| ObjectiveThyroid hormone is a major physiological regulator of mitochondrial function. The role of thyroid hormone on mitochondria can be completed through acting on specific effects on the rate of gene expression in nuclear region. The maintenance of mitochondrial function and phenotype depends on the existence of a system for the synthesis and import of mitochondrial proteins. The precursors of mitochondrial-targeting proteins interact with translocases located in both outer and inner mitochondrial membranes (Toms and Tims, respectively). These translocases help guide the precursors into the mitochondria.Previous works have demonstrated the influence of TH on translocases in mitochondria occurs in the striated muscles. Thus the purpose of this study was to investigate the possibility that the TH-related modifications in the mitochondrial translocases happen in nerve tissue, which could be partly attributable to alterations in the protein import pathway. Both of the translocases, Tom40 and Tim23, are designed to be measured in mRNA and protein levels of neuroblastoma cell line and cardiac muscle cell line following the administration of different pattern of TH. To investigate the effect of TH on translocases in vivo, mRNA and protein expressions of the above two translocases in brains and hearts were also assayed respectively not only derived from iodine deficiency Balb/c mice but also from iodine deficiency mice accepting exogenous T3 injection. The results of this study are believed to be involved in understanding of pathogenesis in disorders due to mitochondrion dysfunction, such as Parkinson’s disease, Alzheimer’s disease, metabolic syndrome and so on.MethodsA total of 120 weaning Balb/c mice (half male and half female) were selected and randomly divided into two groups according to their body mass and gender (n=60). Mice of control group were fed with standard forage, while mice of low-iodine treated group received iodine-insufficient forage with iodine concentration of 20-40 μg/kg (iodine-intake about 0.25μg/d). All mice received deionized water and were fed ad libitum. After 3 months,40 mice (20 mice of each group) were selected and their blood was collected. Then these mice were sacrificed, brains and hearts were collected. The rest of them (40 mice of each group) were kept feeding until 6 months.15 mice were selected randomly from low-iodine group and performed intraperitoneal injection of T3 (100 μg/kg BW per day) for 24hs,48hs or 72hs respectively (n=5). All mice were collected blood samples and their brains and hearts were dissected right after sacrificed.Serum TH and TSH, as well as brain or heart local TH levels were determined by chemiluminescence immunoassay (CIA); brains and hearts were collected to assay the Tom40 and Tim23 mRNA expressions by SYBR GREEN fluorescent real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR); the protein expression levels of two translocases were detected by western blotting (WB).Tom40 and Tim23 mRNA expressions were also examined through RT-PCR in LA-N-5 neuroblastoma and H9C2 cardiac muscle cell lines respectively following the administration of T3, T4 or T2; the protein expression levels of two translocases in these two cell lines were also tested by WB assay; the changes of mitochondria membrane potential caused by T3, T4 or T2 were found by fluorescent probe JC-10.ResultsThree months later, the serum TT4 (nmol/L), FT4 (pmol/L), TT3 (nmol/L) and FT3 (pmol/L) levels in LI group mice (0.47 ± 0.70,2.41 ± 0.28,0.76 ± 0.08,4.01 ± 0.40) were significantly lower than those of NC group mice (55.2 ± 3.68,32.72 ± 1.02,1.10 ± 0.06,5.40 ± 0.38, P <0.001). The serum TSH (mlU/L) strongly raised in LI group mice (35.67 ± 17.39), contrasting to that of NC group mice (0.24 ± 0.10, P< 0.001).Six months later, compared to NC group mice, TT4 and FT4 levels (4.60 ± 2.62, 4.72 ± 0.78) in LI group mice were still lower than those of NC group mice (46.74 ± 10.22,28.80 ± 4.81, P< 0.001), and there was no statistical differences in TT3, FT3 levels between the two groups. The serum TSH in LI group mice (3.22 ± 1.68) was still much higher than that of NC group mice (0.08 ± 0.03, P< 0.001).After T3 administration, TT4, TT3, FT4, FT3 levels were steadly increased and had been higher than LI group mice since 24 hours after injection, equal to NC group mice at 48 hours and higher than NC group mice at 72 hours after the injection. TSH had been suppressed to NC group level since 48h after T3 injection.Expressions of the mitochondria translocases in mice altered according to the changes of serum or local hormone levels. For example, Tom40 and Tim23 mRNA expressions in brains of 3rd month LI group were nearly 0.006-fold and 0.48-fold than NC group (P<0.05), and Tom40 protein contents were about 0.82 times lower than control group. In mice hearts, at the end of 3rd month, Tom40 mRNA and protein levels also decreased, which were equal to 0.41-fold and 0.68-fold (P<0.05) of the NC group quantity. Same result happened in heart Tim23 expressions, mRNA arrived at 0.81-fold (P<0.05) of the NC group.Up to 6 months, serum T4 of LI mice remained lower level. In brain, expressions of Tom40 and Tim23 were down-regulated continously. FT3 and FT4 didn’t decrease in LI mice heart, so the translocase mRNA and protein levels showed no statistical difference between LI and NC group mice. After providing exogenous T3 to iodine dificient mice, T3 and T4 increased gradually, whereas Tom40 mRNA and protein both increased to that of the level of NC group. Same circumstance occurred in Tim23 of brain and heart. It is remarkable that the increasement of translocases expressions in heart was relatively greater than in brain.RT-PCR consequences showed that Tom40 and Tim23 mRNA expressions elevated in neuroblastoma cell line due to T3, as well as rises in protein expressions according to WB results. T3 induced increase of mitochondrial membrane potential in neuroblastoma cell line. On contrast, there were no statistical differences in mRNA level between NC group and that following administration of T4 (P> 0.05). To be consistent with real time PCR result, T4 caused no change in protein contents of both translocases.In cardiac muscle cell line, Tom40 expression in protein level also rose because of adding T3 to it.Conclusions1 Iodine is a vital material for synthesis of thyroid hormone. Mice couldn’t produce enough TH for requirement on account of low iodine feeding as long as several months. The tendency of TH levels alteration is regulated by the classic HPT axis feedback reaction.2 With decreasing of TH level (induced by low iodine diet), the translocases reduced their expressions in brain mitochondria; the expressions of cerebral Tom40 and Tim23 resumed going up for the reason of exogenous T3. As a consequence, TH serves as a critical factor regulating the expressions of mitochondria translocases in brain.3 Exogenous T3 supplying also evoked up-regulation in heart mitochondrial translocases. Moreover, the regulative effects in cardiac muscle were even stronger than in brain. We deduce the reason is that there are some regulation mechanisms in brain to maintain local TH levels, such as TH transporters, modulation of deiodinase activity and so on. On the contrary, cardiac muscle cell utilizes circulatory T3 immediately, so the local T3 level of heart changes drastically according to the serum hormone level.4 T3 promoted the expressions of mitochondria translocases in LA-N-5 cell line, while T4 plays no role on mRNA or protein levels of translocases. It is demonstrated that DII is important for local production of T3 in nerve tissue, so neuron couldn’t accomplish conversion from T4 to T3 without neuroglia.5 T3 also enhanced the expression of mitochondria translocases in H9C2 cell line, which suggests that striated muscle fiber is under the control of TH, and the mitochondrion is target of the thyroid hormone. |
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