DOI: 10.4231/jb0q-dr06
title: Circadian Disruption Decreases Gluconeogenic Flux

description: &lt;p&gt;The data provide here are &lt;sup&gt;13&lt;/sup&gt;C enrichment ratios of amino acids following incubation in [U-&lt;sup&gt;13&lt;/sup&gt;C] propionate to evaluate carbon flux towards glucose or tricarboxylic acid (TCA) cycle intermediates. These data are presented and discussed in a manuscript submitted to JDS-Communications.&lt;/p&gt;

&lt;p&gt;Previous studies from our lab showed that when late gestation Holstein cows are exposed to circadian rhythm disrupting environments glucose homeostasis and liver transcriptome are altered (PMID: 34643103, PMID: 31980225, PMID: 33309361). Glucose production through gluconeogenesis in the cow is tightly controlled by the liver. Thus, the objective of the current study was to determine how carbon flux from propionate towards gluconeogenesis was affected by exposure to circadian rhythm disruption; and the relationship of carbon flux to the hepatic transcriptome. Non-lactating dairy cows were assigned to one of two treatments, control (C) or phase-shifted (PS), 35 d before expected calving. Both C and PS groups received the same amount of light, 16 h, and dark, 8 h, but the PS group&rsquo;s light dark cycle was shifted forward 6 hours every 3 days to disrupt circadian clocks. Hepatic biopsies were collected on d 21 before expected calving. Liver explants prepared and incubated in [U-&lt;sup&gt;13&lt;/sup&gt;C] propionate for 2 h, and flash frozen in liquid nitrogen. Gas-chromatography mass spectrometry was used to measure mass isotopologue distribution of aspartate (ASP), serine (SER), alanine (ALA), and glutamate (GLU) to calculate &lt;sup&gt;13&lt;/sup&gt;C enrichment of the amino acids and the ratio of those amino acids to one another.  Amino acids were used as proxies for TCA cycle metabolites with the assumption of free carbon exchange between the pools of the amino acids and TCA cycle intermediates. Specifically, enrichment of ASP (eASP) was a proxy for oxaloacetate (OAA), the enrichment of SER (eSER) was a proxy for 3-phosphoglycerate, enrichment of ALA (eALA) was a proxy for pyruvate, and enrichment of GLU (eGLU) for &alpha;-ketoglutarate (aKG).&lt;/p&gt;

&lt;p&gt;Supplemental Table S1 represents the molar enrichment of each amino acid by cow and treatment, as well as the ratio of amino acids to one another. Metabolic flux ratios of amino acids were also calculated by summing M+1 to M+3 for the individual amino acid and dividing by the isotopomer sum of the comparator amino acid. The ratio of eSER:eASP represents the flow of carbons from OAA toward glucose, the ratio of eALA:eASP describes carbon flow through phosphoenolpyruvate carboxykinase and the reverse ratio describing carbon flow through pyruvate carboxylase reaction, and the ratio of eALA/eGLU describing TCA cycle flux. Additionally, propionate contribution to OAA, pyruvate, and aKG is shown. This was calculated by dividing the M+3 isotopomer for the individual amino acid that is a proxy for OAA, pyruvate, and aKG by the enrichment of propionate which is 99.5%, Data presented herein were collected from four C animals and four PS animals. Overall, the ratio of eSER/eASP in PS (0.75) was decreased (&lt;em&gt;P&lt;/em&gt; &lt; 0.05) relative to C (0.81) cows, indicating that exposure to circadian disruption reduced gluconeogenesis.&lt;/p&gt;

&lt;p&gt;Supplemental Table S2 lists protein coding genes with significant (&lt;em&gt;P&lt;/em&gt; &lt; 0.05, |r |&ge; 0.70) relationships between RNA-seq measured log base 2 of normalized read counts with eSER/eASP. Linear correlation analysis found that normalized read counts of 781 genes correlated to eSER:eASP. Of those 781 genes, 366 were negatively correlated and 415 were positively correlated.&lt;/p&gt;

&lt;p&gt;Supplemental Table S3 is the functional annotation output from Database for Annotation, Visualization, and Integrated Discovery (DAVID) for the genes that negatively correlated with the eSER:eASP.&lt;/p&gt;

&lt;p&gt;Supplemental Table S4 is the functional annotation output from DAVID for genes that positively correlated with eSER:eASP.&lt;/p&gt;

&lt;p&gt;Unique sample identifier; 22PS-BEC-LIVER2: 22=cow number; PS=phase shifted/C=control; BEC=21 d before expected calving which is the time point of sample collection; LIVER=liver biopsy&lt;/p&gt;
description: The data provide here are 13C enrichment ratios of amino acids following incubation in [U-13C] propionate to evaluate carbon flux towards glucose or tricarboxylic acid (TCA) cycle intermediates. These data are presented and discussed in a manuscript.