The Effect of Chromium and Selenium Ions on Mitochondrial Trans membrane Transport of Fatty Acids in Healthy and Diabetic Rats

Mitochondrial degradation of fatty acid (FA) in β-oxidation pathway requires delivery of substrates. In blood fatty acid are transported by albumins. In the cell fatty acids must be transferred across the mitochondrial membrane. In this process two enzymes, as well as carnitine are engaged: carnitine-palmitoyl transferase 1 (CPT-1) and carnitine-palmitoyl transferase 2 (CPT-2). First of these enzymes takes a part in transport of palmitic acid across external mitochondrial membrane, while second across internal mitochondrial membrane to mitochondrial matrix. Carnitine, [(-)-β-hydroxyl-γ-[trimethylamino-] butyrate] is an essential component of the fatty acids transporting system. Activity of CPT-1 is responsible not only for the activity of fatty acids utilization but also for the hepatic level of triglycerides [1,2]. On the other hand CPT-2 deficiency should lead to myopathies accompanied with hypoglycemia [3] or neuromuscular diseases [4]. Disorders at this step of fatty acids metabolism and disturbances in production of FADH2 and NADH + H + (substrates for oxidative phosphorylation chain) as well as acyl-CoA (the substrate for Krebs’ cycle) should lead to energy production disorders.


Introduction
Mitochondrial degradation of fatty acid (FA) in β-oxidation pathway requires delivery of substrates. In blood fatty acid are transported by albumins. In the cell fatty acids must be transferred across the mitochondrial membrane. In this process two enzymes, as well as carnitine are engaged: carnitine-palmitoyl transferase 1 (CPT-1) and carnitine-palmitoyl transferase 2 (CPT-2). First of these enzymes takes a part in transport of palmitic acid across external mitochondrial membrane, while second -across internal mitochondrial membrane to mitochondrial matrix. Carnitine, [(-)-β-hydroxyl-γ-[trimethylamino-] butyrate] is an essential component of the fatty acids transporting system. Activity of CPT-1 is responsible not only for the activity of fatty acids utilization but also for the hepatic level of triglycerides [1,2]. On the other hand CPT-2 deficiency should lead to myopathies accompanied with hypoglycemia [3] or neuromuscular diseases [4]. Disorders at this step of fatty acids metabolism and disturbances in production of FADH 2 and NADH + H + (substrates for oxidative phosphorylation chain) as well as acyl-CoA (the substrate for Krebs' cycle) should lead to energy production disorders.
Chromium in active form of chromodulin [7,8] optimizes insulin action [9], via membrane phosphotyrosine phosphatase [10] and tyrosine kinase [9]. Selenium ions, Se +4 and Se -2 , are components of several enzymes as glutathione peroxidase, tri iodotyrosine deiodase and selenoprotein P. These ions affect glucose uptake in RBC and betaoxidation activity in WBC obtained from healthy rats [11]. It seems interesting to check if microelements such chromium (Cr +3 ), used in therapy of type 2 Diabetes mellitus and selenium (Se +4 and Se -2 ) should modify fatty acids transport into the mitochondria [12]. Experiments were performed on 54 healthy and 54 diabetic Wistar rats, 4-weeks old, weighting 135 -150 g. Animals were fed ad libitum with a standard laboratory diet, having free access to drinking water. Rats were killed by cervical dislocation and blood was collected on standard EDTA and livers were excised and immediately frozen in liquid nitrogen for further studies. Diabetes was provoked by intraperitoneal injection of streptozotocin dissolved in physiological saline. After increasing of blood glucose concentration from 5.35 ± 0.54 mmoles/L to 28.74 ± 4.05 mmoles/L and falling down the concentration of insulin from 3.16 ± 0.99 ng/ml to 0.26 ± 0.10 ng/ml the animals were kept 1 week at this state before killing.

Materials and Methods
The assays of the activity of CPT-1 and CPT-2 were performed according to Ventura et al. [13] method. Livers were homogenized in buffer: 250 mmol/L mannitol, 5 mmol/L TRIS-HCl, pH 7.4 and 0.5 mmol/L EGTA. Homogenates were centrifuged 10 min. at 600 x g. Supernatant was recentrifuged second time for 10 min. at 3600 x g. The pellet was resuspended in homogenizing buffer and centrifuged third time for 10 min. at 2700×g. Final pellet was resuspended in buffer: 50 mmol/L HEPES -150 mmol/L KCl, pH 7.4 and sonicated 3-times for 15 seconds in 45 seconds intervals at the temperature of +4°C. Activities of palmitoyl transferases were measured in incubation mixtures contained: 50 mmol/L HEPES, 150 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L DTT, 20 µmol/L BSA, 0.5 mmol/L carnitine and 0.1μCi of radioactive L-methyl -3 H-carnitine, 100 µmmol/L palmitoyl-CoA and 50 µl of mitochondial preparation in a final volume of 500 µL. Samples were supplemented with chromium acetate to a final concentration of chromium ions of 96.15 µmol/L, selenomethionine or sodium selenite to a final concentration of selenium ions of 6.33 µmol/L or combinations of Cr +3 with Se +4 or Se -2 . Ions were added in a volume of 1 µL what had no effect on final concentrations of components of incubation mixtures. Activity of CPT-2 was measured after blocking of the CPT-1 with 200 µmol/L M malonyl-CoA. All other components of the reaction mixture were the same. Incubation was carried out at 37 0 C for 10 minutes and terminated by addition of 0.5 ml of 1.2 mol/L HCl and 1 mL of n-butanol saturated with distilled water. Samples were vigorously shaken and organic phase was collected. Water fraction was extracted again with water-saturated butanol and organic phases were combined and counted in scintillation β-spectrometer. Subtraction of the results from unblocked and blocked samples allows estimating the activity of CPT-1. The results were expressed as nmoles of transferred fatty acid per 1 min per 1 mg of mitochondrial protein.The results were statistically evaluated using ANOVA test.

Results
Activities of carnitine-palmitoyl transferase 1 and carnitine-palmitoyl transferase 2in mitochondrial preparations from rat's livers are presented in Table 1 (for healthy animals) and in Table 2 for diabetic rats.
Chromium ions as well as selenium Se +4 ions exhibit slight inhibitory effect on transmembrane transport of fatty acids. This effect should be reversed by supplementation of selenium Se -2 ions. The data indicate, that selenium Se -2 ions had no effect alone and in combination with chromium Cr +4 ions on total activity of transmembrane transport of fatty acids. (44.48 ± 0.94 nmoles . min -1 mg -1 vs 45.56 ± 1.93 nmoles. min -1 mg -1 as compare to control 46.04 ± 1.43 nmoles . min -1 . mg -1 , respectively).
In healthy rats all examined ions, alone and/or in combinations suppress the activity of CPT-1. Transmembrane transport of fatty acids across external mitochondrial membrane was inhibited, especially by chromium ions in all experimental variants, at least in combination with selenium (5.55 ± 0.32, 5.29 ± 0.42 and 9.5 ± 0.32 nmoles . min -1 mg -1 as compare to control -19.62 ± 1.12 nmoles . min -1 mg -1 , respectively). Inhibitory effect should be partially reversed by selenium Se -2 ions, but not by Se +4 . Selenium ions, when used alone were also inhibitors of this enzyme, but the effect was much lower than those observed for chromium ions (8.55 ± 0.58 for Se +4 and 11.09 ± 0.98 nmoles min -1 mg -1 for Se -2 ions as compare to control -19.62 ± 1.12 nmoles min -1 mg -1 ).
On the other hand, chromium Cr +3 as well selenium Se +4 and Se -2 stimulated the activity of transmembrane transport of fatty acids across internal mitochondrial membrane. The activity of CPT-2 increased from 26.42 ± 0.75 for control to 33.45 ± 1.79 for Cr +4 , 33.00 ± 1.55 for Se +4 and 33.39 ± 0.64 nmoles min -1 mg -1 for Se -2 ions.
In diabetic rats all examined ions, alone and/or in combinations also suppress the activity of both enzymes. Only Se -2 and its combination with chromium did not exhibit inhibitory effect (41.72 ± 3.96 and 44.24 ± 3.10 nmoles . min -1 mg -1 as compare to 45.51 ± 3.71 nmoles min -1 mg -1 for control). Transmembrane transport of fatty acids across external mitochondrial membrane was stimulated by all combinations of supplements, especially by chromium and selenium Se -2 ions (24.26 ± 4.64 for chromium and 21.75 ± 2.13 for selenium Se -2 nmoles min -1 mg -1 as compare to control -15.94 ± 2.83 nmolesmin -1 mg -1 ). Inhibition of the activity of CPT-2 should be partially reversed by combination of chromium with selenium Se -2 ions (24.44 ± 3.81) or Se +4 (22.08 ± 1.69 nmoles min -1 mg -1 ).

Discussion
Chromium is known as a stimulator of glucose transport into the cells and modulator of insulin receptor signaling [14]. It is possible, that observed earlier increased oxidation of fatty acids in the presence of chromium Cr +3 ions should be, in part, the effect of increased permeability of cellular membranes for fatty acids [10]. As indicated in presented data chromium activates fatty acids degradation in the absence, as well as in the presence of exogenous carnitine (63.95% and 72.19%, respectively). Stimulation of carnitine acyltransferases was reported earlier by Karlic et al. [15]. Observed activation of β-oxidation by trace elements should result in increased concentration of acetyl-CoA, which may serve as a substrate for Krebs' cycle, as well as for resynthesis of fatty acids. In this process first reaction is carboxylation of acetyl-CoA to malonyl-CoA, which was recognized as a strong inhibitor of CPT-1. Acetyl-CoA carboxylase should be activated by a number of factors, such as substrate (Acetyl-CoA), leptin or glucose (in central nervous system) [16][17][18]. Methodological pitfalls, causing the observed differences can be mostly excluded for the following reason: (a) dietary effects should be excluded because all animals had the same diet and free access to them; (b) CPT-1 activity should be affected by inhibitors due to changes in membranes fluidity. This may be omitted when experiments performed in in vitro, using isolated, sonicated mitochondria. That why differences observed in such experiments seems to be caused by added trace elements, as well as exogenous carnitine.
The role of selenium is not yet well recognized. Low level of this trace element should result in myopathies and cardiomyopathies [19], disturbances in spermatogenesis [20] and in elevated risk of cancerogenesis [21], as well as bacterial [22] and viral infections [23]. Other function of selenium ions is participation in red-ox processes as the structural element of glutathione peroxidases. Glutathione peroxidases are the family of four distinct mammalian selenoproteins. The most popular is classical enzyme cGPx. Other proteins of this family are gastrointestinal isoenzyme (GI-GPx), the most related to cGPx, plasma (pGPx) and glutathione peroxidase of phospholipids peroxides (PHGPx). Other proteins showing activity modified by the selenium ions are 5'-iodotyronin deiodase, thioredoxin reductase and selenoprotein P. [24][25][26]. Observed differences in the activity of fatty acids decomposition following chromium and/or selenium supplementation Means ± SD All vales given in one column with unlike superscript letter differ at p ≥0.05 may suggest that these micronutrients should play a role in some steps of energetical processes. Moderatory effect of investigated trace elements on efficiency of fatty acids β-oxidation was observed in the presence of exogenous carnitine. It is possible that these ions are a part of carnitine shuttle of fatty acids across the mitochondrial membranes. Increased degradation of fatty acids should be caused by its increased concentration in mitochondrial matrix. However, supplementation with ions resulted in modification of the activity of CPT-1. It seems that chromium ions are inhibitors of this enzyme when used alone (5.55 ± 0.32 nmoles min -1 mg -1 as compare to 19.66 ± 1.12 nmoles min -1 mg -1 for control), as well as in the presence of selenium (5.29 ± 0.42 nmoles min -1 . mg -1 and 9.51 ± -0.32 nmoles min -1 mg -1 for Se +4 and Se -2 , respectively). Its inhibitory effect should be partially ameliorated by addition of Se -2 ions. Slight difference in the action of Se +4 and Se -2 should be observed. Selenium Se +4 are less effective in stimulation of CPT-1 as compare to Se -2 alone (8.55 ± 0.58 nmoles min -1 mg -1 and 11.09 ± 0.98 nmoles min -1 mg -1 , respectively), as well as in the presence of Cr +4 . These results indicate that the activity of the mitochondrial degradation of fatty acid is rather regulated by the activity of CPT-1 than of CPT-2 and should be, at least in part, controlled by selenium ions. Karlic et al. [15] found that expression of CPT-1 and CPT-2 genes in rats is age-related and is less effective in adult animals because of accumulation of oxidatively damaged products. Selenium is known as anti-oxidant agent, what should reflect in protection of membrane lipids against ROS oxidation and increased activity of β-oxidation as well as carnitine-palmitoyltransferases CPT-1 and CPT-2.