Insulin temporal sensitivity and its signaling pathway in the rat pineal gland.
OBJECTIVE
In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern.


METHODS
Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3:AANAT complex, respectively.


RESULTS
The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced "night". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IRbeta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity.


CONCLUSIONS
The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland.
Peliciari-Garcia
Rodrigo A
RA
University of São Paulo, Institute of Biomedical Sciences-I, Department of Physiology and Biophysics, São Paulo, SP, Brazil.
Marçal
Anderson C
AC
Silva
Jéssica A
JA
Carmo-Buonfiglio
Daniella
D
Amaral
Fernanda G
FG
Afeche
Solange Castro
SC
Cipolla-Neto
José
J
Carvalho
Carla R O
CR
D048948:14-3-3 Proteins / Q000378:metabolism; D000818:Animals; D047068:Arylalkylamine N-Acetyltransferase / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D000078790:Insulin Secretion; D008297:Male; D008550:Melatonin / Q000096:biosynthesis*; D009638:Norepinephrine / Q000378:metabolism / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010870:Pineal Gland / Q000187:drug effects* / Q000378:metabolism; D011874:Radiometry; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D013997:Time Factors
D048948:14-3-3 Proteins; D007328:Insulin; D047068:Arylalkylamine N-Acetyltransferase; D019869:Phosphatidylinositol 3-Kinases; D008550:Melatonin; D009638:Norepinephrine