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Yina Sun et al

Tianjin Institute of Endocrinology Tianjin, China

EXPRESSIONS OF Mct8 AND Oatp1c1 UP-REGULATED BY T3 AND T4 IN

ASTROCYTES OF PRIMARY CULTURE

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Background

• Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins.

• Mct8 ---- T3 ---- neurons

• Oatp1c1----T4 ---- Choroid plexus

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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.

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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

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Hypothesis

Oatp1c1 and Mct8

• express in astrocytes.

• are related to transferring thyroid hormones in astrocytes.

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Objective

the effect of T3 and T4 on gene expression of Mct8 and Oatp1c1 in

primary cultures of astrocytes of rat brain.

To investigate-----

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Methods & results

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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.

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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).

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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 T3 or T4 of 500 nM , respectively

Mct8 and Oatp1c1 expressions ----- Western Blot

Experiments in vitro

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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

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Figure 4 Mct8 and Oatp1c1 immunofluorescence staining in astrocytes of primary culture.

Mct8 and Oatp1c1 proteins expressed in rat astrocytes.

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Figure 5 Transporter expression in primary astrocytes.

Immunocytochemistry for Mct8 and Oatp1c1 in astrocytes by confocal microscopy. GFAP (green) served as astrocytic marker.

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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 T3 or T4, respectively, was determined by immunoblotting. 30μg total protein was loaded per lane. β-actin served as control. U251 cell line was shown for comparison.

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• Oatp1c1 is coexpressed with Mct8 in astrocytes.

• Our results indicated earlier up-regulated Oatp1c1 might intake T4 into cells and later up-regulated Mct8 could export T3 which is converted by D2 in astrocytes.

Conclusions

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Thank you for your attention!