DOI: 10.6076/d14s37
title: Growth hormone pulses are differentially regulated by the circadian clock gene Bmal1

description: In this study, we found that loss of the circadian clock gene Bmal1 causes
 disruptions throughout the growth hormone (GH) axis, from hepatic gene
 expression to production of urinary pheromones and pheromone-dependent
 behavior. First, we show that Bmal1 knockout (KO) males elicit reduced
 aggressive responses from WT males and secrete lower levels of major
 urinary proteins (MUPs); however, we also found that a liver-specific KO
 of Bmal1 (Liver-Bmal1-KO) produces a similar reduction in MUP secretion
 without a defect in aggressive behavior, indicating that the decrease in
 elicited aggression arises from another factor. We then shifted our
 investigation to determine the cause of MUP dysregulation in Bmal1 KO
 animals. As the pulse pattern of GH drives sexually dimorphic expression
 of hepatic genes including MUPs, we examined GH pulsatility. We found that
 Bmal1 KO males have a female-like pattern of GH release, while
 Liver-Bmal1-KO mice are not significantly different from either WT or
 Bmal1 KO. Since differential patterns of GH release regulate the
 transcription of many sexually dimorphic genes in the liver, we then
 examined hepatic gene transcription in Bmal1 KO and Liver-Bmal1-KO mice.
 We found that while some female-predominant genes increase, there was no
 decrease in male-predominant genes in the Bmal1 KO, and little change in
 the Liver-Bmal1-KO. We also found disrupted serum IGF-1 and liver Igf1
 mRNA in the Bmal1 KO mice, which may underlie the disrupted GH release.
 Overall, our findings differentiate between GH-pulse-driven and
 circadian-driven effects on hepatic genes, and the functional consequences
 of altered GH pulsatility.
description: Mice were handled daily for four weeks prior to the day of assay
 to acclimate to sampling stress. Mice were also provided enrichment
 including nestlets and a small house provided within the cage. To measure
 endogenous growth hormone pulses, we collected 4 µl of blood through the
 tail vein every 10 minutes for six hours. The blood was collected by
 making a small nick at the tail tip and then a capillary tube was held
 gently to the tail to collect blood into a capillary tube (Drummond
 microcaps 40 μL) and the end was sealed with Critoseal. If a scab formed
 during the course of the experiment, it was gently removed with sterile
 gauze to renew blood flow. The volume of blood collected over the
 experiment was under 150 μl, and fluid replacement was not necessary.
 Blood was collected from ZT 4-10, during the light phase. Capillary tubes
 were incubated at room temperature for 20 minutes, followed by
 centrifugation at 2000 x g for 20 minutes and then the serum was removed
 and frozen with serum matrix buffer until assay. Serum was assayed for
 growth hormone concentration with a Luminex Magpix. Briefly, 1.5 μL of
 serum was added to 23.5 μL of serum matrix buffer provided in the kit and
 incubated with antibody beads overnight at 4 degrees C°. GH was measured
 by Luminex assay (Milipore MPTMAG-49K) according to manufacturer
 instructions, with an inter- and intra-assay variation of 14.3% and 8.2%,
 respectively.)
description: Data are presented as ng/ul GH. These are the data generated
 after 5-parameter logistic curve fitting by the Luminex software. We have
 also included the standard curve for each animal. Because plates were
 assayed with two animals per plate, and one standard curve, identical
 standard curves indicate both animals were run on the same plate. Standard
 curves were run in duplicate (displayed horizontally), whereas time point
 samples were in singlicate due to the low serum volumes required by serial
 sampling.