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Differential effects of vanadium, tungsten and molybdenum on inhibition of glucose formation in renal tubules and hepatocytes of control and diabetic rabbits: Beneficial action of melatonin and N-acetylcysteine


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Effect of vanadyl acetylacetonate (VAc), tungstate and molybdate on gluconeogenesis has been studied in isolated hepatocytes and kidney-cortex tubules. In renal tubules of control and alloxan-diabetic animals, the rank order of the metal-compounds-induced (i) inhibition of glucose formation from alanine+glycerol+octanoate or aspartate+glycerol+octanoate, (ii) decrease in the mitochondrial membrane potential (delta psim), (iii) increase in the hydroxyl free radicals (HFR) generation and (iv) decline in glucose-6-phosphatase activity was the following: VAc > tungstate > molybdate. Moreover, in contrast to VAc, both tungstate and molybdate at 100 microM concentration did not practically decrease glucose production in hepatocytes isolated from diabetic rabbits, and significantly increased the rate of lactate formation in renal tubules. N-acetylcysteine at 2 mM concentration partially attenuated vanadium-induced alterations in glucose formation, delta psim and the cellular glutathione redox state, whereas 0.1 mM melatonin did not abolish vanadium-induced changes in gluconeogenesis despite attenuation of vanadium effects on HFR formation and delta psim decline. However, similarly to control rabbits, following 6 days of intraperitoneal administration of both VAc (1.275 mg V/kg body weight daily) and melatonin (1 mg/kg body weight daily) to alloxan-diabetic animals, vanadium-induced elevated serum creatinine and urea levels were decreased, indicating the beneficial effect of melatonin on diabetes- and vanadium-induced nephrotoxicity in rabbits. As serum glucose levels were also significantly diminished by vanadium+melatonin treatment of diabetic animals, the combination therapy of vanadium compounds and melatonin needs a careful evaluation.
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Molecular and Cellular Biochemistry 261: 9–21, 2004.
2004 Kluwer Academic Publishers. Printed in the Netherlands.
Differential effects of vanadium, tungsten and
molybdenum on inhibition of glucose formation
in renal tubules and hepatocytes of control and
diabetic rabbits: Beneficial action of melatonin
and N-acetylcysteine
A. Kiersztan, K. Winiarska, J. Drozak, M. Przedlacka,
M. Wegrzynowicz, T. Fraczyk and J. Bryla
Department of Metabolic Regulation, Institute of Biochemistry, Warsaw University, Warszawa, Poland
Effect of vanadyl acetylacetonate (VAc), tungstate and molybdate on gluconeogenesis has been studied in isolated hepa-
tocytes and kidney-cortex tubules. In renal tubules of control and alloxan-diabetic animals, the rank order of the metal-
compounds-induced (i) inhibition of glucose formation from alanine + glycerol + octanoate or aspartate + glycerol + octanoate,
(ii) decrease in the mitochondrial membrane potential (
), (iii) increase in the hydroxyl free radicals (HFR) generation and
(iv) decline in glucose-6-phosphatase activity was the following: VAc > tungstate > molybdate. Moreover, in contrast to VAc,
both tungstate and molybdate at 100 µM concentration did not practically decrease glucose production in hepatocytes iso-
lated from diabetic rabbits, and significantly increased the rate of lactate formation in renal tubules. N-acetylcysteine at 2 mM
concentration partially attenuated vanadium-induced alterations in glucose formation, 
and the cellular glutathione redox
state, whereas 0.1 mM melatonin did not abolish vanadium-induced changes in gluconeogenesis despite attenuation of vana-
dium effects on HFR formation and 
decline. However, similarly to control rabbits, following 6 days of intraperitoneal
administration of both VAc (1.275 mg V/kg body weight daily) and melatonin (1 mg/kg body weight daily) to alloxan-diabetic
animals, vanadium-induced elevated serum creatinine and urea levels were decreased, indicating the beneficial effect of mela-
tonin on diabetes- and vanadium-induced nephrotoxicity in rabbits. As serum glucose levels were also significantly diminished
by vanadium + melatonin treatment of diabetic animals, the combination therapy of vanadium compounds and melatonin needs
a careful evaluation. (Mol Cell Biochem 261: 9–21, 2004)
Key words: diabetes, gluconeogenesis, glutathione, melatonin, molybdate, N-acetylcysteine, nephrotoxicity, renal tubules,
tungstate, vanadyl acetylacetonate
Vanadium derivatives, tungstate and molybdate have
recently been identified as possessing insulin-mimetic
Address for offprints:J.Bryla, Department of Metabolic Regulation, Institute of Biochemistry, Warsaw University, Ul. Miecznikowa 1, 02-096 Warszawa,
Poland (E-mail:
properties when given orally to either diabetic patients or
diabetic animals [1]. However, the mechanisms by which
these compounds induce their metabolic effects in vivo re-
main poorly understood, despite intensive research.
Forseveral years there has been a great interest in devel-
oping new vanadium compounds as potential oral insulin
alternatives in diabetes treatment. VAc, organically chelated
vanadium, is more potent than inorganic vanadium deriva-
tives in correcting the hyperglycaemia [2, 3]. Moreover, it is
of a greater hydrolytic and redox stability in comparison with
other vanadium compounds [4]. The increased phosphoryla-
tion of proteins in the insulin signalling pathway appears to
be related to the inhibition of protein tyrosine phosphatase
activity by vanadium salts [5]. However, recently it has also
been reported that vanadium may act by enhancing insulin
effects rather than mimicking the action of this hormone
[5, 6], while improvement of glucose homeostasis induced
by molybdate seems to be exerted distally to the insulin
receptor tyrosine kinase step [7].
Despite an impressive anti-diabetic action, vanadium com-
pounds have been associated with several toxic effects, as
concluded from elevated serum urea and creatinine levels in
vanadium-treated rabbits [8] and rats [9]. Some studies have,
however, failed to detect changes in the level of urea, creati-
nine and transaminases, indicating no toxic action on kidney
and liver functions [10, 11]. In contrast to vanadium deriva-
tives, tungstate has been shown to evoke no side effects dur-
ing a long-term application [12]. However, as for any metals,
tissue accumulation and potential toxicity derived from the
chronic use of vanadium compounds, tungstate or molybdate
cannot be dismissed.
In view of observations (i) that kidney in addition to
liver is important to whole body glucose homeostasis [13],
(ii) that renal glucose production in type 1 diabetic patients
is increased proportionately to systemic glucose appearance
[14], (iii) that there are differences in intracellular mecha-
nisms responsible for regulation of gluconeogenesis in hep-
atocytes and renal tubules [15], and (iv) that sensitivity of
kidney-cortex tubules to vanadium and metformin appears to
be higher than in hepatocytes [8], the action of drugs on gluco-
neogenesis in both hepatocytes and renal tubules merits fur-
ther scientific attention and may be of importance in diabetes
therapy. The aim of the present investigation was to study the
effect of vanadium, tungsten and molybdenum compounds
on glucose formation in both hepatocytes and kidney-cortex
tubules isolated from control and alloxan-diabetic rabbits,
which exhibit similar to human intracellular localisation of
gluconeogenic enzymes [16].
Materials and methods
Animals and isolation of kidney-cortex tubules, hepatocytes,
cytosol and mitochondria
The experiments were performed with male white Termond
rabbits weighing approximately 1–1.5 kg. Animals were
maintained on the standard rabbit chow with free access to
water and food and starved for 40 h before experiments.
Alloxan-diabetes was induced by the single injection of
alloxan (150 mg/kg body weight) [8]. Only those alloxan-
treated animals that exhibited decreased or stabilised body
weight and blood glucose concentration in excess of
300 mg/100 ml 3 days after treatment were considered di-
abetic and used for experiments. All animal use procedures
were approved by the First Warsaw Local Commission for
the Ethics of Experimentation on Animals.
Rabbit kidney-cortex tubules and hepatocytes were ob-
tained according to the method in [17]. Approximately 95%
of both hepatocytes and renal tubules excluded Trypan blue.
Mitochondria for measurements of the mitochondrial mem-
brane potential (
) were isolated from the kidney-cortex
using solution containing 225 mM mannitol, 75 mM sucrose,
5mM3-(N-morpholino)propanesulphonic acid (MOPS),
0.1 mM ethylenediaminetetraacetate (EDTA) and 1% BSA
(pH adjusted to 7.2 with Tris) [18], but the final wash was
made with 0.3 M mannitol and the mitochondrial precipitate
was suspended in the mixture containing 210 mM sucrose,
70 mM mannitol and 3 mM MOPS (pH 7.2).
Mitochondrial fractions for GSH and GSSG determina-
tions were obtained from isolated kidney-cortex tubules in-
cubated for1hinthepresence of substrates and effectors, fol-
lowing the treatment with digitonin and centrifugation [19].
The pellets were extracted with either 12% perchloric acid
(PCA) or 50 mM N-ethylmaleimide (NEM) in 12% PCA
for measurements of reduced (GSH) and oxidised (GSSG)
forms, respectively [20].
Cytosol for measurement of glucose-6-phosphatase and
glutathione reductase activities was obtained from kidney-
cortex homogenised in 0.9% NaCl solution (5 ml per 1 g of
tissue), following differential centrifugations. The final pre-
cipitate was suspended in 0.9% NaCl and used for glucose-6-
phosphatase determination, while glutathione reductase ac-
tivity was assayed in the supernatant fraction.
Incubation of renal tubules and hepatocytes
Both isolated kidney-cortex tubules and hepatocytes were
incubated at 37
Cin25mlErlenmeyer flask sealed with
rubber stoppers under atmosphere of O
(95%:5%) in
Krebs-Ringer bicarbonate buffer, pH 7.4, in the presence of
substrates indicated in legends to figures and tables. The rates
of gluconeogenesis under all conditions studied were linear
at least for 90 min of incubation. Reactions were stopped
following 60 min of incubation by either the addition of 1 ml
sample to 0.1 ml of 35% PCA or centrifugation of kidney-
cortex tubules suspension through the silicon oil layer to 1 ml
of 12% PCA [21]. To avoid non-enzymatic GSH oxidation,
samples used for GSSG determinations were centrifuged into
50 mM NEM in 12% PCA [20]. Excess of NEM was removed
by hexane extraction. Samples tested for GSH determination
were stored as PCA extracts, while the others were neutralised
immediately after deproteinisation.
Measurement of the mitochondrial membrane potential
was assessed with 5,5
tetraethylbenzimidazolocarbocyanine iodide (JC-1), exhibit-
ing the membrane potential-dependent accumulation in mi-
tochondria accompanied by a shift of fluorescence emission
from green (525 nm) to red (595 nm). Fluorescence mea-
surements were performed using Shimadzu spectrofluorime-
ter RF-5301 PC under gentle stirring, with excitation wave-
length at 490 nm and emission wavelength at 595 nm [22].
Mitochondria (0.1 mg of protein/ml) were added to the ther-
mostated (at 37
C) cuvette, containing 110 mM KCl, 1 mM
EGTA, 20 mM MOPS/K (pH 7.4), 5 mM succinate and 2 µM
rotenone. JC-1 (5 mg/ml) dissolved in 40% dimethyl sulfox-
ide (DMSO) was diluted in PBS buffer up to 7.7 µM and
added at 2 µM final concentration, causing an increase in the
fluorescence within 10–15 min, resulting from J-aggregate
Determination of the hydroxyl free radicals
Hydroxyl free radicals (HFR) were estimated as 2,3-
dihydroxybenzoic acid (DHBA) generated in the presence
of sodium salicylate (SAL) [23]. Isolated renal tubules were
pre-incubated for 10 min with 5 mM pyruvate and 1 mM SAL
in the absence or presence of 0.1 mM melatonin or 2 mM N-
acetylcysteine (NAC) before the addition of VAc, tungstate
or equivalent volume of vehicle. After 60 min of incubation,
1mlsamples were withdrawn and acidified with 0.1 ml
of 35% PCA, containing 1 mM EDTA and 4 mM sodium
metabisulphite (Na
), and centrifuged. Supernatants
were placed on ice and analysed on the day of experiment.
Analytical methods
Glucose-6-phosphatase activity was assayed spectrophoto-
metrically [24], while glutathione reductase activity was de-
termined fluorimetrically [25].
Glucose, lactate, pyruvate, malate, phosphoenolpyruvate,
triose phosphates, 3-phosphoglycerate + 1,3-bisphospho-
glycerate, fructose-1,6-bisphosphate, fructose-6-phosphate,
glucose-6-phosphate and GSSG were estimated either spec-
trophotometrically or fluorimetrically [25 Bergmeyer 1983].
GSH levels were determined by HPLC (Beckman Instru-
ments) after derivatization with N-(1-pyrenyl)maleimide
(NPM) [26]. Blood glucose was analysed with hexokinase
and glucose-6-phosphate dehydrogenase [25], while urea was
measured in blood plasma as ammonium [27], following
treatment of neutralised samples with urease. Creatinine was
determined by the reaction of Jaffe as described by Michalik
et al. [28].
2,3-DHBA assays were performed by HPCL using Beck-
man Ultrasphere ODS column. The mobile phase consisted
of 50 mM NaH
, 1.125 mM octanesulphonic acid, 0.2
mM EDTA, 3% methanol and 5.5% acetonitrile (v/v). pH
was adjusted to 2.8 with1Mortophosphoric acid. The Beck-
man 110B System Gold HPLC (Beckman, USA) equipped
with a Bio-Rad 1640 electrochemical detector (Bio-Rad,
USA) and a glassy carbon working electrode operating at
+0.75 V against Ag/AgCl reference electrode and a detec-
tion range of 2 nA was used. The flow rate was 1 ml/min and
all separations were performed at 30
C. Quantification was
achieved using external standard of 2,3-DHBA. Data from the
detector were collected and integrated by PC equipped with
appropriate interface and chromatography software. Mela-
tonin and NAC did not interfere with the measurement of
Protein content in both mitochondrial and cytosolic frac-
tions was determined spectrophotometrically according to
[29] and [30], respectively.
Enzymes, coenzymes and nucleotides for metabolites deter-
mination were from Roche (Mannheim, Germany). VAc and
JC-1 were from Aldrich (Milwaukee, WI, USA) and Molecu-
lar Probes (Leiden, The Netherlands), respectively. All other
chemicals were from Sigma (St. Louis, MO, USA).
Expression of results
Data shown are means ± S.D. for at least three separate
experiments, each one obtained from a different animal.
The statistical evaluation was performed using ANOVA two-
factor unrepeated test. Values of p < 0.05 were considered
Glucose and lactate synthesis
As rabbit kidney-cortex tubules efficiently produce glucose
from amino acids only in the presence of glycerol and either
fatty acids [31] or ketone bodies [32], in all experiments with
the use of amino acids (at 2 mM concentration) rabbit renal
tubules were incubated with both 2 mM glycerol and 0.5 mM
octanoate. Pyruvate was added at 5 mM concentration.
Fig. 1.Effect of increasing concentrations of VAc, tungstate and molybdate on glucose formation in rabbit kidney-cortex tubules incubated in the presenceof
2mMalanine + 2mMglycerol + 0.5 mM octanoate. Values are means ± S.D. for 8–10 separate experiments.
The data presented in Fig. 1 show that increasing concen-
trations of VAc were more potent than those of tungstate and
molybdate in inhibition of glucose formation in kidney-cortex
tubules incubated with alanine + glycerol + octanoate. At
100 µM concentration VAc decreased this process by about
60%, while tungstate and molybdate achieved 60–70% of in-
hibition at 500 µM concentration. Moreover, in contrast to
VAc, tungstate and molybdate caused a significant increase
in lactate production (from 105.8 ± 9.6 to 136.9 ± 12.2 and
117.4 ± 10.5 nmol/h per mg dry weight in the absence and
presence of 200 µM tungstate or molybdate and up to 184.5 ±
14.8 and 136.1 ± 8.2 in the presence of 500 µM tungstate and
molybdate, respectively). As in the presence of 200 µMVAc
the intracellular ATP (adenosine triphosphate) level was sig-
nificantly decreased [8], in following experiments VAc was
used at 100 µM concentration, while tungstate and molybdate
were added at either 100 or 200 µM concentrations.
Inhibition of gluconeogenesis by 100 µMVAc was much
less significant in hepatocytes (by about 20–30%) than in re-
nal tubules (by about 40–60%), depending on the substrate
used. The action of tungstate was similar in both renal tubules
and hepatocytes, while molybdate did not practically affect
this process in hepatocytes (Fig. 2). Moreover, in diabetic rab-
bit renal tubules, glucose formation was increased by about
30–40% in comparison with values determined for control an-
imals, whereas the rate of this process in diabetic hepatocytes
was overtwofold higher than in control animals. However,
the inhibitory effect of VAc on glucose formation was sim-
ilar in tubules and hepatocytes of both control and diabetic
rabbits, whereas tungstate and molybdate practically did not
affect glucose production in hepatocytes of alloxan-treated
animals at 100 µMaswell as 200 µM concentrations (data
not shown), indicating a lower sensitivity of liver than kidney
towards tungstate and molybdate treatments.
Gluconeogenic intermediates and enzyme activities
In order to identify the steps responsible for inhibition of
glucose synthesis pathway in renal tubules we have mea-
sured both tungstate- and molybdate-induced changes in glu-
coneogenic intermediate levels in kidney-cortex tubules in-
cubated with alanine + glycerol + octanoate. Both tungstate
and molybdate resulted in the most significant accumula-
tion of glucose-6-phosphate contents accompanied by dimin-
ished intracellular glucose levels (Fig. 3), indicating a de-
crease in glucose-6-phosphatase activity. A potent inhibitory
effect of vanadium and tungstate on this enzyme activity
was also established using the microsomal fraction of kid-
ney cortex, while molybdate action was less pronounced
(Fig. 4). Although on the addition of 0.05% Triton X-100
the glucose-6-phosphatase activity was decreased, however,
the inhibitory effect of vanadium was significantly attenuated
(about 50% inhibition at 100 µMVAc) while tungstate and
molybdate at 100 µM concentrations increased the enzyme
activity for about 10 and 100%, respectively, indicating that
in the intact microsomes these metals might also affect trans-
porters of glucose-6-phosphate, phosphate and/or glucose,
Fig. 2.Effect of 100 µMVAc, tungstate and molybdate on glucose formation in hepatocytes and kidney-cortex tubules isolated from control and diabetic
rabbits. Renal tubules were incubated in the presence of either 2 mM alanine or 2 mM aspartate in the presence of 2 mM glycerol and 0.5 mM octanoate, where
indicated, while isolated hepatocytes were incubated with either 5 mM alanine + 2mMglycerol + 0.5 mM octanoate or 5 mM pyruvate, where indicated.
Values are means ± S.D. for 5–9 separate experiments. Statistical significance:
p < 0.05,
p < 0.01,
p < 0.005, compared with control values determined
in the absence of VAc, tungstate and molybdate.
which are integral constituents of multicomponent glucose-
6-phosphatase enzyme system [33].
Mitochondrial membrane potential measurements
As in kidney-cortex tubules incubated with 100 µMVAcor
200 µM tungstate, the intracellular ATP level was reduced
by about 30% (data not shown), measurements of 
the use of JC-1 have been performed in the absence and pres-
ence of VAc, tungstate and molybdate. The addition of VAc
resulted in the concentration-dependent reduction of the red
fluorescence, indicating dissociation of the J-aggregates and a
decrease in the mitochondrial membrane potential. As shown
in Fig. 5, at both 50 µMVAcand 200 µM tungstate the flu-
orescence value diminished much less (by about 85 arbitrary
units) than at 100 µMVAc (by almost 350 units). In addition,
molybdate, which at 200 µM concentrations practically did
not affect the cellular ATP content, also produced a small
decrease in 
(by about 20 units of fluorescence). How-
ever, following pre-incubation of mitochondria with 100 µM
melatonin, the scavenger of reactive oxygen species (ROS)
and/or metal-chelating agent [34], VAc- or tungstate-induced
decrease in 
was attenuated as concluded from the re-
duced fluorescence decrease by about 50%. NAC, another
antioxidative agent [35], appeared to be less effective than
melatonin. The addition of cyclosporine A, inhibitor of the
mitochondrial permeability transition pore [36], which has
also been reported to protect against decrease in 
did not reduce the fluorescence decline induced by VAc, de-
spite elevation of the basal level of fluorescence above that
determined for the control conditions (data not shown). Thus,
it seems that VAc action related to decrease in 
is not as-
sociated with the mitochondrial permeability transition pore
Changes in HFR generation
As shown in Table 1, in contrast to tungstate, VAc in-
duced an increase (by about fourfold) in the concentration
Fig. 3. Influence of 100 µMVAc, tungstate and molybdate on changes
in the intracellular levels of gluconeogenic intermediates in renal tubules
incubated for 1 h in the presence of 2 mM alanine + 2mMglycerol +
0.5 mM octanoate. The concentrations of intracellular metabolites in the
presence of VAc, tungstate and molybdate are expressed as percentage of
control values measured with no metals added to the incubation mixture.
The control values (expressed in nmol · mg
dry weight) for metabolites
listed from left to right are pyruvate (Pyr) 0.29 ± 0.04, malate (Mal) 0.46 ±
0.04, phosphoenolpyruvate (PEP) 0.27 ± 0.03, 3-phosphoglycerate + 1,3-
bisphosphoglycerate (PGA) 1.66 ± 0.09, 3-phosphoglyceraldehyde + phos-
phodihydroksyacetone (TP) 0.29 ± 0.04, fructose-1,6-bisphosphate (FBP)
0.17 ± 0.03, fructose-6-phosphate (F6P) 0.06 ± 0.03, glucose-6-phosphate
(G6P) 0.26 ± 0.02 and glucose (G) 5.56 ± 0.30. Values are means ± S.D.
for 6–8 separate experiments. Statistical significance:
p < 0.05,
p < 0.01,
p < 0.005 compared with control values measured in the absence of VAc,
tungstate and molybdate.
of 2,3-DHBA, a marker of HFR generation. Following pre-
incubation of renal tubules with melatonin, 2,3-DHBA accu-
mulation was significantly decreased both in the absence (by
about 25%) and presence (by about 40%) of these metals.
Surprisingly, NAC did not change the metal-induced HFR
Table 1. Effect of VAc, melatonin and NAC on 2,3-DHBA production
in rabbit kidney-cortex tubules incubated with pyruvate
2,3-DHBA production
Additions (pmol · h
· mg
dry weight)
None 75.3 ± 3.7
VA c 303.2 ± 20.9
Tungstate 79.9 ± 7.0
Melatonin 57.3 ± 3.9
NAC 83.4 ± 3.1
VA c + melatonin 183.8 ± 39.4
VA c + NAC 286.3 ± 35.5
Tungstate + melatonin 46.1 ± 5.5
Tungstate + NAC 85.3 ± 9.7
Renal tubules were pre-incubated for 10 min with 5 mM pyruvate,
1mMSALand 100 µM melatonin or 2 mM NAC before addition of
100 µMVAcor 200 µM tungstate, where indicated. Further incubation
was carried out for 60 min as described in Materials and Methods.
Values are means ± S.D. for 3 experiments. Statistical significance:
p < 0.005 compared with control values determined in the absence of
any addition,
p < 0.05 compared with value measured in the presence
of VAc or tungstate, respectively.
Alterations in glutathione content and redox state
VAc-induced inhibition of gluconeogenesis from aspartate
+ glycerol + octanoate was accompanied by about a twofold
decline in the intracellular GSH level and about 40% increase
in GSSG content, resulting in about 60% decrease in the in-
tracellular GSH/GSSG ratio (Table 2). Despite lowering the
rate of the glucose synthesis by about 30%, both tungstate
and molybdate changed neither the intracellular glutathione
content nor the glutathione redox state (data not shown). Dif-
ferences in vanadium, tungsten and molybdenum actions on
the intracellular glutathione status might be due to differen-
tial effects of these metals on glutathione reductase activity,
as only VAc turned out to be a potent inhibitor of this enzyme,
resulting in 20% inhibition of the enzyme activity at 100 µM
concentration (Fig. 6). This suggestion is supported by a par-
tial attenuation of vanadium-induced effects on both glucose
synthesis and the intracellular glutathione redox state in the
presence of 2 mM NAC, an ant-ioxidant and cysteine precur-
sor that increased the intracellular glutathione content [35].
Moreover, in the presence of NAC the intracellular GSH con-
tent was not altered upon the addition of VAc, while decrease
in the intracellular GSH/GSSG ratio was due to elevation of
GSSG content. In contrast to NAC, melatonin failed to re-
verse the effects of vanadium on both gluconeogenesis and
glutathione redox state (data not shown).
The action of VAc on the mitochondrial glutathione
changes was similar to that observed in renal tubules
(Table 3). In the presence of VAc the mitochondrial GSH
content was diminished by 25%, while GSSG level was in-
creased by about 40%, resulting in decrease in GSH/GSSG
ratio by about 35%.
Fig. 4.Effect of increasing concentrations of VAc, tungstate and molybdate on glucose-6-phosphatase activity in the kidney-cortex microsomal fraction.
Glucose-6-phosphate was added at 5 mM concentration. Values are means ± S.D. for 5–7 separate experiments performed in duplicate.
Fig. 5.Effect of VAc, tungstate, molybdate, NAC and melatonin on the mitochondrial membrane potential. At indicated concentrations, VAc, tungstate or
molybdate was added into kidney-cortex mitochondria (0.1 mg of protein/ml) following loading with JC-1 for about 10–15 min. Melatonin (100 µM) and NAC
(2 mM) were pre-incubated with mitochondria prior to the addition of VAc or tungstate, where indicated.
Table 2. Effect of VAc and NAC on glucose formation, intracellular GSH and GSSG contents and GSH/GSSG ratio in rabbit kidney-cortex tubules
incubated with aspartate, glycerol and octanoate
Glucose formation
Additions (nmol · h
· mg
dry weight) GSH (nmol · mg
dry weight) GSSG (nmol · mg
dry weight) GSH/GSSG
None 109.2 ± 8.3 2.96 ± 0.18 0.049 ± 0.006 60.9 ± 8.2
VA c 60.0 ± 8.0
1.58 ± 0.21
0.068 ± 0.008
23.2 ± 3.8
NAC 107.8 ± 9.1 3.84 ± 0.19
0.074 ± 0.008
54.7 ± 8.0
VA c + NAC 82.6 ± 9.9
3.70 ± 0.24
0.093 ± 0.010
39.0 ± 5.2
Renal tubules were incubated for 60 min in the presence of 2 mM aspartate + 2mMglycerol + 0.5 mM octanoate. VAc and NAC were added at 100
µM and 2 mM concentrations, respectively. Values are means ± S.D. for 3–5 experiments. Statistical significance:
p < 0.05 compared with control
values determined in the absence of both VAc and NAC,
p < 0.05 compared with values measured in the presence of VAc.
Table 3. Effect of VAc on mitochondrial GSH and GSSG contents and GSH/GSSG ratio in rabbit kidney-
cortex tubules incubated with aspartate, glycerol and octanoate
Additions GSH (nmol · mg
dry weight) GSSG (nmol · mg
dry weight) GSH/GSSG
None 0.434 ± 0.074 0.016 ± 0.003 17.5 ± 2.3
VA c 0.325 ± 0.056
0.023 ± 0.005
11.4 ± 3.3
Renal tubules were incubated for 60 min in the presence of 2 mM aspartate + 2mMglycerol + 0.5 mM
octanoate. VAc was added at 100 µM concentration. Mitochondria were isolated as described in Material
and Methods. Values are means ± S.D. for 3–5 experiments. Statistical significance:
p < 0.05 compared
with control values determined in the absence of VAc.
Blood glucose, urea and creatinine levels in vanadium- and
melatonin-treated rabbits
As melatonin has been reported (i) to exhibit metal-chelating
and ROS scavenging effects [34] and (ii) to attenuate sig-
nificantly VAc action on the 
(cf. Fig. 7), the insulin-
mimetic effect of VAc was investigated in alloxan-treated
rabbits in comparison with that exhibited in control animals
in the absence and presence of melatonin. At the dose of
Fig. 6.VAc-induced inhibition of glutathione reductase activity in the cy-
tosolic fraction of kidney cortex isolated from control rabbits. Values are
means ± S.D. for 3–5 separate experiments. All determinations were per-
formed in duplicate.
1.275 mg of vanadium per kg body weight, VAc was adminis-
tered intraperitoneally to rabbits for 6 days to bypass gastroin-
testinal tract [8] with or without melatonin (at the dose of 1 mg
per kg body weight [38]). As reported previously [8], during
6 days of VAc treatment the blood glucose content in con-
trol animals did not change despite 30% decrease in the rabbit
body weight. On the contrary, blood glucose levels of diabetic
animals were progressively falling down to achieve control
values on 1–2 days following VAc withdrawal regardless of
no differences in the body weight [8]. It is necessary, however,
to point out that in the absence of melatonin, plasma urea and
creatinine levels were markedly elevated following 6 days of
VA c treatment of control animals (from 18.7 ± 1.7 and 1.27
± 0.2 mg/dl up to 132.8 ± 9.7 and 2.2 ± 0.3 mg/dl, respec-
tively, N = 5, p < 0.05), while administration of both mela-
tonin and VAc protected against vanadium-induced elevation
of both creatinine and urea levels (cf. Fig. 7). In diabetic rab-
bits, in the absence of melatonin, plasma urea and creatinine
concentrations were also higher than in control rabbits (122.0
± 2.7 and 3.68 ± 0.3 mg/dl, respectively, N = 5). Follow-
ing VAc administration serum creatinine level did not change,
while urea concentration was increased up to 232 ± 10 mg/dl
(N = 5). However, melatonin administration resulted in a
significant decline in urea and creatinine levels in diabetic
rabbits both untreated and treated with VAc (cf. Fig. 7).
Attenuation by melatonin of VAc-induced elevation of serum
urea and creatinine concentrations was accompanied by a sig-
nificant decrease in serum glucose level (from 620 ± 55 to
372 ± 33 mg/dl, N = 5), while melatonin administration in
Fig. 7.Time-course changes in serum creatinine and urea levels of control and diabetic rabbits following VAc and/or melatonin treatments. Melatonin was
dissolved in a small volume of ethanol and diluted to 1.6 mg/ml with isotonic saline. The concentration of ethanol in the final melatonin solution was 3.5%.
Both compounds were administered intraperitoneally for 6 days at the dose of 1.275 mg of vanadium and/or 1 mg of melatonin per kg body weight, where
indicated, while control rabbits were treated with saline containing 3.5% alcohol. Values are means ± S.D. for five rabbits in each group.
the absence of VAc did not reduce serum glucose level (557
± 42 and 630 ± 54 mg/dl before and after melatonin admin-
istration to diabetic animals, respectively, N = 3). In view of
these observations it seems likely that melatonin might have
beneficial effects during diabetes therapy, protecting against
vanadium-induced acute renal injury.
In agreement with earlier report for metformin [8], VAc,
tungstate and molybdate appeared to be more potent in-
hibitors of gluconeogenesis in renal tubules than in hepa-
tocytes isolated from both control and diabetic rabbits (cf.
Fig. 2). Moreover, although VAc exerted inhibitory action
on glucose production in hepatocytes of diabetic animals,
both tungstate and molybdate showed practically no effect,
suggesting their low potential possibilities to be applied
as hypoglycaemic agents in comparison with the vanadium
compounds. Thus, their marked blood glucose lowering ac-
tion in alloxan-diabetic rats might be due to restoration of
mRNA levels and activities of liver glucokinase, hexoki-
nase, pyruvate kinase, phosphoenolpyruvate carboxykinase
(PEPCK), glucose-6-phosphatase and glycogen synthase
[7, 39, 40] rather than their direct inhibitory effects on the
cellular enzymes.
Vanadium compounds exert their insulin-mimetic effects
through an inhibition of tyrosine phosphatases acting on the
insulin receptor and/or at points distal to the receptor in
the insulin signalling pathway [41]. Besides, vanadium has
also been shown to produce direct inhibitory effects on a
number of various cellular enzymes [8, 42, 43]. In view of
measurements of crossover plots of gluconeogenic intermedi-
ates, the metabolic action of vanadium on glucose formation
in renal tubules could be due to inhibition of pyruvate car-
boxylase, PEPCK, FBPase and G6Pase activities (cf. Fig. 3).
An inhibitory action of vanadium has been reported in terms
of decrease in the liver PEPCK activity as well as mRNA level
of this enzyme [41], while inhibitory effect of VAc on kidney-
cortex G6Pase is in agreement with that reported for hepatic
enzyme [44]. In contrast to VAc, tungstate and molybdate
affected mainly glucose-6-phosphatase activity (cf. Figs. 3
and 4). A smaller effect of VAc on hepatic than renal gluco-
neogenesis (cf. Fig. 2) may be due to an increased vanadium
accumulation in renal tubules than in hepatocytes [8].
As oxidative stress plays a crucial role in the development
of diabetes-related complications, drugs applied in diabetes
therapy are expected to normalize blood glucose concentra-
tion as well as to exhibit antioxidative properties, like it is
attributed to metformin [45]. However, our present knowl-
edge concerning vanadium action on the cellular redox state
is rather controversial. Vanadate(V) has been reported as a
pro-oxidative compound promoting lipid peroxidation in
many tissues [46–49], diminishing GSH level in rat
adipocytes [50] and lowering the intracellular GSH/GSSG
ratio rather than glutathione content in murine macrophages
[51]. Vanadate-induced changes in the intracellular glu-
tathione status are not surprising, as in biological systems
the reduction of vanadium(V) to vanadium(IV) seems to oc-
cur via glutathione-mediated mechanism [50, 52]. The action
of vanadyl(IV) remains more obscure. Pro-oxidative vanadyl
action [48, 53] as well as no effect on lipid peroxidation [54]
have been reported. Surprisingly, in contrast to the markedly
diminished intracellular GSH levels in isolated rabbit renal
tubules incubated with VAc (cf. Table 2), vanadium-induced
increase in hepatic and renal GSH contents have been ob-
served in vivo [55].
VAc-induced decline in the intracellular glutathione redox
state (cf. Table 2) might result from the inhibition of glu-
tathione reductase (cf. Fig. 6). As (i) VAc added to incuba-
tion medium at 100 µM concentration raises the intracellular
vanadium concentration up to 82 µg/g dry weight [8], i.e.
to about 1 mM (calculated in terms of the intracellular wa-
ter content in renal tubules equal to 1.53 ± 0.20 µ l/mg dry
weight [56]), and (ii) at 100 µM and 1 mM VAc concen-
trations glutathione reductase is inhibited by 25 and 75%,
respectively (cf. Fig. 6), it is likely that in renal tubules in-
cubated in the presence of 100 µMVAc the activity of this
enzyme may be markedly decreased, resulting in a decline in
GSH/GSSG ratio. A diminished intracellular GSH content is
also in agreement with this suggestion, as under conditions of
oxidative stress and/or limited glutathione reductase activity
GSSG is removed outside the cell, leading to the depletion of
the intracellular glutathione pool [57]. Moreover, NAC, a pre-
cursor of cysteine [35], which limits the rate of glutathione
synthesis under physiological conditions [58], partially re-
verses VAc-induced decline in the intracellular glutathione
redox state (cf. Table 2). Therefore, it seems likely that the
combined administration of vanadium and NAC might be
beneficial for diabetes therapy, although a partial reversal
of vanadium-induced inhibition of gluconeogenesis would
be undesirable. In contrast to NAC, melatonin produced no
changes in the intracellular glutathione status in renal tubules
incubated for 1 h both in the presence and absence of VAc,
probably due to a short term of these experiments, not per-
mitting the prolonged effects involving changes in gene ex-
pression level [59].
VAc-induced rise in the HFR generation (cf. Table 1) sup-
ports the suggestion that vanadium compounds might dam-
age mitochondria via HFR production, resulting in the 
decline, which is attenuated in the presence of melatonin
and NAC (cf. Fig. 5). As pre-treatment of mitochondria with
cyclosporine A, the inhibitor of the mitochondrial perme-
ability transition pore [36], does not reduce the VAc-induced
decrease in 
,itislikely that the VAc action is not as-
sociated with the mitochondrial permeability transition pore
In most studies, vanadium compounds were typically ad-
ministered in the drinking water to experimental animals,
resulting in a diminished food consumption, weight loss and
even death [9]. In order to overcome gastrointestinal toxic-
ity, i.e. diarrhoea, in the present study, rabbits were treated
with vanadium by intraperitoneal injection of VAc at a dose
equal to 1.275 mg per kg per day [2], i.e. much lower than
that administered orally [60]. In contrast to Reul et al. [2],
who have observed blood glucose decrease by about 25%
after one intraperitoneal injection of VAc to diabetic rats,
six injections to diabetic rabbits were required to normalise
blood glucose level [8]. Unfortunately, although in diabetic
rabbits increased blood glucose levels were reduced follow-
ing vanadium treatment, elevated serum urea and creatinine
concentrations were measured in vanadium-treated control
rabbits (cf. Fig. 7). Increased levels of urea and creatinine
are in agreement with those reported for vanadium-treated
rats (reviewed in [9]), while other authors failed to detect
changes in levels of these compounds [10, 11]. Moreover,
vanadium accumulates in rat kidney [9] and rabbit kidney-
cortex [8].
Similarly to vanadium derivatives, melatonin is typically
applied orally. However, in this investigation it was adminis-
tered intraperitoneally since after 1 h, a rapid and marked
increase in melatonin concentration has been reported in
serum of rodents [61]. The data presented in this study show
that melatonin protects against elevation of serum urea and
creatinine levels in VAc-treated control rabbits as well as
significantly diminishes the urea and creatinine concentra-
tions in serum of untreated and VAc-treated diabetic animals
(cf. Fig. 7). This compound has also been found to protect
against nephrotoxicity evoked by variety of toxic agents: adri-
amycin and constant light exposure [38], chronic cyclosporin
A [62–66], cisplatin [67] and gentamicin [68–70] as well
as several other drugs [71]. Attenuation of hypoglycaemic
vanadium action by melatonin in diabetic rabbits might be
due to chelating melatonin properties [34] or direct action of
this compound on the liver, causing elevation of the plasma
glucose level [72]. On the other hand, melatonin has been
reported to either decrease [73] or normalise hyperglycaemia
in diabetic rats [74].
Despite the beneficial action of melatonin during
nephropathy, the mechanism of its action is not completely
understood. It is commonly accepted that melatonin pre-
vents free-radical-mediated injury during drug-induced tox-
icity and that the primary cause of the reduced oxidative in-
jury in melatonin-treated animals might not be a change in
antioxidant enzyme levels but rather altered production of
free radicals or activity of the defence system [65]. Protec-
tive properties of melatonin could also result from regulation
of mitochondrial homeostasis (cf. Fig. 5 and [75]) and de-
crease in the lipid peroxidation, which is considered as an
early important event in the pathogenesis of nephrotoxicity
[64]. Moreover, synergistic effects exerted by melatonin and
NAC, when administered in combination [76], merit consid-
eration of these compounds as potential therapeutic agents in
drug-induced nephrotoxicity. In view of (i) the low toxicity
of melatonin and (ii) its ability to reduce the side effects and
increase the efficacy of several drugs [71], its use as a com-
bination therapy with these agents seems important and wor-
thy of evaluation. Moreover, as decrease in 
may lead
to programmed cell death [77], it seems also likely to apply
vanadium derivatives in the cancer therapy since apoptosis-
inducing activity of these compounds is beneficial. Metvan, a
novel oxovanadium(IV) complex, has already been identified
as the most promising multi-targeted anti-cancer compound
The authors would like to thank Dr A.K. Jagielski for assay
of nucleotides and Ms I. Lukasinska, R. Doroszewska and
D. Malinska for determinations of serum glucose, urea and
creatinine. We are also grateful to Professor J. Singh (Univer-
sity of Central Lancashire, UK) for the discussion of the data
presented in this investigation, which was possible due to a
Travel Award offered to J.B. by the Wellcome Trust (UK).
Technical assistance of Miss B. Dabrowska is acknowledged.
We are also very indebted to Dr Bartoszewicz (Medical Uni-
versity of Warsaw) for making available the electrochemical
detector for HFR measurements as well as to Professor L.
Wojtczak (Nencki Institute of Experimental Biology), Pro-
fessor A. Mostowska (Warsaw University) and Mr. L. Lipiec
(Brenntag-Polska) for generous gifts of cyclosporin A, JC-1
and silicon oil, respectively. This investigation was supported
by grants of the State Committee for Scientific Research
(1601/56, 1601/57, 1601/59) and the Ministry of Scientific
Research and Information Technology (Project No. 3 PO5A
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11: 1829–1836, 2002
... Позже были получены данные, что ввиду плохой биодоступности (низкое всасывание в сочетании с высокой скоростью экскреции ванадия) токсические и терапевтические дозы ванадия не перекрываются. Типичными клиническими проявлениями токсичности ванадия являются: диарея (легкая), рвота, спазмы гладкой мускулатуры кишечника, зеленый язык, бронхоспазм (тяжелый), неврологические расстройства и необратимое повреждение почек [57,58]. Ванадий (V) оказывает токсическое действие на печень, индуцируя окислительный стресс и гибель клеток печени. ...
... Одновременное введение комплексообразующих агентов может уменьшить потенциально токсичные эффекты ванадия. Например, совместное введение ванадия с тироном (4,5-дигидроксибензол-1,3-дисульфонат натрия), не уменьшая гипогликемического эффекта, снижало накопление V в почках, костях, печени и сердце [59], а совместное введение с мелатонином снижало токсические эффекты микроэлемента [57]. ...
At this stage in the development of nutriciological science, it has not been established if biologically active substances are essential to the human body; however, an explanation of the physiological role of minor biologically active substances is necessary to clarify the qualitative composition of Nutrioma. Of particular interest is the transition metal, vanadium. Adding vanadium to the diet of animals with induced or genetically determined type 2 diabetes mellitus normalizes glucose and blood insulin levels, reduces insulin resistance, promotes β-cell regeneration, and has a beneficial effect on lipid metabolism. Clinical studies of the effectiveness of vanadium are not convincing, in most part, because of their insufficient duration. The review briefly discusses the main mechanisms of the action of vanadium compounds. Therapeutic doses of vanadium compounds may overlap with toxic doses. Organic vanadium compounds could be used in significantly lower doses. The main problem with the possible use of vanadium compounds in antidiabetic therapy is the balance between their beneficial effects and the connected risks of side effects.
... A decrease in glucose level was observed in all vanadium treated groups. This buttresses the anti-diabetic property of vanadium as vanadium has been reported to normalize glucose level in insulin-dependent diabetic rats and humans as well as inhibit glucose-6-phosphatase which is a key enzyme involved in the final step in gluconeogenesis and glycogenolysis (Cam et al., 2000;Kiersztan et al., 2004;Saima 2013). Urea concentration was seen to be increased in vanadium group treated with 100ppm and 200ppm sodium metavanadate and was significant at 200ppm. ...
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The interest in the role of vanadium compounds in living organisms has grown tremendously especially since the report of its glycemic normalization activity in the 1980s. There has been reports of both its toxic as well as positive effects, thus there is a paucity of information on the essentiality of this element in biological systems. In this study, the effect of different doses of sodium metavanadate on the haematological and biochemical variables of male Wistar rats was investigated. Twenty male Wistar rats were divided into four groups of five each and were given tap water containing various concentrations of sodium metavanadate (0ppm- group 1, 50ppm- group 2, 100ppm- group 3, or 200ppm- group 4) for 10weeks. Weekly body changes were noted and blood was collected at the end of 10 weeks by retro orbital puncture for haematological and serum biochemical variables. Histological sections were also performed on liver and kidney tissues. There was a significant increase in body weight in the 50ppm group compared with control. Sodium metavanadate at 200ppm caused a significant decrease in packed cell volume (PCV), red blood cell count (RBC), white blood cell count (WBC) and Lymphocytes with significant increases in neutrophils and neutrophil-lymphocyte ratio when compared with control values. There was also a significant decrease in ALP, ALT and a significant increase in urea concentration in the 200ppm group when compared with control values. All doses of sodium metavanadate significantly reduced blood glucose level. Sections of liver and kidney revealed severed damage at 200ppm compared with control. The results from this study showed that vanadium affects both haematological and biochemical parameters and could be toxic at higher concentrations, while at low concentration could be beneficial as seen with the enhanced body weight.
... In a previous study, Rio et al. [43] also found that a high dosage of MO can result in decreased activity of antioxidant enzymes and a decline in the antioxidant capacity of the organism. Studies in rabbits have shown that high MO accumulation generates free radical processes or reactive intermediates, resulting in the alteration of MDA and GSH-Px levels [44,45]. In ducks exposed to high levels of MO, an increase in MDA levels and a decrease in XOD and CAT activities in serum and spleen tissue has also been observed [28,37]. ...
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High dietary levels of molybdenum (MO) can negatively affect productive performances and health status of laying hens, while tea polyphenol (TP) can mitigate the negative impact of high MO exposure. However, our understanding of the changes induced by TP on MO challenged layers performances and oxidative status, and on the microbiota, remains limited. The aim of the present study was to better understand host (performances and redox balance) and microbiota responses in MO-challenged layers with dietary TP. In this study, 200 Lohmann laying hens (65-week-old) were randomly allocated in a 2 × 2 factorial design to receive a diet with or without MO (0 or 100 mg/kg), and supplemented with either 0 or 600 mg/kg TP. The results indicate that 100 mg/kg MO decreased egg production (p = 0.03), while dietary TP increased egg production in MO challenged layers (p < 0.01). Egg yolk color was decreased by high MO (p < 0.01), while dietary TP had no effect on yolk color (p > 0.05). Serum alanine transaminase (ALT), aspartate aminotransferase (AST), and malonaldehyde (MDA) concentration were increased by high MO, while total antioxidant capacity (T-AOC), xanthine oxidase (XOD) activity, glutathione s-transferase (GSH-ST), and glutathione concentration in serum were decreased (p < 0.05). Dietary TP was able to reverse the increasing effect of MO on ALT and AST (p < 0.05). High MO resulted in higher MO levels in serum, liver, kidney, and egg, but it decreased Cu and Se content in serum, liver, and egg (p < 0.05). The Fe concentration in liver, kidney, and eggs was significantly lower in MO supplementation groups (p < 0.05). High MO levels in the diet led to lower Firmicutes and higher Proteobacteria abundance, whereas dietary TP alone and/or in high MO treatment increased the Firmicutes abundance and the Firmicutes/Bacteroidetes ratio at phylum level. High MO increased the abundance of Proteobacteria (phylum), Deltaproteobacteria (class), Mytococcales (order), and Nanocystaceae (family), whereas dietary TP promoted the enrichment of Lactobacillus agilis (species). Dietary TP also enhanced the enrichment of Bacilli (class), Lactobacillates (order), Lactobacillus (family), and Lactobacillus gasseri (species). Microbiota analysis revealed differentially enriched microbial compositions in the cecum caused by MO and TP, which might be responsible for the protective effect of dietary TP during a MO challenge.
... Many studies reported that when bones were in growth and development stage, ALP activity was significantly [40]. High dietary of Mo considerably increased the activity of ALP [41,42], and different levels of Cd could disturb the metabolism of ALP [43], which affected the function of osteoblast and inhibited the degree of bone mineralization [44]. In this study, ALP activity was increased in ducks when coexposure to Mo and Cd, which suggested Mo combined with Cd could disrupt the bone salt deposition. ...
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Cadmium (Cd) and high molybdenum (Mo) can lead to adverse reactions on animals, but the coinduced toxicity of Mo and Cd to bone in ducks was not well understood. The objective of this study was to investigate the changes in trace elements’ contents and morphology in bones of duck exposed to Mo or/and Cd. One hundred twenty healthy 11-day-old male ducks were randomly divided into six groups and treated with commercial diet containing Cd or/and Mo. On the 60th and 120th days, the blood, excretion, and metatarsals were collected to determine alkaline phosphatase (ALP) activity and the contents of Mo, Cd, calcium (Ca), phosphorus (P), copper (Cu), iron (Fe), zine (Zn), and selenium (Se). In addition, metatarsals were subjected to histopathological analysis with the optical microscope and radiography. The results indicated that Mo and Cd contents significantly increased while Ca, P, Cu, and Se contents remarkably decreased in metatarsals in coexposure groups (P < 0.01). Contents of Fe and Zn in metatarsals had no significant difference among groups (P > 0.05). Ca content in serum had no significant difference among experimental groups (P > 0.05), but P content was significantly decreased in HMo and HMo + Cd groups (P < 0.05). Contents of Ca and P in excretion and ALP activity were significantly increased in coinduced groups (P < 0.05). Furthermore, osteoporotic lesions, less and thinner trabecular bone were observed in combination groups. The findings suggested that dietary of Cd or/and Mo could lead to bone damages in ducks via disturbing the balance of Ca and P in body and homeostasis of Cu, Fe, Zn, and Se in bones; moreover, the two elements showed a possible synergistic relationship.
... In the body, a high dosage of Mo can result in decreased activity of antioxidant enzymes and a decline in the antioxidant capacity of the organism [23]. A few studies in rabbits have shown that high Mo accumulation generates free radical processes or reactive intermediates, resulting in alteration of the levels of MDA and GSH-Px [3,14]. In the present study, a duck-based model for exposure to Mo and/or Cd was created. ...
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The present study is designed to research the effects of molybdenum (Mo) or/and cadmium (Cd) on antioxidant function and the apoptosis-related genes in duck spleens. Sixty healthy eleven-day-old ducks were randomly divided into to six groups with equal number (control group, LMo group, HMo group, Cd group, LMo+Cd group, HMo+Cd group) which were fed with basal diet containing variable doses of Mo or/and Cd. Relative weight of spleen, antioxidant indexes, apoptosis-related genes mRNA expression levels and ultrastructural changes were evaluated on 120 days. The results showed that relative weight of spleen was decreased in treatment groups. Malondialdehyde (MDA) level was increased, while the activities of xanthine oxidase (XOD) and catalase (CAT) were decreased in Mo or/and Cd groups compared with control group. Bak-1 and Caspase-3 expressions were up-regulated in high dose of Mo combined with Cd group, while Bcl-2 was down-regulated. What's more, mitochondrial crest fracture, swelling, vacuolation, deformed nuclei and karyopyknosis in Mo+Cd combination treated groups were more severe. The results suggested that Mo or/and Cd could induce oxidative stress, apoptosis of spleen associated with mitochondrial intrinsic pathway, and the two elements show possible synergistic relationship.
Based on the similar mechanism shared by diabetes mellitus (DM) with Alzheimer's disease (AD), we previously demonstrated that insulin enhanceing vanadyl complexes could preserve cognitive function in an AD model of APP/PS1 mice after emergence of β-amyloid (Aβ) plaque. Herein, we further studied the preventive effects of vanadyl complexes. Vanadyl acetylacetonate (VAC) was given to 3-month-old APP/PS1 mice, whose brain Aβ level started to rise with unobservable deposits. Results showed that VAC could ameliorate Aβ pathogenesis on APP/PS1 mice but was less effective than that administrated at 5 months of age. Investigation on the molecular mechanism revealed that compared with vanadium treatment after Aβ plaque formation, earlier VAC administration showed increased clearance of Aβ deposition in cerebral cortex, however, was much less effective in neuronal protection due to failure to activate the Grp75 chaperone. These results suggest that neural cells under Aβ burden might exhibit different metabolic pattern and intracellular signal transduction before and after Aβ plaque formation, thus responding differently to vanadyl complexes on different intervention stages. Further works clarifying the mechanism of the stage-dependent actions of vanadium would be helpful for rational design of effective anti-AD vanadium agents and better understanding of the AD pathogenesis.
Association of Alzheimer’s disease (AD) with cerebral glucose hypometabolism, likely due to impairments of insulin signaling, has been reported recently, with encouraging results when additional insulin is provided to AD patients. Here, we tested the potential effects of the anti-diabetic vanadium, vanadyl (IV) acetylacetonate (VAC), on AD in vitro and in vivo models. The experimental results showed that VAC at sub-micromolar concentrations improved the viability of neural cells with or without increased β-amyloid (Aβ) burden; and in APP/PS1 transgenic mice, VAC treatment (0.1 mmol kg⁻¹ d⁻¹) preserved cognitive function and attenuated neuron loss, but did not reduce brain Aβ plaques. Further studies revealed that VAC attenuated Aβ pathogenesis by (i) activation of the PPARγ-AMPK signal transduction pathway, leading to improved glucose and energy metabolism; (ii) up-regulation of the expression of glucose-regulated protein 75 (Grp75), thus suppressing p53-mediated neuronal apoptosis under Aβ-related stresses; and (iii) decreasing toxic soluble Aβ peptides. Overall, our work suggested that vanadyl complexes may have great potential for effective therapeutic treatment of AD.
The influence of the crystal structure and chemical nature of some ionic liquid/phosphomolybdate hybrids on their catalytic activity in the epimerization of glucose is studied. A clear evidence for structure-activity relationship is found. The inorganic part of the hybrid assures the active sites for the reaction; meanwhile the organic cation nature organizes the structure and controls the diffusion of the reactants. This study can be used as a first approach to predict the symmetry, long range order and active sites availability of the presented class of imidazolium based polyoxometalate hybrids.
Aim: To observe the effect of melatonin on proliferation of lymphocyte and secretion of cytokine which were cultured in vitro. Methods: The experiment was performed from October 2004 to October 2006 in the Laboratory of Center of Liver Disease, the 123 Hospital of Nanjing Military Area Command of Chinese PLA. 1 Male Wistar rats aged 3 months with the body mass of (230±20) g were bought from Shanghai SLAKE Experimental Animal Limited Company. Melatonin was bought from Sigma Biotechnology Company of America.2 The rat models of autoimmune hepatitis (AlH) were made by Freund's complete adjuvant and liver-specific protein. The peripheral blood of normal and model rats was collected and conventionally cultured in vitro. 2 mg/L melatonin was added in the culture medium of melatonin group. 2 mg/L hepatocyte growth-promoting factors (pHGF) were added in the culture medium of pHGF group. No cell division stimulator was added in the blank control group. 3 Peripheral blood lymphocyte (PBL) of rats were conventionally counted and counts per minute (CPM) was recorded with liquid scintillation counter after 48-hour culture to observe the stimulatory function of melatonin and pHGF on PBL. The cytokine in supernatant of culture fluid was detected to study the effect of MT and pHGF on cytokine secretion of PBL. Results: 1 Melatonin had the stimulatory function on PBL of normal rats. The PBL number and CPM in melatonin group were obviously higher than those in blank control group (P < 0.05). The pHGF had no stimulatory function on PBL fo normal rats. 2 Melatonin had the stimulatory function on PBL of AlH rats. The PBL number and CPM in melatonin group were markedly higher than those in blank control group (P < 0.05). The pHGF had no stimulatory function on PBL of AIH rats. 3 The levels of interleukin ([Q-2 and immunoreactive fibronectin (IFN)-γ (Th1 cytokine) in supernatant of culture fluid of melatonin group were higher than those of blank control group and pHGF group (P < 0.01). The level of 1L-6 (Th2 cytokine) of melatonin group was much higher (P < 0.05), but the level of IL-4 (Th2 cytokine) had no significant deviation (P > 0.05). No significant difference was found in Th1 cytokine and Th2 cytokine between the pHGF group and blank control group (P > 0.05). 4 The levels of IL-2 and IFN-γ(Th1 cytokine) in supernatant of culture fluid of melatonin group were markedly higher than those of blank control group and pHGF group (P < 0.05). The levels of IL-4 and IL-6 (Th2 cytokine) in supernatant of culture fluid of melatonin group had no significant deviation compared with the blank control group and pHGF group (P > 0.05). Conclusion: Melatonin has a strong effect to stimulate PBL of rats with AIH.
Diabetes is a chronic metabolic disease which leads to several acute and chronic complications, morbidity and mortality, and decreased lifespan and quality of life. Therefore, in research studies that aim to enlighten the pathogenesis of diabetes and investigate possible treatment strategies, experimental animal models of diabetes provide many advantages to the investigator. Models of diabetes obtained by chemical induction, diet, surgical manipulations or combination thereof and also new genetically modified animal models are some of the experimental models. Alloxan and streptozotocin (STZ), which are toxic glucose analogues that preferentially accumulate in pancreatic beta cells, are widely used toxic agents to induce experimental diabetes in animals. This review gives an overview on the use of alloxan and STZ to induce chemical diabetes models with reference to their mechanisms, utilizable doses, advantages and disadvantages in diabetes research.
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Vanadate solutions as ‘metavanadate’ (containing ortho and metavanadate species) and ‘decavanadate’ (containing manly decameric species) (5 mM; 1 mg/kg) were injected intraperitoneously in Halobatrachus didactylus (toadfish), in order to evaluate the contribution of decameric vanadate species to vanadium (V) intoxication on the cardiac tissue. Following short-term exposure (1 and 7 days), different changes on antioxidant enzyme activities—superoxide dismutase (SOD), catalase (CAT), selenium-glutathione peroxidase (Se-GPx), total glutathione peroxidase (GPx), lipid peroxidation and subcellular vanadium distribution were observed in mitochondrial and cytosolic fractions of heart ventricle toadfish. After 1 day of vanadium intoxication, SOD, CAT and Se-GPx activities were decreased up to 25%, by both vanadate solutions, except mitochondrial CAT activity that increased (+23%) upon decavanadate administration. After 7 days of exposure, decavanadate versus metavanadate solutions promoted different effects mainly on cytosolic CAT activity (−56% versus −5%), mitochondrial CAT activity (−10% versus +10%) and total GPx activity (+1% versus −35%), whereas lipid peroxidation products were significantly increased (+82%) upon 500 μM decavanadate intoxication. Accumulation of vanadium in total (0.137±0.011 μg/g) and mitochondrial (0.022±0.001 μg/g) fractions was observed upon 7 days of metavanadate exposure, whereas for decavanadate, the concentration of vanadium increased in cytosolic (0.020±0.005 μg/g) and mitochondrial (0.021±0.009 μg/g) fractions. It is concluded that decameric vanadate species are responsible for a strong increase on lipid peroxidation and a decrease in cytosolic catalase activity thus contributing to oxidative stress responses upon vanadate intoxication, in the toadfish heart.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The turbidity produced when protein is mixed with low concentrations of any of the common protein precipitants can be used as an index of protein concentration. The resulting turbidity is maximum after about 10 minutes and may be measured spectrophotometrically in the wavelength region of 600 m. Standardization may be effected by comparison with the turbidity produced by a suspension of a dried protein precipitate, or reference may be had to the methyl acrylate-styrene polymer. Turbidimetric techniques are rapid and convenient, but they yield different values with different proteins. They do not permit differentiation between protein and acid-insoluble compounds such as nucleic acids. Protein estimation with the Folin-Ciocalteu reagent include (1) biuret reaction of protein with copper ion in alkali, and (2) reduction of the phosphomolybdic-phosphotungstic reagent by the tyrosine and tryptophan present in the treated protein. Protein estimation by ultraviolet absorption takes advantage of the fact that nucleic acid, however, absorbs much more strongly at 260 mμ than at 280 mμ, whereas with protein the reverse is true. This advantage is used to eliminate, by calculation, the interference of nucleic acids in the estimation of protein.
Molybdate (Mo) exerts insulinomimetic effects in vitro. In this study, we evaluated whether Mo can improve glucose homeostasis in genetically obese, insulin-resistant ob/ob mice. Oral administration of Mo (174 mg/kg molybdenum element) for 7 weeks did not affect body weight, but decreased the hyperglycaemia (approximately 20 mM) of obese mice to the levels of lean (L) (+/+) mice, and reduced the hyperinsulinaemia to one-sixth of pretreatment levels. Tolerance to oral glucose was improved: total glucose area was 30% lower in Mo-treated mice than in untreated ob/ob mice (O), while the total insulin area was halved. Hepatic glucokinase (GK) mRNA level and activity were unchanged in O mice compared with L mice, but the mRNA level and activity of L-type pyruvate kinase (L-PK) were increased in O mice by 3.5- and 1.7-fold respectively. Mo treatment increased GK mRNA levels and activity (by approximately 2.2-fold and 61% compared with O values), and had no, or only a mild, effect on the already increased L-PK variables. mRNA levels and activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK) were augmented in O liver (sixfold and by 57% respectively), and these were reduced by Mo treatment. Insulin binding to partially purified receptors from liver was reduced in O mice and restored by Mo treatment. Despite this correction, overall receptor tyrosine kinase activity was not improved in Mo mice. Moreover, the overexpression (by two- to fourfold) of the cytokine tumour necrosis factor alpha (TNF alpha) in white adipose tissue, which may have a determinant role in the insulin resistance of the O mice, was unaffected by Mo. Likewise, overexpression of the ob gene in white adipose tissue was unchanged by Mo. In conclusion, Mo markedly improved glucose homeostasis in the ob/ob mice by an insulin-like action which appeared to be exerted distal to the insulin receptor tyrosine kinase step. The blood glucose-lowering effect of Mo was unrelated to over-expression of the TNF alpha and ob genes in O mice, but resulted at least in part from attenuation of liver insulin resistance by the reversal of pre-translational regulatory defects in these mice.
The chemistry of vanadium compounds that can be taken orally is very timely since a vanadium(IV) compound, KP-102, is currently in clinical trials in humans, and the fact that human studies with inorganic salts have recently been reported. VO(acac)2 and VO(Et-acac)2 (where acac is acetylacetonato and Et-acac is 3-ethyl-2,4-pentanedionato) have long-term in vivo insulin mimetic effects in streptozotocin induced diabetic Wistar rats. Structural characterization of VO(acac)2 and two derivatives, VO(Me-acac)2 and VO(Et-acac)2, in the solid state and solution have begun to delineate the size limits of the insulin-like active species. Oral ammonium dipicolinatooxovanadium(V) is a clinically useful hypoglycemic agent in cats with naturally occurring diabetes mellitus. This compound is particularly interesting since it represents the first time that a well-characterized organic vanadium compound with the vanadium in oxidation state five has been found to be an orally effective hypoglycemic agent in animals.
The possible use of vanadium compounds in the treatment of diabetic patients is now being evaluated. However, previously to establish the optimal maximum dose for diabetes therapy, it should be taken into account that vanadium is a highly toxic element to man and animals. The toxic effects of vanadium are here reviewed. The tissue vanadium accumulation, which would mean an additional risk of toxicity following prolonged vanadium administration is also discussed. Recently, it has been shown that coadministration of vanadate and TIRON, an effective chelator in the treatment of vanadium intoxication, reduced the tissue accumulation of this element, decreasing the possibility of toxic side effects derived from chronic vanadium administration without diminishing the hypoglycemic effect of vanadium. However, previously to assess the effectiveness of this treatment in diabetic patients, a critical reevaluation of the antidiabetic action of vanadium and its potential toxicity is clearly needed.