Ia

KD

a

ARAA

KNADGKR

1

tfNieniNN

0d

Chemico-Biological Interactions 189 (2011) 119–126

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

nhibition of renal gluconeogenesis contributes to hypoglycaemicction of NADPH oxidase inhibitor, apocynin

atarzyna Winiarska ∗, Michal Grabowski, Maciej K. Rogackiepartment of Metabolic Regulation, Institute of Biochemistry, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland

r t i c l e i n f o

rticle history:eceived 27 August 2010ccepted 30 September 2010vailable online 8 October 2010

eywords:ADPH oxidasepocyniniabetesluconeogenesisidneyabbit

a b s t r a c t

NADPH oxidase, catalysing superoxide radical (O2•−) formation, is considered as a main source of reactive

oxygen species in kidneys and its increased activity is supposed to be involved in the development ofdiabetic nephropathy. The aim of this study was to investigate the effect of NADPH oxidase inhibitor,apocynin, on renal gluconeogenesis, which is an important source of endogenous glucose under diabeticconditions.

The following observations were made during the experiments performed on isolated renal proxi-mal tubules of control and alloxan diabetic rabbits: (1) apocynin (200 �M) inhibited the rate of glucosesynthesis by 45–80%, depending on the substrate applied; (2) the rate of glucose production was also sig-nificantly diminished in the presence of TEMPOL (5 mM), a superoxide radical scavenger, suggesting thatthe decrease in O2

•− formation might be involved in apocynin-evoked gluconeogenesis inhibition; (3) theactivities of phosphoenolpyruvate carboxykinase (PEPCK) and/or aldolase were lowered in the presenceof either apocynin or TEMPOL, as concluded from the intracellular levels of gluconeogenic intermediates.The data from in vivo experiments indicated that apocynin treatment (2 g/l of drinking water): (1) sig-nificantly (by about 30%) attenuated serum glucose concentration in diabetic rabbits and did not affectglycaemia in control animals; (2) normalized diabetes-stimulated rate of glucose synthesis and slightlyinhibited gluconeogenesis in control rabbits; (3) normalized diabetes-increased activity of mitochondrial

PEPCK and lowered cytosolic PEPCK activity by about 20% below the value for untreated control animals;(4) slightly decreased the activity of mitochondrial PEPCK and did not change the activity of cytosolic onein control rabbits.

Thus, it is concluded that: (1) the inhibition of NADPH oxidase might contribute to lowered rate ofrenal gluconeogenesis, probably due to decreasing PEPCK activity; (2) inhibition of renal gluconeogen-esis is involved in apocynin hypoglycaemic action in vivo; (3) apocynin might be beneficial for diabetes

treatment.

. Introduction

NADPH oxidase (EC 1.6.2.4, Nox) catalyses one electron reduc-ion of molecular oxygen, leading to superoxide radical (O2

•−)ormation, which begins the cascade of free radical reactions.ox was originally discovered in phagocytes, where its activity is

nvolved in pathogen elimination. However, it soon turned out thatnzymes exhibiting high homology to phagocyte NADPH oxidase,amed Nox2, are present in many other tissues. Currently the fam-

ly of Nox enzymes consists of seven oxidases: Nox1, Nox2, Nox3,ox4, Nox5, Duox1 and Duox2 [1]. Three of them: Nox1, Nox2 andox4 (Renox) are expressed in kidney cortex [2].

∗ Corresponding author. Tel.: +48 22 5543208; fax: +48 22 5543221.E-mail address: k.winiarska@biol.uw.edu.pl (K. Winiarska).

009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2010.09.033

© 2010 Elsevier Ireland Ltd. All rights reserved.

NADPH oxidase reaction tends to be a main source of reactiveoxygen species in kidneys and increased activity of the enzyme isconsidered as an important cause of nephropathies, including dia-betic nephropathies [2,3]. It has been reported that renal NADPHoxidase is activated by angiotensin II, high salt diet, hyperlipidemia,hyperglycaemia [2,3] and glycated albumin [4]. Elevated NADPHoxidase activity has been observed in kidneys of streptozotocindiabetic rats [5,6], diabetic PKC-beta (−/−) mice [7] and alloxandiabetic rabbits [8,9]. Thus, inhibition of NADPH oxidase seems tobe a promising therapeutic strategy.

Apocynin (acetovanillone, 4′-hydroxy-3′-methoxyacetophe-none) was originally derived from the roots of a Himalayan plant,

Picrorhiza kurroa, which in local traditional medicine has a long his-tory as a remedy applied to patients with liver and cardiovasculardisorders [10,11]. According to the present knowledge, apocynin isthought to be one of the most selective NADPH oxidase inhibitors,effectively attenuating superoxide formation. The proposed gen-

1 ogical

epsmtae

odiah[u

Ncuf

2

2

wonTpwtotoom

WA

2

ai2sts1omotgNwtop

rs

20 K. Winiarska et al. / Chemico-Biol

ral mechanism of its action includes the inhibition of regulatory47phox subunit (or its homologue) aggregation with the otherubunits of the enzyme. The data from experiments on animalodels of diseases suggest that apocynin might be useful in the

reatment of arterial hypertension [10], ischemic injuries, asthmand arthritis [11]. It is also worth emphasizing that no adverse sideffects of apocynin have been reported [3].

As it is commonly accepted that hyperglycaemia-evokedxidative stress plays a crucial role in the development ofiabetic complications [12,13], it appears reasonable that the

deal antidiabetic therapy should combine both hypoglycaemicnd antioxidative effect. There are some reports on apocyninypoglycaemic action in animals with experimental diabetes14,15], but the mechanism of this phenomenon remainsnknown.

The aim of the present study was to investigate the effect ofADPH oxidase inhibitor, apocynin, on the regulation of renal glu-oneogenesis, which is an important source of endogenous glucosender diabetic conditions [16] and could be a considerable targetor therapeutic intervention.

. Materials and methods

.1. Animals

The experiments were performed with male Termond rabbitseighing approximately 2.0–2.2 kg. Rabbits are useful for studies

n glucose metabolism as the intracellular localization of gluco-eogenic enzymes in their kidneys is similar to that in humans [17].he animals were purchased from a licensed laboratory animal sup-lier and maintained on standard rabbit chow with free access toater and food. Diabetes was induced by a single intravenous injec-

ion of alloxan (175 mg/kg body weight) freshly dissolved in 1 mlf sterile 10 mM citrate buffer (pH 4.5) [18]. Only these alloxan-reated animals that 3 days after the treatment exhibited decreasedr stabilized body weight and blood glucose concentration in excessf 300 mg/dl were considered diabetic and used for the experi-ents.All animal treatment procedures were approved by the First

arsaw Local Commission for the Ethics of Experimentation onnimals.

.2. Isolation and incubation of renal proximal tubules

Renal proximal tubules were isolated according to Jarzyna etl. [19]. Freshly isolated tubules (about 10 mg dry weight) werencubated at 37 ◦C, under the atmosphere of 95% O2 + 5% CO2, inml of Krebs-Ringer bicarbonate buffer containing gluconeogenic

ubstrates, indicated in tables and figures legends, in 25 ml plas-ic Erlenmeyer flasks sealed with rubber stoppers. Reactions weretopped following 60 min of incubation by either the addition ofml sample to 0.1 ml of 35% perchloric acid (PCA) or, in casef samples intended for intracellular metabolite level measure-ents, centrifugation of tubules suspension through the silicone

il layer to 1 ml of 12% PCA [20]. To avoid non-enzymatic oxida-ion of reduced glutathione (GSH), samples intended for oxidizedlutathione (GSSG) determinations were centrifuged into 50 mM-ethylmaleimide (NEM) in 12% PCA and then the excess of NEMas removed by hexane extraction [21]. Samples used for glu-

athione determinations were stored as PCA extracts, while the

thers were immediately neutralized with 3 M K2CO3. All the sam-les were stored at −80 ◦C.

Proximal tubules intended for the estimation of hydroxyl freeadicals (HFR) [22] were incubated with 5 mM pyruvate and 1 mModium salicylate, both in the absence and in the presence of

Interactions 189 (2011) 119–126

apocynin. After 60 min of incubation, 1 ml samples were with-drawn and acidified with 0.1 ml of 35% PCA, containing 1 mMethylenediaminetetraacetate (EDTA) and 4 mM sodium metabisul-phite (Na2S2O5), and centrifuged. Supernatants were placed on iceand analysed on the day of the experiment.

In order to measure the rate of superoxide radical production,after 60 min of incubation the suspension of proximal tubules inincubation medium containing 5 mM pyruvate, without or withapocynin, was centrifuged (20 × g, 1 min). The pellet was homog-enized in Krebs-Ringer buffer (0.1 g/0.5 ml) and then processedaccording to Chen et al. [23]. The rate of superoxide radical gen-eration was determined as described in Section 2.7.4.

2.3. Experimental design of in vivo studies

During in vivo studies four groups of rabbits, each consistingof 5 animals, were used: (1) untreated control rabbits; (2) controlrabbits treated with apocynin; (3) untreated diabetic rabbits; (4)diabetic rabbits treated with apocynin. NADPH oxidase inhibitorwas applied as a solution in drinking water (2 g/1 l [24]).

After 3 weeks of the experiment animals were euthanized byintravenous injection of pentobarbital (30 mg/kg body weight).Right kidneys were immediately collected for enzymatic activitymeasurements and then proximal tubules were isolated from theleft ones, according to Section 2.2.

2.4. Preparation of blood and urine samples

Blood (0.7 ml) was withdrawn from ear marginal vein and col-lected into heparinized tubules placed on ice and then centrifugedin order to separate blood cells. The supernatants dedicated for glu-cose, urea and creatinine determination were deproteinized with1% Na2WO4 in 30 mM H2SO4 (6:1, v/v).

Urine for albumin determination was withdrawn from bladderimmediately after animal killing.

2.5. Preparation of tissue samples for enzyme activitiesmeasurements

Kidney cortex homogenates for NADPH oxidase activitydeterminations were prepared according to Chen et al. [23]. Mito-chondria for phosphoenolpyruvate (PEPCK) activity determinationwere obtained as described by Harris et al. [25]. Cytosolic frac-tion for PEPCK activity measurement was prepared according toMacDonald et al. [26]. Kidney cortex samples intended for aldolaseactivity measurements were homogenized in 0.15 M KCl, pH 7.4adjusted with KHCO3 (5 ml/1 g of the tissue). The homogenate wascentrifuged at 70,000 × g (20 min, 4 ◦C) and the supernatant wasimmediately used for the determinations.

2.6. Measurements of enzyme activities

2.6.1. NADPH oxidaseSuperoxide anion production by renal NADPH oxidase was

determined using lucigenin-enhanced chemiluminescence method[6,23]. Chemiluminometric measurements were performed usingThriatler 425-014 luminometer (Hidex Ltd., Turku, Finland).

2.6.2. Gluconeogenic enzymesMitochondria for PEPCK activity measurements were incu-

bated in thermostatted glass containers at 30 ◦C and under the

atmosphere of 95% O2 + 5% CO2. The incubation medium con-sisted of 75 mM Tris–HCl buffer, pH 7.4, containing 50 mM glucose,15 mM KCl, 5 mM MgCl2, 15 mM K3PO4, 5 mM malate, 5 mM 2-oksoglutarate and 3 units of hexokinase. The reaction was startedby addition of ADP to final 200 �M concentration and conducted

ogical Interactions 189 (2011) 119–126 121

fwPa

td

2

2

r[owM

2

i3bgmpK

2

wdIp

2

(acsarRdeas

2

fm

2

Ci

2

i

Table 1Apocynin (200 �M) action on superoxide radicals (O2

•−) and hydroxyl radicals (HO•)generation and glutathione redox state in isolated rabbit proximal tubules.

Control +Apocynin

O2•− (RLU × mg−1 d.w. × min−1) 1900 ± 110 1305 ± 187a

HO• (fmol DHBA × mg−1 d.w. × min−1) 1670 ± 30 1013 ± 155a

GSH (nmol × mg−1 d.w.) 1.88 ± 0.17 1.81 ± 20GSSG (nmol × mg−1 d.w.) 0.038 ± 0.005 0.034 ± 0.005GSH/GSSG 49.6 ± 6.4 53.1 ± 8.2

Renal tubules isolated from control rabbits were incubated for 60 min in the pres-

cose synthesis in control rabbit proximal tubules incubated withpyruvate as a glucose precursor. This effect was concentration-dependent. 200 �M apocynin, which caused 60% inhibition, wasapplied throughout the further experiments.

801

40

60

x m

g-1

d.w

.x h

-1

a

a

0

20

0 50 100 200 400

nm

olx

a

K. Winiarska et al. / Chemico-Biol

or 10 min. Samples for phosphoenolpyruvate (PEP) determinationere withdrawn at the beginning and every 2 min of the incubation.

EP concentration was measured spectrophotometrically applyingstandard enzymatic technique [27].

PEPCK activity in cytosolic fraction was measured accordingo Bentle and Lardy [28]. Aldolase activity was determined asescribed by Yeltman and Harris [29].

.7. Analytical methods

.7.1. GlucoseGlucose serum concentration was analysed spectrophotomet-

ically with hexokinase and glucose-6-phosphate dehydrogenase27]. The same method was applied while determining the ratef renal gluconeogenesis. All spectrophotometrical measurementsere performed using Cary 50Bio spectrophotometer (Varian Ltd.,elbourne, Australia).

.7.2. Gluconeogenic intermediatesThe intracellular levels of gluconeogenic intermediates, includ-

ng pyruvate, malate, phosphoenolpyruvate, phosphotrioses,-phosphoglycerate + 1,3-bisphosphoglycerate, fructose-1,6-isphosphate, fructose-6-phosphate, glucose-6-phosphate andlucose, were measured fluorimetrically applying standard enzy-atic techniques [27]. All fluorimetrical measurements were

erformed using RF-5301PC spectrofluorimeter (Shimadzu Corp.,yoto, Japan).

.7.3. GlutathioneOxidized glutathione (GSSG) was estimated fluorimetrically

ith glutathione reductase [27]. Reduced glutathione (GSH) wasetermined by HPLC (110B System Gold®, Beckman Instruments

nc., San Ramon, CA, USA) following derivatization with N-(1-yrenyl) maleimide, as described by Ridnour et al. [30].

.7.4. Hydroxyl free radicalsHydroxyl free radicals were estimated as dihydroxybenzoic acid

DHBA) generated in the presence of sodium salicylate [22]. DHBAssays were performed by HPLC using Beckman Ultrasphere® ODSolumn. The mobile phase consisted of 50 mM NaH2PO4, 1.125 mModium octanesulphonic acid, 0.2 mM EDTA, 3% methanol and 5.5%cetonitryl (v/v). pH was adjusted to 2.8 with 1 M ortophospho-ic acid. 110B System Gold® HPLC (Beckman Instruments Inc., Sanamon, CA, USA) was equipped with Bio-Rad 1640 electrochemicaletector (Bio-Rad, Hercules, CA, USA) and a glassy carbon workinglectrode operating at +0.75 V against Ag/AgCl reference electrodend detection range of 2 nA. The flow rate was 1 ml/min and alleparations were performed at 30 ◦C.

.7.5. Urea and creatinineUrea was measured spectrophotometrically as ammonium

ollowing sample treatment with urease [31]. Creatinine was deter-ined by Jaffe’s reaction as described by Michalik et al. [32].

.7.6. Urinary albuminUrinary albumin level was determined with an assay kit (Sigma

hemicals, St Louis, MO, USA) according to the manufacturer’snstructions.

.7.7. ProteinProtein content was evaluated spectrophotometrically accord-

ng to Layne [33].

ence of 5 mM pyruvate. In case of renal tubules intended for HFR measurements1 mM sodium salicylate acid was added to the incubation medium. Values aremeans ± SD for 5 experiments. Statistical significance: aP < 0.05 versus correspond-ing values in the absence of apocynin.

2.8. Chemicals

Enzymes, coenzymes and nucleotides for metabolite determina-tions were purchased from Roche (Mannheim, Germany). All otherchemicals were from Sigma Chemicals (St Louis, MO, USA).

2.9. Expression of results

The significance of the differences was estimated using ANOVA.Values are expressed as means ± SD for three to five separate exper-iments.

3. Results

3.1. Apocynin action on reactive oxygen species generation andglutathione redox state in isolated renal proximal tubules

As presented in Table 1, 60 min incubation of renal proxi-mal tubules with 200 �M apocynin resulted in diminished rateof both superoxide anions and hydroxyl radicals (by 31% and39%, respectively) generation. Upon apocynin addition to incuba-tion medium neither glutathione content nor its redox state waschanged (Table 1).

3.2. Apocynin in vitro action on the rate of gluconeogenesis inrenal proximal tubules isolated from control and diabetic rabbits

As presented in Fig. 1, apocynin, added to the incubationmedium at 50–400 �M concentrations, inhibited the rate of glu-

Apocynin concentration (µM)

Fig. 1. The effect of increasing apocynin concentrations on the rate of gluconeo-genesis in control rabbit renal tubules incubated for 60 min with 5 mM pyruvate.Values are means ± SD for 5 experiments. Statistical significance: aP < 0.05 versuscorresponding values in the absence of apocynin.

122 K. Winiarska et al. / Chemico-Biological Interactions 189 (2011) 119–126

Table 2The effect of apocynin (200 �M) on the rate of glucose formation in kidney cortex tubules isolated from control and diabetic rabbits and incubated with various substrates.

Substrates Glucose synthesis (nmol × mg−1 d.w. × h−1)

Control Control + apocynin Diabetes Diabetes + apocynin

Pyruvate 63.7 ± 4.0 25.1 ± 1.9a 110.7 ± 19.2b 73.5 ± 8.9a,b

Malate 123.8 ± 17.6 68.4 ± 3.6a 193.8 ± 23.8b 129.0 ± 9.8a,b

Ala + glycerol + octanoate 66.6 ± 5.5 12.5 ± 2.5a 65.2 ± 8.2 15.8 ± 3.1a

Asp + glycerol + octanoate 121.0 ± 7.25 63.4 ± 7.5a 217.0 ± 26.0b 105.8 ± 12.7a,b

R min.0 atisticb .

wctt

t(2t

3gg

tmatg

iagpem

Fliwpp0pb6ftp

enal tubules isolated from either control or diabetic rabbits were incubated for 60.5 mM concentrations, respectively. Values are means ± SD for 5 experiments. StP < 0.05 versus corresponding values for renal tubules isolated from control rabbits

Similar apocynin (200 �M) inhibitory action was observedhen control renal tubules were incubated with other glu-

oneogenic substrates (Table 2), including malate (45% inhibi-ion), alanine + glycerol + octanoate (80% inhibition) and aspar-ate + glycerol + octanoate (48% inhibition).

Diabetes resulted in accelerated glucose synthesis from allhe substrates tested, excluding alanine + glycerol + octanoateTable 2). However, the extent of gluconeogenesis inhibition by00 �M apocynin was comparable with that reported for renalubules isolated from control animals.

.3. Apocynin in vitro effect on the intracellular level ofluconeogenic intermediates and the activities of selectedluconeogenic enzymes

In order to establish gluconeogenic steps affected by apocynin,he intracellular levels of the intermediates of the process were

easured in renal tubules incubated with pyruvate, both in thebsence and in the presence of 200 �M apocynin. Fig. 2 depic-ures relative changes in the intracellular content of the particularluconeogenic metabolites.

The addition of apocynin resulted in a significant decreasen phosphoenolpyruvate and fructose-1,6-bisphosphate levels,ccompanied by augmented phosphotrioses content. This sug-

ested that acetovanillone might lower the activity of phos-hoenolpyruvate carboxykinase (PEPCK) and/or aldolase, i.e. thenzymes catalysing the reactions leading to the formation of theetabolites, which diminished content was observed in the pres-

160a

80

100

120

140ab

ab

a

aa

a

0

20

40

60a

aa

%o

f co

ntr

ol

PYR MAL PEP PGA TP FBP F6P G6P GLC

ig. 2. Apocynin- (�) and TEMPOL-induced (©) relative changes in the intracel-ular levels of gluconeogenic intermediates. Renal tubules of control rabbits werencubated for 60 min in the presence of 5 mM pyruvate. Apocynin and TEMPOL

ere added at 200 �M and 5 mM concentrations, respectively. Control values forarticular metabolites, expressed as nmol × mg−1 dry weight, were the following:yruvate (PYR) 2.30 ± 0.21; malate (MAL) 0.76 ± 0.08; phosphoenolpyruvate (PEP).25 ± 0.03; 3-phosphoglycerate + 1,3-bis-phosphoglycerate (PGA) 0.83 ± 0.10; 3-hosphoglyceraldehyde + phosphodihydroxyacetone (TP) 0.32 ± 0.04; fructose-1,6-isphosphate (FBP) 0.16 ± 0.01; fructose-6-phosphate (F6P) 0.06 ± 0.01; glucose--phosphate (G6P) 0.20 ± 0.03; glucose (GLC) 4.74 ± 0.52. Values are means ± SDor 5 experiments. Statistical significance: aP < 0.05 versus corresponding values inhe absence of apocynin and TEMPOL; bP < 0.05 versus corresponding values in theresence of apocynin.

Pyruvate, malate, glycerol, amino acids and octanoate were added at 5, 5, 2, 2 andal significance: aP < 0.05 versus corresponding values in the absence of apocynin.

ence of apocynin. Thus, apocynin direct effects on PEPCK andaldolase activities were examined.

Upon the addition of apocynin to the test cuvettes, no changesin the activities of either PEPCK, both cytosolic (20.71 ± 3.80and 19.06 ± 3.92 nmol × min−1 × mg−1 protein in the absence andin the presence of apocynin, respectively) and mitochondrial(14.64 ± 0.37 and 14.77 ± 0.40 nmol × min−1 × mg−1 protein in theabsence and in the presence of apocynin, respectively), or aldolase(85.15 ± 6.85 and 76.82 ± 7.12 nmol × min−1 × mg−1 protein in theabsence and in the presence of apocynin, respectively) wereobserved, indicating that the mechanism of the inhibitory actionof acetovanillone could involve rather a diminished rate of super-oxide radical production via NADPH oxidase than a direct effect onthe activities of the gluconeogenic enzymes.

3.4. Comparison with TEMPOL, a superoxide radical scavenger,effect on the rate of gluconeogenesis

To verify the hypothesis that the inhibitory action of acetovanil-lone on glucose formation in rabbit renal tubules might result fromdecreased NADPH oxidase activity and lowered superoxide radicallevel, apocynin action on gluconeogenesis was compared with theeffect of TEMPOL (4-hydroksy-2,2,6,6-tetramethylpiperidine-1-oksyl), a potent superoxide radical scavenger that easily penetratescellular membranes [34].

As presented in Table 3, TEMPOL applied at 5 mM concentration,i.e. at the concentration commonly used in ex vivo experiments[34], effectively inhibited glucose formation in rabbit proximaltubules incubated with all the substrates tested. Moreover, themeasurements of the intracellular level of gluconeogenic inter-mediates (Fig. 2) clearly indicated that TEMPOL inhibited theprocess at the same steps of PEPCK and/or aldolase reactions asapocynin did. In contrast to renal tubules incubated with aceto-vanillone, these incubated in the presence of TEMPOL exhibitedintracellular concentrations of pyruvate and malate exceeding the

control value. However, such changes, accompanied with loweredPEP content, are still in agreement with the postulated inhibi-tion of PEPCK. As in case of apocynin, no direct TEMPOL actionon PEPCK (19.91 ± 2.88 and 15.02 ± 1.43 nmol × min−1 × mg−1 pro-

Table 3The effect of TEMPOL (5 mM) on the rate of glucose synthesis in kidney cortex tubulesincubated with various substrates.

Substrates Glucose synthesis (nmol × mg−1 d.w. × h−1)

Control +TEMPOL

Pyruvate 69.8 ± 5.3 30.0 ± 2.6a

Malate 141.2 ± 13.5 95.9 ± 9.8a

Ala + glycerol + octanoate 59.1 ± 2.3 28.9 ± 4.6a

Asp + glycerol + octanoate 139.1 ± 5.5 79.6 ± 4.1a

Renal tubules isolated from control rabbits were incubated for 60 min. Pyruvate,malate, glycerol, amino acids and octanoate were added at 5, 5, 2, 2 and 0.5 mMconcentrations, respectively. Values are means ± SD for 5 experiments. Statisticalsignificance: aP < 0.05 versus corresponding values in the absence of TEMPOL.

K. Winiarska et al. / Chemico-Biological Interactions 189 (2011) 119–126 123

700

300

400

500

600

mg

/dl

0

100

200

2120191817161514131211109876543210

aa

a

day

Fig. 3. Serum glucose concentrations in diabetic rabbits untreated (�) and treatedw2td

ta

lcN

3

daaf3yu

tcruyw

tioban

gr

0

20

40

60

80

100

mg/dl

0

0,5

1

1,5

2

2,5

3

2120191817161514131211109876543210

day

mg/ml

Urea

Creatinine

a aa

a a a

aa

Fig. 4. Serum creatinine and urea concentrations in diabetic rabbits untreated (�)and treated with apocynin (©). Apocynin was administered to animals as described

TT

Aga

u

ith apocynin (©). Apocynin was administered to animals as described in Section.3. Values are means ± SD for 5 animals. The dotted line depicts glucose concentra-ion value for control rabbits (126 ± 10 mg/dl). aP < 0.05 versus values for untreatediabetic rabbits.

ein for cytosolic and mitochondrial one, respectively) or aldolasectivity (86.01 ± 9.16 nmol × min−1 × mg−1 protein) was observed.

In view of these observations, it might be suggested, that theowered level of superoxide radical might affect the rate of glu-ose formation and be involved in the inhibitory effect of apocynin,ADPH oxidase inhibitor.

.5. In vivo hypoglycaemic action of NADPH oxidase inhibitor

Following 3 weeks of apocynin treatment renal NADPH oxi-ase activity was lowered by 80% in both control (12,880 ± 645nd 2170 ± 213 RLU × min−1 × mg−1 protein for untreated andpocynin treated animals, respectively; P < 0.05 versus the valueor untreated rabbits) and diabetic rabbits (16,705 ± 588 and436 ± 1617 RLU × min−1 × mg−1 protein for untreated and apoc-nin treated animals, respectively; P < 0.05 versus the value forntreated rabbits).

As depicted in Fig. 3, in alloxan diabetic rabbits apocyninreatment markedly (by about 30%) attenuated serum glucoseoncentration. Hypoglycaemic action of NADPH oxidase inhibitorevealed during the second week of the treatment and continuedntil the end of the experiment. Control rabbits treated with apoc-nin exhibited normoglycaemia (120 ± 26 mg/dl) throughout the 3eeks of the experiment.

Table 4 presents the effect of the 3-week apocynin treatment onhe rate of renal gluconeogenesis. In case of diabetic animals drink-ng apocynin solution normalisation of the diabetes-stimulated ratef glucose synthesis was observed in renal proximal tubules incu-ated in the presence of all the substrates tested. In control rabbits

pocynin treatment also resulted in the inhibition of renal gluco-eogenesis (by 25–45%, depending on the substrate applied).

As the data from the in vitro experiments (cf. Section 3.3) sug-ested that apocynin affects gluconeogenesis at the stages of theeactions catalysed by PEPCK and/or aldolase, the activities of these

able 4he effect of apocynin treatment on the rate of glucose formation in kidney cortex tubule

Substrates Glucose synthesis (nmol × mg−1 d.w. × h−1)

Control rabbits

Untreated Treated with ap

Pyruvate 63.7 ± 4.0 47.7 ± 3.4a

Malate 123.8 ± 17.6 84.8 ± 6.6a

Asp + glycerol + octanoate 121.0 ± 7.2 65.6 ± 14.8a

pocynin was administered to animals as described in Section 2.3. Renal tubules isolatedlycerol, amino acids and octanoate were added at 5, 5, 2, 2 and 0.5 mM concentrationP < 0.05 versus corresponding values for renal tubules isolated from corresponding untrentreated control rabbits.

in Section 2.3. Values are means ± SD for 5 animals. The dotted lines depict valuesfor control rabbits (23.0 ± 5.1 mg/dl and 1.3 ± 0.2 mg/dl for creatinine and urea con-centrations, respectively). aP < 0.05 versus the values for untreated diabetic rabbits.

enzymes were measured in kidney cortex of rabbits, both controland diabetic, untreated and treated with apocynin. As shown inTable 5, diabetes resulted in an approximately two-fold increasein the activities of both, cytosolic and mitochondrial, isoforms ofPEPCK. Apocynin application to diabetic rabbits normalized theactivity of mitochondrial PEPCK and lowered the activity of cytoso-lic one by 55%, i.e. about 20% below the value for untreated controlanimals. In control rabbits apocynin treatment slightly (by about20%) decreased the activity of mitochondrial PEPCK but did notaffect the activity of the cytosolic isoform of the enzyme. Neitherdiabetes nor apocynin treatment changed the activity of aldolasein kidney cortex (Table 5).

3.6. The effect of apocynin treatment on renal functionparameters

In order to estimate if apocynin treatment affects renal func-tion parameters of both control and alloxan diabetic animals, serumcreatinine and urea concentrations were measured. As presentedin Fig. 4, apocynin administration to diabetic rabbits resulted ina marked attenuation of the disease-evoked increase in serumurea concentration. Moreover, it also slightly lowered serum crea-

tinine level in diabetic animals (Fig. 4). Control rabbits treated withapocynin exhibited normal serum urea and creatinine concentra-tions (22.5 ± 6.2 and 1.3 ± 0.3 mg/dl, respectively) throughout the3 weeks of the experiment.

s of both control and diabetic rabbits.

Diabetic rabbits

ocynin Untreated Treated with apocynin

110.7 ± 19.2b 58.1 ± 5.7a

193.8 ± 23.8b 106.3 ± 11.2a

217.0 ± 26.0b 115.5 ± 14.3a

from either control or diabetic rabbits were incubated for 60 min. Pyruvate, malate,s, respectively. Values are means ± SD for 5 experiments. Statistical significance:ated rabbits. bP < 0.05 versus corresponding values for renal tubules isolated from

124 K. Winiarska et al. / Chemico-Biological Interactions 189 (2011) 119–126

Table 5The effect of apocynin treatment on the activities of PEPCK and aldolase in kidney cortex of both control and diabetic rabbits.

Enzyme Activity (nmol × min−1 × mg−1 protein)

Control rabbits Diabetic rabbits

Untreated Treated with apocynin Untreated Treated with apocynin

Mitochondrial PEPCK 14.64 ± 0.37 11.76 ± 2.12a 30.89 ± 9.14b 12.90 ± 3.39a

Cytosolic PEPCK 20.71 ± 3.80 23.06 ± 2.98 37.11 ± 8.11b 16.71 ± 4.49a,b

12

A s ± SDf valu

dircmvu

4

toTsawaaikyatk

gphitssspsttppatic

–ernteK

Aldolase 85.15 ± 6.85 76.82 ± 7.

pocynin was administered to animals as described in Section 2.3. Values are meanor kidney cortex of corresponding untreated rabbits. bP < 0.05 versus corresponding

Apocynin treatment-evoked changes in serum parameters ofiabetic rabbits were accompanied by abated albuminuria. Follow-

ng 3 weeks of diabetes urinary albumin level raised four times,eaching 41.3 ± 5.2 mg/l (P < 0.05 versus the value for untreatedontrol rabbits, i.e. 11.3 ± 4.8 mg/l). Apocynin administration nor-alized albuminuria in diabetic rabbits (8.9 ± 2.7 mg/l; P < 0.05

ersus the value for untreated diabetic rabbits) and did not affectrinary albumin level in control animals (10.7 ± 2.9 mg/l).

. Discussion

It is commonly accepted that hyperglycaemia results in oxida-ive stress which contributes to the development of a wide rangef diabetic complications, including diabetic nephropathy [12,13].herefore, it seems reasonable that effective antidiabetic therapyhould comprise both strategies – hypoglycaemic treatment as wells counteraction to tissue oxidative damages. In the present studye have investigated the postulated hypoglycaemic properties of

pocynin in alloxan diabetic rabbits. This compound is widely useds a potent inhibitor of NADPH oxidase [10,11], the enzyme thats known as the main source of reactive oxygen species in diabeticidneys [2,3]. Although there are some controversies about apoc-nin impact on the activity of Nox4, the oxidase that was originallyttributed to kidneys [35], we have found that acetovanillone effec-ively (by about 80%) decreases the total NADPH oxidase activity inidney cortex of both control and alloxan diabetic rabbits.

Since Castor et al. [36] have postulated that apocynin radical,enerated in the presence of horseradish peroxidase and hydrogeneroxide, acts as a pro-oxidant and oxidizes glutathione (GSH), weave examined if acetovanillone does not exhibit similar effects

n renal proximal tubules. In view of unaffected glutathione con-ent and redox state as well as diminished generation of bothuperoxide radicals and hydroxyl free radicals, commonly con-idered as highly reactive and the most harmful reactive oxygenpecies [37], observed upon apocynin addition (cf. Table 1), thero-oxidative action of this compound in rabbit proximal tubuleshould be excluded. The observations presented above confirmhat the inhibition of NADPH oxidase by apocynin might result inhe attenuation of oxidative stress in kidneys and extend nephro-rotective effects. Such properties of acetovanillone have beenreviously reported, also under diabetic conditions [38–40], andre in agreement with our present findings, which have indicatedhat diabetic animals treated with this compound exhibit normal-zed urinary albumin concentration and lowered serum urea andreatinine levels (cf. Fig. 4).

Two phenomena are to be blamed for diabetic hyperglycaemiaimpaired glucose utilization by peripheral tissues and accel-

rated glucose synthesis de novo. Moreover, the participation of

enal gluconeogenesis to the whole body glucose production sig-ificantly increases under diabetic conditions [16], suggesting thathis process might be a promising therapeutic target. Furukawat al. [14] have found that apocynin lowers blood glucose level ofKAy diabetic mice, but they have excluded the possibility that

89.20 ± 9.81 95.12 ± 8.89

for 3–5 experiments. Statistical significance: aP < 0.05 versus corresponding valueses for kidney cortex of untreated control rabbits.

hypoglycaemic effect of this compound results from the increase inglucose utilization by muscles. Thus, we have investigated if apoc-ynin affects the rate of renal glucose formation. The results of invitro experiments on isolated rabbit proximal tubules clearly indi-cate that apocynin lowers both control and diabetes-acceleratedrate of gluconeogenesis (cf. Table 2). While interpreting results pre-sented in Table 2, it is also worth noticing that diabetes stimulatedglucose formation from all the substrates tested, excluding ala-nine + glycerol + octanoate. This phenomenon has been observed inour previous studies on gluconeogenesis in rabbit proximal tubules[41] and suggests that under physiological conditions alanine mightnot be an important gluconeogenic precursor in rabbit kidneys,which is in agreement with the data obtained for human kidneys[42].

In view of our findings (cf. Fig. 2), it seems likely that apoc-ynin inhibits renal gluconeogenesis via decreasing flux throughPEPCK and aldolase. In contrast to aldolase, catalyzing a reversiblereaction common for both glycolysis and gluconeogenesis, PEPCKhas been reported as a highly regulated key enzyme of gluconeo-genesis, which activity is known to be stimulated under diabeticconditions [43,44]. As the direct apocynin effect on the activitiesof the above mentioned enzymes has been excluded (cf. Section3.3), the mechanism of gluconeogenesis inhibition seems to beassociated with the diminished activity of NADPH oxidase and thelowered intracellular concentration of reactive oxygen species. Inorder to confirm this hypothesis, the experiments applying TEMPOL(cf. Table 3 and Fig. 2), a compound known to effectively scav-enge superoxide radical [34], have been conducted. Similarly toapocynin, TEMPOL turned out to decrease the rate of renal glu-coneogenesis (cf. Table 3) via inhibition of PEPCK and/or aldolaseactivities, as concluded from the measurements of the intracel-lular levels of gluconeogenic intermediates in isolated proximaltubules (cf. Fig. 2). The tubules incubated with TEMPOL not onlyexhibited lowered intracellular PEP content but it was accompa-nied by the accumulation of pyruvate and malate, suggesting thatthe inhibitory effect of this compound on PEPCK activity might beeven stronger than that observed upon addition of apocynin intothe incubation medium. However, the idea of TEMPOL applicationin vivo would raise serious controversies, as its double-edged actionin diabetic nephropathy has been described [45].

Our hypothesis that decreased level of reactive oxygen speciesmight lead to the diminished gluconeogenic activity of the cellseems to be in agreement with the recent findings emphasizingthe important role of redox state in the regulation of cellu-lar metabolism [46,47]. However, considering the short time ofresponse (1 h of incubation in the presence of apocynin), theobserved inhibition of glucose formation should not be contributedto the changes in gene expression level, but rather in the activitiesof already existing gluconeogenic enzymes.

In our in vivo studies (cf. Fig. 3), in agreement with the previ-ous observations of the other authors [14,15], apocynin, applied asthe solution in drinking water, has turned out to attenuate hyper-glycaemia in diabetic animals. In view of the data from the in

ogical

vTigtoMhttrtNoi

otc[oiaebloic[rabifc

5

tsfcabatttii

C

A

Pshso

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

K. Winiarska et al. / Chemico-Biol

itro experiments performed on isolated renal proximal tubules (cf.able 2), we have postulated that apocynin hypoglycaemic action

n vivo is the consequence of the lowered rate of renal gluconeo-enesis. The comparison of the rate of glucose production in renalubules isolated from apocynin-treated rabbits with that in tubulesf untreated animals (cf. Table 4) has confirmed this hypothesis.oreover, we made similar findings previously, while studying

ypoglycaemic effects of taurine [9]. In case of diabetic rabbitsreated with this amino acid, known for its antioxidative proper-ies, the lowered glycaemia has also resulted from the inhibition ofenal gluconeogenesis and, which seems to be the most intriguing,hose animals have also exhibited the diminished activity of renalADPH oxidase, additionally suggesting that this enzyme might bef importance for the regulation of the process of glucose formationn kidney cortex.

We have chosen alloxan-treated rabbits as an animal modelf diabetes used throughout the experiments, since, in contrasto rodents, this species exhibits the same intracellular, i.e. bothytosolic and mitochondrial, localization as occurs in humans17]. In agreement with the other reports [43,44,48], we havebserved a diabetes-evoked increase in the activity of both PEPCKsoenzymes, which has turned out to be effectively attenuated bypocynin administration (cf. Table 5). It is worth noticing that thexpression of rat hepatic PEPCK gene has been demonstrated toe up-regulated by reactive oxygen species [49,50]. The postu-

ated mechanism of this phenomenon comprises the involvementf redox sensitive p38 MAP kinase [49], which is known to benfluenced by Nox activity [1,51]. Moreover, some plant-derivedompounds of antioxidative properties, including cinnamaldehyde52] and lemon balm (Melissa officinalis) essential oil [53], have beeneported to diminish diabetes-stimulated PEPCK expression and/orctivity. Summarizing, it seems likely that apocynin-evoked inhi-ition of renal NADPH oxidase might affect PEPCK expression level

n these organs and that this phenomenon might be of importanceor lowering the rate of renal glucose formation in vivo and, in aonsequence, for ameliorating hyperglycaemia.

. Conclusions

In view of the data presented in this paper, it is concludedhat: (1) apocynin-evoked decrease in renal gluconeogenesis rateeems to result from the inhibition of NADPH oxidase rather thanrom the direct action of the compound on the activities of glu-oneogenic enzymes; (2) the steps of gluconeogenesis that areffected in the presence of apocynin include the reactions catalysedy PEPCK and/or aldolase; (3) inhibition of renal gluconeogenesisppears to contribute to apocynin hypoglycaemic action in vivo andhe decrease in PEPCK activity might be of crucial importance forhis phenomenon; (4) apocynin exhibits nephroprotective proper-ies in diabetic animals. Thus, it is suggested, that NADPH oxidasenhibitor, apocynin, might be considered as a promising compoundn terms of diabetes treatment.

onflict of interest

The authors declare that there are no conflicts of interest.

cknowledgements

The authors would like to express their deepest gratitude to

rofessor J. Bryla, the Director of Institute of Biochemistry (Univer-ity of Warsaw), for her scientific supervision and comprehensiveelp. They are also very grateful to Professor A. Rychter (Univer-ity of Warsaw) for making available the luminometer for NADPHxidase activity determinations and to Mr M. Usarek for his par-

[

[

Interactions 189 (2011) 119–126 125

ticipation in HFR determinations. The technical assistance of Ms B.Dabrowska and is acknowledged too. The study was supported byState Committee for Scientific Research, KBN, through Faculty ofBiology, University of Warsaw intramural grant, BW 179161/08.

References

[1] M. Ushio-Fukai, Aspects of Nox/Duox signaling, in: C. Jacob, P.G. Winyard (Eds.),Redox Signaling and Regulation in Biology and Medicine, Wiley–VCH VerlagGmbH & Co. KGaA, Weinheim, 2009, pp. 317–349.

[2] P.S. Gill, C.S. Wilcox, NADPH oxidases in the kidney, Antioxid. Redox Signal. 8(2006) 1597–1607.

[3] A. Tojo, K. Asaba, M.L. Onozato, Suppressing renal NADPH oxidase to treatdiabetic nephropathy, Expert Opin. Ther. Targets 11 (2007) 1011–1018.

[4] Y. Li, S. Wang, Glycated albumin activates NADPH oxidase in rat mesangialcells through up-regulation of p47phox, Biochem. Biophys. Res. Commun. 397(2010) 5–11.

[5] T. Etoh, T. Inoguchi, M. Kakimoto, N. Sonoda, K. Kobayashi, J. Kuroda, H.Sumimoto, H. Nawata, Increased expression of NAD(P)H oxidase subunits,NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic ratsand its reversibity by interventive insulin treatment, Diabetologia 46 (2003)1428–1437.

[6] Y Gorin, K. Block, J. Hernandez, B. Bhandari, B. Wagner, J.L. Barnes, H.E. Abboud,Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression inthe diabetic kidney, J. Biol. Chem. 280 (2005) 39616–39626.

[7] Y. Ohshiro, R.C. Ma, Y. Yasuda, J. Hiraoka-Yamamoto, A.C. Clermont, K. Isshiki, K.Yagi, E. Arikawa, T.S. Kern, G.L. King, Reduction of diabetes-induced oxidativestress, fibrotic cytokine expression, and renal dysfunction in protein kinaseCbeta-null mice, Diabetes 55 (2006) 3112–3120.

[8] K Winiarska, D. Malinska, K. Szymanski, M. Dudziak, J.J. Bryla, Lipoic acid ame-liorates oxidative stress and renal injury in alloxan diabetic rabbits, Biochimie90 (2008) 450–459.

[9] K. Winiarska, K. Szymanski, P. Gorniak, M. Dudziak, J. Bryla, Hypoglycaemic,antioxidative and nephroprotective effects of taurine in alloxan diabetic rab-bits, Biochimie 91 (2009) 261–270.

10] H.C. Williams, K.K. Griendling, NADPH oxidase inhibitors: new antihyperten-sive agents? J. Cardiovasc. Pharmacol. 50 (2007) 9–16.

11] J. Stefanska, R. Pawliczak, Apocynin: molecular aptitudes, Mediat. Inflamm.2008 (2008) 106507.

12] J.M. Forbes, M.T. Coughlan, M.E. Cooper, Oxidative stress as a major culprit inkidney disease in diabetes, Diabetes 57 (2008) 1446–1454.

13] W. Wei, Q. Liu, Y. Tan, L. Liu, X. Li, L. Cai, Oxidative stress, diabetes, and diabeticcomplications, Hemoglobin 33 (2009) 370–377.

14] S Furukawa, T. Fujita, M. Shimabukuro, M. Iwaki, Y. Yamada, Y. Nakajima,O. Nakayama, M. Makishima, M. Matsuda, I. Shimomura, Increased oxidativestress in obesity and its impact on metabolic syndrome, J. Clin. Invest. 114(2004) 1752–1761.

15] D.S. Hutchinson, R.I. Csikasz, D.L. Yamamoto, I.G. Shabalina, P. Wikström, M.Wilcke, T. Bengtsson, Diphenylene iodonium stimulates glucose uptake inskeletal muscle cells through mitochondrial complex I inhibition and activationofAMP-activated protein kinase, Cell. Signal. 19 (2007) 1610–1620.

16] J.E. Gerich, C. Meyer, H.J. Woerle, M. Stumvoll, Renal gluconeogenesis: itsimportance in human glucose homeostasis, Diabetes Care 24 (2001) 382–391.

17] M.S. Usatenko, Hormonal regulation of phosphoenolpyruvate carboxykinaseactivity in liver and kidney of adult animals and formation of this enzyme indeveloping rabbit liver, Biochem. Med. 3 (1970) 298–310.

18] A. Kiersztan, A. Modzelewska, R. Jarzyna, J. Bryla, Inhibition of gluconeogenesisby vanadium and metformin in kidney-cortex tubules isolated from controland diabetic rabbits, Biochem. Pharmacol. 63 (2002) 1371–1382.

19] R. Jarzyna, A. Kiersztan, O. Lisowa, J. Bryła, The inhibition of gluconeogenesisby chloroquine contributes to its hypoglycaemic action, Eur. J. Pharmacol. 428(2001) 381–388.

20] J. Zaleski, K. Zablocki, J. Bryla, Short-term effect of glucagon on gluconeogenesisand pyruvate kinase in rabbit hepatocytes, Int. J. Biochem. 14 (1982) 733–739.

21] K. Winiarska, J. Drozak, M. Wegrzynowicz, A.K. Jagielski, J. Bryla, Relationshipbetween gluconeogenesis and glutathione redox state in rabbit kidney cortextubules, Metabolism 52 (2003) 739–746.

22] F.C. Cheng, J.F. Jen, T.H. Tsai, Hydroxyl radical in living systems and its separa-tion methods, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 781 (2002)481–496.

23] Y. Chen, P.S. Gill, W.J. Welch, Oxygen availability limits renal NADPH-dependentsuperoxide production, Am. J. Physiol. Renal Physiol. 289 (2005) 749–753.

24] Y.I. Chirino, D.J. Sánchez-González, C.M. Martínez-Martínez, C. Cruz, J. Pedraza-Chaverri, Protective effects of apocynin against cisplatin-induced oxidativestress and nephrotoxicity, Toxicology 245 (2008) 18–23.

25] E.J. Harris, C. Tate, J.R. Manger, J.A. Bangham, The effects of colloids on theappearance and substrate permeability of rat liver mitochondria, J. Bioenerg. 2

(1971) 221–232.

26] M.J. MacDonald, L.A. Bentle, H.A. Lardy, P-enolpyruvate carboxykinase ferroac-tivator. Distribution, and the influence of diabetes and starvation, J. Biol. Chem.253 (1978) 116–124.

27] H.U. Bergmeyer, Methods in Enzymatic Analysis, Verlag Chemie GmbH, Wein-heim, Basel, 1983.

1 ogical

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[of cinnamaldehyde on transcriptional regulation of pyruvate kinase, phospho-

26 K. Winiarska et al. / Chemico-Biol

28] L.A. Bentle, H.A. Lardy, Interactions of anions and divalent metal ions withphosphoenolpyruvate carboxykinase, J. Biol. Chem. 251 (1976) 2916–2921.

29] D.R. Yeltman, B.G. Harris, Fructose-bisphosphate aldolase from human erythro-cytes, Methods Enzymol. 90 (1982) 251–254.

30] L.A. Ridnour, R.A. Winters, N. Ercal, D.R. Spitz, Measurement of glu-tathione, glutathione disulfide, and other thiols in mammalian cell and tissuehomogenates using high-performance liquid chromatography separation ofN-(1-pyrenyl)maleimide derivatives, Methods Enzymol. 299 (1999) 258–267.

31] F Da Fonseca-Wollheim, K.G. Heinze, The influence of pCO2 on the rate ofammonia formation in blood, Eur. J. Clin. Chem. Clin. Biochem. 30 (1992)867–869.

32] M. Michalik, I. Biedermann, T. Lietz, J. Bryla, Recovery of impaired gluconeoge-nesis in kidney cortex tubules of gentamicin-treated rabbits, Pharmacol. Res.23 (1991) 259–269.

33] E. Layne, Spectrophotometric and turbidimetric methods for measuring pro-tein, in: S.P. Colowick, N.O. Kaplan (Eds.), Methods in Enzymology, AcademicPress, New York, 1957, pp. 447–454.

34] C.S. Wilcox, Effects of tempol and redox-cycling nitroxides in models of oxida-tive stress, Pharmacol. Ther. 126 (2010) 119–145.

35] J.D. Lambeth, T. Kawahara, B. Diebold, Regulation of Nox and Duox enzymaticactivity and expression, Free Radic. Biol. Med. 43 (2007) 319–331.

36] L.R. Castor, K.A. Locatelli, V.F. Ximenes, Pro-oxidant activity of apocynin radical,Free Radic. Biol. Med. 48 (2010) 1636–1643.

37] C. Jacob, M. Doering, T. Burkholz, The chemical basis of biological redox control,in: C. Jacob, P.G. Winyard (Eds.), Redox Signaling and Regulation in Biology andMedicine, Wiley–VCH Verlag GmbH & Co. KGaA, Weinheim, 2009, pp. 63–122.

38] K. Asaba, A. Tojo, M.L. Onozato, A. Goto, M.T. Quinn, T. Fujita, C.S. Wilcox, Effectsof NADPH oxidase inhibitor in diabetic nephropathy, Kidney Int. 67 (2005)1890–1898.

39] K. Susztak, A.C. Raff, W. Schiffer, E.P. Böttinger, Glucose-induced reactive oxy-gen species cause apoptosis of podocytes and podocyte depletion at the onsetof diabetic nephropathy, Diabetes 55 (2006) 225–233.

40] S.M. Nam, M.Y. Lee, J.H. Koh, J.H. Park, J.Y. Shin, J.G. Shin, S.B. Koh, E.Y. Lee, C.H.Chung, Effects of NADPH oxidase inhibitor on diabetic nephropathy in OLETFrats: The role of reducing oxidative stress in its protective property, DiabetesRes. Clin. Pract. 83 (2009) 176–182.

41] K. Winiarska, J. Drozak, M. Wegrzynowicz, T. Fraczyk, J. Bryla, Diabetes-inducedchanges in glucose synthesis, intracellular glutathione status and hydroxyl free

[

Interactions 189 (2011) 119–126

radical generation in rabbit kidney-cortex tubules, Mol. Cell. Biochem. 261(2004) 91–98.

42] M. Stumvoll, C. Meyer, G. Perriello, M. Kreider, S. Welle, J. Gerich, Human kid-ney and liver gluconeogenesis: evidence for organ substrate selectivity, Am. J.Physiol. 274 (1998) E817–E826.

43] S.D. Nandan, E.G. Beale, Regulation of phosphoenolpyruvate carboxykinasemRNA in mouse liver, kidney, and fat tissues by fasting, diabetes, and insulin,Lab. Anim. Sci. 42 (1992) 473–477.

44] A. Barthel, D. Schmoll, Novel concepts in insulin regulation of hepatic gluco-neogenesis, Am. J. Physiol. Endocrinol. Metab. 285 (2003) E685–E692.

45] K. Asaba, A. Tojo, M.L. Onozato, A. Goto, T. Fujita, Double-edged action ofSOD mimetic in diabetic nephropathy, J. Cardiovasc. Pharmacol. 49 (2007)13–19.

46] Y.M. Janssen-Heininger, B.T. Mossman, N.H. Heintz, H.J. Forman, B. Kalyanara-man, T. Finkel, J.S. Stamler, S.G. Rhee, A. van der Vliet, Redox-based regulationof signal transduction: principles, pitfalls, and promises, Free Radic. Biol. Med.45 (2008) 1–17.

47] C. Jacob, P.G. Winyard (Eds.), Redox Signaling and Regulation in Biology andMedicine, Wiley–VCH Verlag GmbH & Co. KGaA, Weinheim, 2009.

48] P.G. Quinn, D. Yeagley, Insulin regulation of PEPCK gene expression: a model forrapid and reversible modulation, Curr. Drug. Targets Immune. Endocr. Metabol.Disord. 5 (2005) 423–437.

49] Y. Ito, S. Oumi, T. Nagasawa, N. Nishizawa, Oxidative stress induces phos-phoenolpyruvate carboxykinase expression in H4IIE cells, Biosci. Biotechnol.Biochem. 70 (2006) 2191–2198.

50] K. Kimura, S. Tawara, K. Igarashi, A. Takenaka, Effect of various radical gen-erators on insulin-dependent regulation of hepatic gene expression, Biosci.Biotechnol. Biochem. 71 (2007) 16–22.

51] D.I. Brown, K.K. Griendling, Nox proteins in signal transduction, Free Radic. Biol.Med. 47 (2009) 1239–1253.

52] P. Anand, K.Y. Murali, V. Tandon, P.S. Murthy, R. Chandra, Insulinotropic effect

enolpyruvate carboxykinase, and GLUT4 translocation in experimental diabeticrats, Chem. Biol. Interact. 186 (2010) 72–81.

53] M.J. Chung, S.Y. Cho, M.J. Bhuiyan, K.H. Kim, S.J. Lee, Anti-diabetic effects oflemon balm (Melissa officinalis) essential oil on glucose- and lipid-regulatingenzymes in type 2 diabetic mice, Br. J. Nutr. 140 (2010) 180–188.