Tightly regulated insulin secretion is a pre-requisite for maintaining appropriate blood glucose level. Failure to do so results in hypoglycemia or in diabetes. It has been reported that treatment of pancreatic b-cells with TGF-
b1 induces insulin secretion. However, the molecular mechanism for TGF-b signaling in insulin secretion is largely unknown. We used immunoprecipitation followed by mass spectrometry to identify partner molecules for DAF-8/R-Smad in TGF-b signaling, including a nuclear hormone receptor (NHR-69), which is a strong ortholog of mammalian hepatocyte nuclear factor 4a (HNF4a). HNF4a is a human MODY (Maturity Onset Diabetes of the Young) gene, and its mutation results in decreased insulin secretion. RNAi against
nhr-69 in a sensitized genetic background enhanced dauer formation.
nhr-69 mutants not only enhanced dauer formation of TGF-b pathway Daf-c (constitutive dauer formation) mutants, but also suppressed dauer recovery. Stably integrated
nhr-69p::
nhr-69::gfp was expressed strongly in the intestinal nuclei, hypodermis, and ASI neurons. Nuclear retention of NHR-69 in the intestine was reduced in pre-dauer (L2d) larvae and barely detectable in dauer larvae. When starvation-induced dauers were exposed to food for 30 mins, ASI expression resumed within 6 hrs even without food.
daf-2/IGF1-R, but not
daf-7/TGF-b, was required for expression in ASI neurons. NHR-69 was associated with DAF-8 in vivo and in vitro, but not with DAF-14/Smad or DAF-3/Co-Smad.
daf-8(
m85)
nhr-69(
ok1926) double mutants showed defects in neuropeptide secretion and increased expression of marker genes
sod-3 and
gst-10, which are normally repressed by
daf-2 signaling. A yeast-one hybrid analysis had indicated that NHR-69 targets the promoter of
exp-2, which encodes a voltage-gated potassium channel. Expression of
exp-2 was synergistically increased in
daf-8 nhr-69 mutants. Loss-of-function (lof)
exp-2 mutants conferred an increased insulin secretion and gain-of-function mutants showed decreased insulin secretion.
exp-2 (gof) mutants also exhibited upregulated
sod-3 and
gst-10 expression. We propose that DAF-8/R-Smad and NHR-69/HNF4a target the voltage-gated potassium channel gene,
exp-2, to promote insulin secretion in C. elegans. A chromatin IP experiment to examine direct binding of NHR-69 to the
exp-2 promoter is underway.