C. ficifolia) extract and D-chiro-inositol (DCI) on the redox state of 3T3-L1 adipocytes after 48 h of treat-ment. Adipocytes were incubated with C. ficifolia extract (with a DCI concentration of 0.125 mM) and with synthetic DCI (0.125 mM) for 48 h, forthe determination of the concentrations of (a) reduced glutathione (GSH) and(b) oxidised glutathione (GSSG), (c) and the GSH/GSSG ratio. Thereported values are the means ± standard error of the mean (SEM) (n = 5). ØIndicates statistically significant differences between cells exposed to25 mM glucose and 5 mM glucose, P < 0.05 (Student’s t-test). *Indicates statistically significant differences between treated and untreated cells,both cultured with 25 mM glucose, P < 0.05 (analysis of variance (ANOVA)).
Ángeles Fortis-Barrera et al. C. f. and DCI redox state, inflammation
DCI decreased the levels of these cytokines similar tothe effect observed after treatment with rosiglitazone.C. ficifolia extract decreased TNF-α mRNA expression andresistin levels. All of these effects may be associated with areduction in the intracellular level of H2O2. Nevertheless, itis well known that reactive oxygen species such as H2O2
may regulate the activation of NF-κB, a factor that regu-lates the transcription of proinflammatory cytokinesincluding TNF-α.[17,30] Because treatment with C. ficifoliaor with DCI decreases the level of intracellular oxidative
stress, these treatments can prevent the activation ofNF-κB and thereby ameliorate inflammatory states inadipocytes.
Treatment with C. ficifolia extract increased IL-6 secre-tion, unlike treatment with DCI. Consistent with ourresults, in diabetic mice this extract increases the serumlevels of IL-6.[4] We propose that the increase in IL-6 levelsmay have an effect on lipid metabolism similar to thatreported with chito-oligosaccharide. This compoundstimulates the expression of IL-6 in 3T3-L1 cells decreasing
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Figure 4 Effects of Cucurbita ficifolia (C. ficifolia) extract and D-chiro-inositol (DCI) on glutathione peroxidase activity, glutathione reductase activ-ity and the concentration of hydrogen peroxide (H2O2) in 3T3-L1 adipocytes after 24 and 48 h of treatment. Adipocytes were incubated withC. ficifolia extract (with a DCI concentration of 0.125 mM) and with synthetic DCI (0.125 mM) for (a, b, c) 24 and (d, e, f) 48 h for the determinationof the activity of (a, d) glutathione peroxidase (GPX) and (b, e) of glutathione reductase (GR), and (c, f) the concentration of H2O2, indicated as thefluorescence index of 2′,7′-dichlorofluorescein diacetate (DCFH-DA). The reported values are the means ± standard error of the mean (SEM) (n = 4).ØIndicates statistically significant differences between cells exposed to 25 mM glucose and 5 mM glucose, P < 0.05 (Student’s t-test). *Indicates sta-tistically significant differences between treated and untreated cells, both cultured with 25 mM glucose, P < 0.05 (analysis of variance (ANOVA)).
Ángeles Fortis-Barrera et al.C. f. and DCI redox state, inflammation
lipogenesis and adipogenesis.[31] The mechanism of actionof this cytokine involves the activation of mitogen-activated protein kinases.[32] Additionally, IL-6 administra-tion to obese mice results in decreased body weights.[33]
Therefore, it is necessary to study the effect of C. ficifolia
extract on the lipids accumulation and β-oxidation inadipose tissue in vivo. Some additional studies are neededto explain the effects of the extract on the stimulation ofIL-6 and to determine the compounds responsible for thiseffect.
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Figure 5 Effect of Cucurbita ficifolia (C. ficfolia) extract and D-chiro-inositol (DCI) on mRNA expression and protein levels of tumour necrosisfactor-α (TNF-α) in 3T3-L1 adipocytes. Adipocytes in Dulbecco’s modified Eagle’s medium (DMEM) with 25 mM glucose were treated with differentdoses of C. ficifolia with varying DCI contents (0.0125, 0.025, 0.05, 0.125 and 0.25 mM) or with the same concentrations of synthetic DCI.Rosiglitazone (5 μM) was included as a positive control, as well as untreated cells incubated with 5 and 25 mM glucose. (a, b) After 24 h of incuba-tion, the level of excreted TNF-α protein was determined. In other experiments, adipocytes were treated with 0.125 mM C. ficifolia extract or withDCI, and the following measurements were determined: TNF-α mRNA expression and the protein level of TNF-α in the incubation medium (c, d)after 24 h of treatment and (e, f) after 48 h of treatment. The reported values are the means ± standard error of the mean (SEM). (mRNA n = 5 andprotein n = 4). mRNA expression was reported as a ratio over the expression level of the housekeeping gene 36B4. ØIndicates statistically significantdifferences between cells exposed to 25 mM glucose and 5 mM glucose, P < 0.05 (Student’s t-test). *Indicates statistically significant differencesbetween treated and untreated cells, both cultured with 25 mM glucose, P < 0.05 (analysis of variance (ANOVA)).
Ángeles Fortis-Barrera et al. C. f. and DCI redox state, inflammation
We expected that the level of activated PKB would be lowin adipocytes cultured with 25 mm glucose, as demon-strated by Lin et al.[26] However, this effect was not observedin our study, this pathway may not have been alteredbecause adiponectin levels were still normal, and this
adipokine can sensitise cells to insulin.[34] We had expectedan inhibition in the activation of PKB because of anincrease in IL-6 secretion after treatment with C. ficifoliaextract because adipocytes incubated with IL-6 developinsulin resistance, as reported by Lagathu et al.[35] However,
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Figure 6 Effect of Cucurbita ficifolia (C. ficfolia) extract and D-chiro-inositol (DCI) on mRNA expression and protein levels of interkeukin (IL)-6 in3T3-L1 adipocytes. Adipocytes were incubated under the same conditions as in Figure 5. (a, b) The effects of C. ficifolia extract at different doses orof synthetic DCI on the protein levels of interleukin (IL)-6 after 24 h are reported. In other experiments, adipocytes were treated with 0.125 mM
C. ficifolia extract or with synthetic DCI, and the following measurements were determined: IL-6 mRNA expression and the protein levels of IL-6excreted in the incubation medium (c, d) after 24 h of treatment and (e, f) after 48 h of treatment. The reported values are the means ± standarderror of the mean (SEM) (mRNA n = 5 and protein n = 4). mRNA expression was reported as a ratio over the expression level of the housekeepinggene 36B4. ØIndicates statistically significant differences between cells exposed to 25 mM glucose and 5 mM glucose, P < 0.05 (Student’s t-test).*Indicates statistically significant differences between treated and untreated cells, both cultured with 25 mM glucose, P < 0.05 (analysis of variance(ANOVA)).
Ángeles Fortis-Barrera et al.C. f. and DCI redox state, inflammation
Lagathu used IL-6 at concentrations higher than thosesecreted in the presence of C. ficifolia extract, and the incu-bation time used in Lagathu’s report was 8 days, as opposedto 24 h in this study.
In 3T3-L1 adipocytes, treatment with DCI for 30 minresulted in a significant increase in PKB activation in the
absence of stimulation by insulin, demonstrating theinsulin-mimetic effects of DCI. This effect complementsthat shown in fibroblasts, in which treatment with 0.1–1 mm DCI increases the translocation of glucose transport 4and promotes the uptake of glucose.[36] This indicates thatthe mechanism of action of DCI on insulin signalling is
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Figure 7 Effect of Cucurbita ficifolia (C. ficfolia) extract and D-chiro-inositol (DCI) on the mRNA expression and protein levels of resistin in 3T3-L1adipocytes. Adipocytes were incubated under the same conditions as in Figures 5 and 6. (a, b) The effects of C. ficifolia extract at different doses orof synthetic DCI on the protein levels of resistin after 24 h are reported. In other experiments, adipocytes were treated with 0.125 mM C. ficifoliaextract or with DCI, and the following measurements were determined: resistin mRNA expression and the protein levels of resistin in the incubationmedium (c, d) after 24 h of treatment and (e, f) after 48 h of treatment. The reported values are the means ± standard error of the mean (SEM)(mRNA n = 5 and protein n = 4). mRNA expression was reported as a ratio over the expression level of the housekeeping gene 36B4. ØIndicates sta-tistically significant differences between cells exposed to 25 mM glucose and 5 mM glucose, P < 0.05 (Student’s t-test). *Indicates statistically signifi-cant differences between treated and untreated cells, both cultured with 25 mM glucose, P < 0.05 (analysis of variance (ANOVA)).
Ángeles Fortis-Barrera et al. C. f. and DCI redox state, inflammation
rapid and that its effect dissipates after long incubationssuch as that used in our study (24 h). Unlike DCI,C. ficifolia has no insulin-mimetic effect; we propose thatthe extract may act through a pro-oxidant/antioxidantbalance to improve the insulin sensitivity.
This study presents new possibilities for the effects ofaqueous C. ficifolia extract and DCI. To understand themechanism of action of C. ficifolia as a hypoglycaemic anti-oxidant agent and other cellular activity, it is necessary toidentify other compounds that may act synergistically withDCI. Our results support the use of DCI as a therapeuticagent with advantages over other antioxidant compoundsbecause DCI improves insulin sensitivity and has an anti-inflammatory effect.
Conclusion
Comparative analysis of the effects of an aqueous extract ofC. ficifolia and of DCI indicated that the antioxidant andanti-inflammatory effects of C. ficifolia could be explainedin part by its DCI content. Therefore, increasing GPX activ-ity modulates GSH/GSSG and decreases the activity ofproinflammatory pathways. The results of this study suggestthat treatment with C. ficifolia extract and DCI may reducethe cellular damage caused by oxidative stress. This research
provides novel evidence of the pharmacological anti-inflammatory effects of DCI, besides may improve insulinsensitivity because of its insulin-mimetic effects; therefore,this compound may be beneficial for the treatment ofobesity and T2D.
Declarations
Funding
This work was supported by the Fondo de Investigación enSalud del Instituto Mexicano del Seguro Social (FIS/IMSS/PROT/MD11/1004), CONACYT (Scholarship 212872) andIMSS (Scholarship 9909442).
Acknowledgements
We acknowledge Dr Edith Cortés Barberena and Dr RocioOrtíz (Department of Health Sciences, MetropolitanAutonomous University of Iztapalapa) for technical assis-tance in flow cytometry, and Dr Daniel Hernández Saavedra(Department of Medicine and Department of Pediatrics,University of Colorado) and Dr Clara Ortega Camarillo(Hospital of Specialties, CMNSXXI, Mexican Institute ofSocial Security) for advice in this research.
Figure 8 The effect of Cucurbita ficifolia (C. ficifolia) extract and D-chiro-inositol (DCI) on protein kinase B (PKB) ser-473 phosphorylation in 3T3-L1adipocytes. Adipocytes were incubated with 25 mM glucose and with C. ficifolia extract (with 0.125 mM DCI), or with 0.125 mM synthetic DCI for24 h. The cells were then washed as indicated in the methods and incubated for 15 min in the presence or absence of insulin (1 μM). In otherexperiments, adipocytes were incubated with 0.125 mM C. ficifolia extract or with 0.125 mM DCI, in the absence of insulin, for 30 min. Experimentswith adipocytes ± insulin exposure (15 min) without additional treatments were included. The reported values are the means ± standard error of themean (SEM) (n = 3). *Indicates statistically significant differences between treated and untreated cells, both cultured with 25 mM glucose, P < 0.05(analysis of variance (ANOVA)).
Ángeles Fortis-Barrera et al.C. f. and DCI redox state, inflammation
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