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 ...
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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]
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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
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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
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Probl Hig Epidemiol 2011, 92(3): 583-586
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