10.6076/D14S37
Schoeller, Erica
0000-0002-3355-5964
University of California, San Diego
Tonsfeldt, Karen
0000-0001-8044-8857
University of California, San Diego
Sinkovich, McKenna
University of California, San Diego
Shi, Ruijing
University of California, San Diego
Mellon, Pamela
0000-0002-8856-0410
University of California, San Diego
Growth hormone pulses are differentially regulated by the circadian clock
gene Bmal1
Dryad
dataset
2020
Eunice Kennedy Shriver National Institute of Child Health and Human Development
https://ror.org/04byxyr05
HD082567
National Institute of Diabetes and Digestive and Kidney Diseases
https://ror.org/00adh9b73
HD100580
Eunice Kennedy Shriver National Institute of Child Health and Human Development
https://ror.org/04byxyr05
HD072754
Eunice Kennedy Shriver National Institute of Child Health and Human Development
https://ror.org/04byxyr05
HD012303
National Institute of Diabetes and Digestive and Kidney Diseases
https://ror.org/00adh9b73
DK063491
National Institute of Environmental Health Sciences
https://ror.org/00j4k1h63
ES101337
National Cancer Institute
https://ror.org/040gcmg81
CA023100
National Institute of Diabetes and Digestive and Kidney Diseases
https://ror.org/00adh9b73
DK007044
Eunice Kennedy Shriver National Institute of Child Health and Human Development
https://ror.org/04byxyr05
HD007203
Eunice Kennedy Shriver National Institute of Child Health and Human Development
https://ror.org/04byxyr05
HD090837
Lalor Foundation
https://ror.org/03vpdge44
Eunice Kennedy Shriver National Institute of Child Health and Human Development
https://ror.org/04byxyr05
HD028934
2021-02-04T00:00:00Z
2021-02-04T00:00:00Z
en
https://doi.org/10.1210/endocr/bqab023
240971 bytes
4
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.
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.)
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.