Does ghrelin regulate clock genes expression in ...

GHRELIN MODIFIES CLOCK GENES EXPRESSION IN THE LIVER OF GOLDFISH VIA PLC-PKC PATHWAY Aída Sánchez-Bretaño, A.M. Blanco, A.L. Alonso-Gómez, M.J. Delgado, E. Isorna.

Department of Physiology, Faculty of Biology, Complutense University of Madrid, Spain. Experimental designs Introduction LIVER: main link between metabolism and circadian system [1,2,3,4]

ZT 0 ZT2

n=6

GHRELIN

15 mg liver/1 ml DMEM/well 24L (x lux), 21 °C

LIVER SAMPLING Ghrelin concentrationresponse study

Proximal gastrointestinal tract (main site of ghrelin synthesis)

[2,4]

 Does ghrelin regulate clock genes expression in cultured liver?  Which is the intracellular pathway involved?

ZT 0 12L:12D

ZT 12

2h

Which is the intracellular pathway involved?

2h

15’

Stabilization

Drug

1h

1h Drug plus Ghrelin (10 nM)

• [D-Lys3]GHRP-6 → 1, 10, 100 nM

2h

5h

Stabilization

Acylghrelin-17 (0, 0.1, 1, 10 nM)

• H89 → 50, 200 μM • U73122 → 1, 10 μM • Chelerythine (Chel.) → 1, 5 μM

Clock genes expression by RT-qPCR

Results 1 Ghrelin action on clock genes expression in cultured liver gper1a

PKC inhibitor

PLC inhibitor

PKC inhibitor

grev-erbα

gbmal1a

PLC inhibitor

PKA inhibitor

Figure1. Concentration-response effects of clock genes expression in cultured liver treated with ghrelin (GRL) for 1 or 5 h. Data are shown as mean±s.e.m. (n=6) in relative units (ΔΔCt method). Different letters indicate significant differences among groups (p