Effect of carnitine and microelements (chromium and selenium) on

Kuryl Hig
Probl
T et Epidemiol
al. Effect of
2011,
carnitine
92(3):and
583-586
microelements (chromium and selenium) on fatty acids metabolism in healthy ...
583
Effect of carnitine and microelements (chromium and
selenium) on fatty acids metabolism in healthy and type 1
diabetic rats
Wpływ karnityny i mikroelementów (chromu i selenu) na metabolizm kwasów tłuszczowych
u szczurów zdrowych i z cukrzycą typu 1
Tomasz Kuryl 1/, Bogdan Debski 1/, Maria Milczarek 1/, Jerzy Bertrandt 2/, Anna Klos 2/
1/
2/
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland
Dept. of Hygiene and Physiology, Military Institute of Hygiene and Epidemiology, Warsaw, Poland
Wprowadzenie. U szczurów z cukrzycą produkcja energii w wyniku
metabolizmu węglowodanów jest bardzo ograniczona i musi być zastąpiona
innym procesem metabolicznym. Jednym z nich może być β-oksydacja
kwasów tłuszczowych.
Cel pracy. Porównanie aktywności metabolizmu kwasów tłuszczowych
u szczurów zdrowych i z cukrzycą typu I po suplementacji karnityną i jonami
mikroelementów – chromu i selenu.
Materiał i metody. Doświadczenia przeprowadzono wykorzystując
samce szczurów Wistar. Cukrzycę wywoływano przez dootrzewnowe
podanie streptozotocyny. Aktywność β-oksydacji mierzono w limfocytach
izolowanych od szczurów zdrowych i z cukrzycą typu I. Hodowle komórkowe
limfocytów uzupełniano karnityną oraz jonami chromu lub selenu.
Wydajność β-oksydacji określano ilością rozłożonego radioaktywnego
[9,10]–3H – kwasu palmitynowego.
Wyniki. Karnityna i jony mikroelementów bardziej efektywnie stymulują
metabolizm kwasów tłuszczowych u szczurów z cukrzycą niż u zwierząt
zdrowych. W izolowanych układach limfocytów wszystkie zastosowane
warianty suplementacji wykazywały stymulujący efekt na produkcję energii
w procesie β-oksydacji. Karnityna jest znanym stymulatorem metabolizmu
kwasów tłuszczowych. Jej efekt był wzmacniany dodatkiem jonów
mikroelementów: chromu [Cr+3], selenu [Se+4 lub Se–2] lub ich kombinacji.
Obserwowany efekt był zdecydowanie silniejszy w limfocytach zwierząt
chorych w porównaniu do wywoływanego w komórkach izolowanych od
zwierząt zdrowych.
Wnioski. Degradacja kwasów tłuszczowych może u organizmów z cukrzycą
zastąpić glikolizę w procesie dostarczania energii. Efekt ten może być
wzmocniony przez podawanie pacjentom karnityny i jonów chromu lub
selenu w celu zwiększenia efektywności procesu β-oksydacji kwasów
tłuszczowych i związanej z nim syntezy substratów dla cyklu Krebsa.
Introduction. In a diabetic rat the energy production in pathways of
carbohydrate metabolism is very limited and must be substituted by other
metabolic tracts. One of them should be β-oxidation of fatty acids.
Aim. The comparison of fatty acids metabolism in healthy and type I
diabetic rats after supplementation with carnitine and microelements
– chromium and selenium.
Materials and methods. Male Wistar rats were used. Diabetes was
provoked by intraperitoneal infusion of streptozotocin. The activity of fatty
acids degradation was examined in lymphocytes isolated from healthy
and diabetic animals. Cell cultures were supplemented with carnitine and
microelements and the efficiency of fatty acids metabolism was degradation
of tritium labeled [9,10]–3H – palmitic acid.
Results. Carnitine and microelement supplementation improves fatty acids
metabolism in diabetic rats as compared with healthy ones. In isolated
lymphocytes additives in all variants exhibit stimulatory effect on energy
production in β-oxidation pathway. Carnitine is a well-known activator of
fatty acids degradation. The stimulatory effect is increased after additional
supplementation with chromium [Cr+3], selenium [Se+4 or Se–2] or their
combinations. The stimulatory effect of carnitine and used microelements
on activity of the fatty acids metabolism is much more pronounced in cells
from diabetic rats in comparison with healthy subjects.
Conclusions. Fatty acids metabolism should serve as an energy source in
diabetic organisms especially after supplementation with carnitine and
chromium or selenium alone or in combinations.
Key words: rats, β-oxidation, carnitine, microelements, diabetes
Słowa kluczowe: szczury, β-oksydacja, karnityna, mikroelementy,
cukrzyca
© Probl Hig Epidemiol 2011, 92(3): 583-586
www.phie.pl
Nadesłano: 10.06.2011
Zakwalifikowano do druku: 08.07.2011
Adres do korespondencji / Address for correspondence
dr hab. Tomasz Kuryl
Department of Physiological Sciences, Faculty of Veterinary Medicine
Warsaw University of Life Sciences
ul. Nowoursynowska 159, 02-776 Warsaw, Poland
(+48-22) 59-36-242, e-mail: [email protected]
584
Introduction
Diabetes mellitus is one of the most frequent
diseases in humans and animals. Type 1 diabetes
is caused by insulin deficiency, while type 2 is the
effect of limited response to insulin. In both types
of disease an improper uptake of glucose is observed.
Disorders in glucose uptake result in disorders in
energy metabolism and synthesis. Thus, the source of
energetical substrates must be shifted from sugars to
other groups of metabolites, mainly lipids and fatty
acids. These compounds are metabolized by diabetic
patients to acetyl-CoA, ketone bodies, acetoacetate,
β-hydroxyacetate and acetone. Ketone bodies should
serve as an energy source for neurons and nervous
system cells.
Chromium ions were reported as diet components
improving glucose uptake and fat metabolism [1].
Chromium supplement in the diet of broiler chicken
resulted in lowering the concentrations of fatty acids,
cholesterol and glucose in the blood serum [2, 3].
The form of supplementation is less important for the
final effect, as compared to the dose of microelement
used. The lack of sensitive indicators of chromium
nutritional status in humans makes it difficult to
determine the level of chromium intake most likely
to promote optimum health. Therefore, a Daily
Reference Intake was set, based on estimated mean
intakes (balance studies). The DRI was estimated
to be 25-35 μg/day and 20-25 μg/day for men and
women, respectively. Lactation may substantially
increase the requirement for chromium if only 1%
of dietary Cr is absorbed and about 20 μg Cr/day is
secreted in human milk, so DRI for lactating women
was established to 45 μg Cr/day.
Fol l ow i n g t h e L i nu s Pa u l i n g I n s t i t u t e
recommendation to take a multivitamin/ multimineral
supplement containing 100% of the daily values (DV)
of most nutrients will generally provide 60-120 μg
Cr/day of chromium, well above the adequate intake
level. The WHO [4] assumes that Cr supplementation
of 250 μg Cr/day should not be exceeded. Actual
EFSA [5] opinion also suggests that the maximum
intake should not exceed 250 μg/day for supplemental
intake.
The selenium deficiency is manifested in
humans in the form of Keshan disease, Kashin-Back
disease, different kinds of miopaties and an increased
sensitivity to cancerogenesis [6, 7]. The addition of
selenium in the form of Se–2 (as selenocysteine) or Se+4
(as selenium oxide) should not exceed 0.5 ppm, which
may be toxic for animals. In animal cells selenium
is present in the organic form of selenomethionine
or selenocysteine. Metabolic effects of selenium
supplementation result also in lipid and cholesterol
metabolism [8,9].
Probl Hig Epidemiol 2011, 92(3): 583-586
Carnitine is a very important component of
the fatty acids metabolic pathway. This betaine is
the main element of “carnitine shuttle”, member
of the way of fatty acids transportation, in the
form of acyl-carnitines, from the cytoplasm to
mitochondrium across the mitochondrial membrane.
The supplementation of this compound results in
a more effective fatty acids transport to the place of
their decomposition in the process of β-oxidation
[10, 11]. It is possible that in a diabetic organism’s
carnitine may improve the fuel metabolism because in
these conditions the sugar decomposition pathways
are substituted by an increased fatty acids uptake.
Aim
The aim of experiments was to study the effect of
the chromium (Cr+3) and selenium (Se–2 and Se+4)
supplementation in the presence of carnitine on
fatty acids degradation in lymphocytes isolated from
healthy and type 1 diabetic rats.
Materials and methods
Experiments on living mammals were performed
after obtaining the permission of the Ethics Committee
of Warsaw University of Life Sciences.
Animals
Experiments were performed on 9 healthy and
9 type 1 diabetic Wistar rats, 4-weeks old; weighting
135-150 g. Diabetes was provoked by intraperitoneal
injection of streptozotocin in physiological saline
(PBS) in the dose of 50 mg per 1 kg body weight.
Indications of diabetes were levels of blood glucose
and insulin. The animals were kept at 23°C in light
cycle of 12/12 hours. The animals were fed ad libitum
with the standard laboratory diet (AIN-93), having
free access to food and drinking water. The rats were
killed by cervical dislocation and blood obtained by
heart puncture was collected on standard EDTA.
Methods
The activity of β-oxidation was examined in
isolated lymphocytes as described earlier [12]. The
fatty acids decomposition was studied in systems
contained 20-50 µg of lymphocyte protein, carnitine
(50 µmol/L), Cr+3 ions (96.15 µmol/L), selenium Se+4
or selenium Se–2 (6.33 µmol/L) or the combination of
selenium and chromium ions (as presented in table),
52,4 μM palmitic acid and 0.1 μCi of [9,10]–3H
– palmitic acid in a final volume of 200 μl. The samples
were incubated for 120 min at 37° C. The amount
of radioactive water formed during the degradation
of radioactive palmitic acid was the indicator of
β-oxidation activity. The results were expressed as
Kuryl T et al. Effect of carnitine and microelements (chromium and selenium) on fatty acids metabolism in healthy ...
pmoles·min–1·mg–1 protein of decomposed palmitic
acid.
Statistical analysis
The data are presented as the mean ± SD with
variation coefficient (V) of 9 experiments. The
statistical differences were analyzed by the Duncan
test as well as t-test at the level of significance of
=0.001.
Results
The effect of supplementation of isolated
lymphocytes with microelements in the presence
of 50 μM carnitine are studied and the results are
presented in Table I.
The lymphocytes from diabetic animals are more
active in palmitic acid degradation, as compared
to those from healthy objects. Both systems were
stimulated by microelements added. In the lymphocytes
from healthy rats the effect of supplementation
with chromium and selenium was very similar,
independently of the level of oxidation, as well as the
composition of microelement added. In diabetic rats
the most effective were chromium ions used alone,
while the mixture of Cr+3 with Se–2 was less effective
in the stimulation of fatty acids degradation.
The differences between the samples supplemented
with microelements were statistically highly significant
(p≤0.001) as compared with the controls in both
experimental groups.
In diabetic subjects the degradation of
carbohydrates is very limited. This metabolic
pathway is substituted by degradation of fatty acids.
However, as we reported earlier [13], the fatty acids
degradation in lymphocytes obtained from diabetic
rats was slightly lower, as compared to healthy ones
(27.46±0.68 comparing to 24.80±2.02 pmoles
per min per 1 mg of lymphocyte protein). Those
cells were not supplemented with carnitine, one of
the most important components of the “carnitine
shuttle” transporting fatty acids across mitochondrial
membrane to places of their oxidation. An addition of
50 μM carnitine increases β-oxidation of fatty acids
by 15% in lymphocytes from healthy and almost 85%
for lymphocytes from diabetic rats (31.61±0.58 and
45.47±4.75 pmoles per min per 1 mg of lymphocyte
protein, respectively). As presented in Table 1,
in all the examined combinations of experiments
fatty acids were degraded more intensively in cells
obtained from diabetic rats, as compared to those from
healthy animals. It may suggest that supplementation
of the diet of diabetic patients with carnitine and
585
composition of chromium Cr+3 with any form of
selenium should result in an increased energy delivery
in diabetic patients. Exogenous carnitine was already
administered to the patients with dysfunction of the
liver in type 2 diabetes [14], in physical exercises
[11] and, together with selenium, to phenylketonuric
patients [15].
Table I. The effect of supplementation with 50 mM carnitine, Cr+3 and Se+4
or Se–2 on β-oxidation efficiency in lymphocytes isolated from healthy and
type 1 diabetic rats (pmol·min–1·mg–1 lymphocyte protein) (Mean±S.D.;
n=9) [p≤0.001] (V – coefficient of variation)
Tabela I. Wpływ suplementacji 50 mM karnityną, Cr+3 oraz Se+4 lub Se–2 na
wydajność β-oksydacji w limfocytach izolowanych od szczurów zdrowych
i z cukrzycą typu I (pmol·min–1·mg–1 białka limfocytów) (Średnia±odch.
stand.; n=9) [p≤0,001] (V – współczynnik zmienności)
Experimental variant
/Badane osoby
Helthy /Zdrowi
Diabetic /Cukrzycy
pmol·min–1·mg–1 protein
X±SD
V (%)
X±SD
V (%)
Control /Kontrola
31.61±0.58
1.83
45.47±4.75
10.45
+ Cr+3
54.58±1.75
3.17
77.09±7.31
9.48
+ Se+4
52.66±3.19
6.06
74.16±5.66
7.62
54.52±3.00
5.50
73.09±5.87
8.03
54.36±1.99
3.66
70.18±3.33
4.74
52.60±0.87
1.65
68.37±5.63
8.23
+ Cr
+3
+ Se+4
+ Se–2
+ Cr
+3
+ Se
–2
Selenium is still examined as a nutrient and a diet
supplement regarding the point of its toxicity [16],
bioavailability or metabolic effects [17].
The role of chromium is discussed from the
beginning of its use and is not yet clear. Hitherto
presented observations indicate a positive role of this
element on the carbohydrate metabolism both in
healthy and diabetic rats [18]. On the other hand, the
discussions between Cefalu et at [19] and Kleefstra et
al [20] may confirm that the role of chromium and its
influence on the carbohydrate and lipid metabolism
requires further studies.
Summarizing, we found a positive stimulatory
effect of chromium Cr+3 as well as both selenium
Se+4 and Se–2 ions on the activity of β-oxidation in
lymphocytes of healthy and diabetic rats supplemented
additionally with carnitine. In this model, a stronger
effect as compared to the controls, was observed in
healthy rats. However, this was probably the result of a
very high stimulation of control samples with carnitine
alone, especially in diabetic rats. The absolute values
of the activity of fatty acids degradation were highest
in diabetic rats. This leads to the conclusion that
supplementation of the diet of diabetic patients
with carnitine and any combination of chromium
and selenium ions should gradually improve energy
production in patients with a limited possibility of
carbohydrate degradation
586
Probl Hig Epidemiol 2011, 92(3): 583-586
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