1. TIANJIN MEDICAL UNIVERSITY Yina Sun et al Tianjin Institute of Endocrinology Tianjin, China EXPRESSIONS OF Mct8 AND Oatp1c1 UP-REGULATED BY T 3 AND T 4 IN ASTROCYTES OF PRIMARY CULTURE

2. TIANJIN MEDICAL UNIVERSITY Background Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins. Mct8 ---- T 3 ---- neurons Oatp1c1----T 4 ---- Choroid plexus

3. TIANJIN MEDICAL UNIVERSITY Background Mol Endocrinol, 2011, 25(1):1-14 Endocrinology, 2008,149(12): 6251-61. Model of TH regulation in brain. Transporters (paired ovals) are required for uptake and release of T4 and T3. T3 in brain is either derived from the circulation [via the blood-brain barrier(BBB) or indirectly via the blood-cerebrospinal fluid (CSF) barrier] or locally produced from T4 in astrocytes by D2. All principal cells of the brain are T3 targets: neurons, astrocytes, and oligodendrocytes. However ， Oatp1c1 related to T4 import to brain from the blood-brain barrier(BBB) and the blood- cerebrospinal fluid (CSF) barrier.

4. TIANJIN MEDICAL UNIVERSITY J Clin Endocrinol Metab, June 2011, 96(6):E967–E971 Oatp1c1 immunoreactivity was present in glial cells throughout the hypothalamus. In addition, staining was present in capillary walls and in neurons of some nucleus in the hypothalamus. the PVN （ paraventricular nucleus ） IFN （ infundibular nucleus ） Supraoptic nucleus. Background

5. TIANJIN MEDICAL UNIVERSITY Hypothesis Oatp1c1 and Mct8 express in astrocytes. are related to transferring thyroid hormones in astrocytes.

6. TIANJIN MEDICAL UNIVERSITY Objective the effect of T 3 and T 4 on gene expression of Mct8 and Oatp1c1 in primary cultures of astrocytes of rat brain. To investigate-----

7. TIANJIN MEDICAL UNIVERSITY Methods results Methods & results

8. TIANJIN MEDICAL UNIVERSITY Figure 1 Oatp1c1 staining in Cerebral cortex of rat. (A:100×; B:400×; C,D:1000×) Oatp1c1 immunoreactivity was present in neurons and glial cells, choroid plexus and capillary endothelial cells.

9. TIANJIN MEDICAL UNIVERSITY Figure 2 Mct8 staining in Cerebral cortex of rat. (A,B,C,D:1000×) Mct8 immunoreactivity was mainly present in neurons(C,D), choroid plexus(B) and capillary endothelial cells(A).

10. TIANJIN MEDICAL UNIVERSITY Cerebrum from Wistar rat pups aged postnatal day 1 astrocytes were dissociated GFAP staining---- immunofluorescence(IF) Mct8 or Oatp1c1 with GFAP staining---- confocal microscopy the monolayer cell density reached to 80~90%, cells were starved 2 days with the medium without serum 6, 12, 24, 48, 72 hours with T 3 or T 4 of 500 nM, respectively Mct8 and Oatp1c1 expressions ----- Western Blot Experiments in vitro

11. TIANJIN MEDICAL UNIVERSITY Figure 3 GFAP immunofluorescence staining in astrocytes of primary culture. After 3~4 times of passages, the GFAP positive cells were over 95%. GFAP DAPI merge

12. TIANJIN MEDICAL UNIVERSITY Figure 4 Mct8 and Oatp1c1 immunofluorescence staining in astrocytes of primary culture. Mct8 and Oatp1c1 proteins expressed in rat astrocytes.

13. TIANJIN MEDICAL UNIVERSITY Figure 5 Transporter expression in primary astrocytes. Immunocytochemistry for Mct8 and Oatp1c1 in astrocytes by confocal microscopy. GFAP (green) served as astrocytic marker.

14. TIANJIN MEDICAL UNIVERSITY Figure 6 Expression of Mct8 and Oatp1c1 in primary astrocytes. The expression of Mct8 and Oatp1c1 in astrocytes incubated for 6, 12, 24, 48 and 72 hours with 500 nM T 3 or T 4, respectively, was determined by immunoblotting. 30μg total protein was loaded per lane. β-actin served as control. U251 cell line was shown for comparison.

15. TIANJIN MEDICAL UNIVERSITY Oatp1c1 is coexpressed with Mct8 in astrocytes. Our results indicated earlier up-regulated Oatp1c1 might intake T 4 into cells and later up-regulated Mct8 could export T 3 which is converted by D2 in astrocytes. Conclusions

16. TIANJIN MEDICAL UNIVERSITY Thank you for your attention!