Circadian Disruption Decreases Gluconeogenic Flux

<p>The data provide here are <sup>13</sup>C enrichment ratios of amino acids following incubation in [U-<sup>13</sup>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.</p>

<p>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’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-<sup>13</sup>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 <sup>13</sup>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 α-ketoglutarate (aKG).</p>

<p>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 (<em>P</em> < 0.05) relative to C (0.81) cows, indicating that exposure to circadian disruption reduced gluconeogenesis.</p>

<p>Supplemental Table S2 lists protein coding genes with significant (<em>P</em> < 0.05, |r |≥ 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.</p>

<p>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.</p>

<p>Supplemental Table S4 is the functional annotation output from DAVID for genes that positively correlated with eSER:eASP.</p>

<p>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</p>

本数据集提供的是经[U-¹³C]丙酸盐（propionate）孵育后氨基酸的¹³C富集率，用于评估碳流向葡萄糖或三羧酸循环（tricarboxylic acid (TCA) cycle）中间产物的通量。相关数据已在投稿至《JDS-Communications》的手稿中呈现并讨论。

本实验室既往研究表明，妊娠晚期荷斯坦奶牛（Holstein cows）暴露于扰乱昼夜节律（circadian rhythm）的环境中时，其葡萄糖稳态（glucose homeostasis）与肝脏转录组（hepatic transcriptome）会发生改变（PMID：34643103、PMID：31980225、PMID：33309361）。奶牛体内通过糖异生（gluconeogenesis）途径生成葡萄糖的过程严格受肝脏调控。因此本研究的目的是明确暴露于昼夜节律扰乱环境下，丙酸盐的碳流向糖异生途径的通量如何变化，以及碳通量与肝脏转录组之间的关联。研究于预计产犊前35天将非泌乳奶牛（non-lactating dairy cows）随机分为两组：对照组（control，缩写C）与相位偏移组（phase-shifted，缩写PS）。两组均接受相同的光照周期：16小时光照、8小时黑暗，但相位偏移组的光暗周期每3天向前偏移6小时，以扰乱其昼夜节律。于预计产犊前第21天采集肝脏活检（hepatic biopsies）样本。制备肝外植体（liver explants）并置于[U-¹³C]丙酸盐中孵育2小时，随后用液氮快速冷冻样本。采用气相色谱-质谱联用法（gas-chromatography mass spectrometry）测定天冬氨酸（aspartate，缩写ASP）、丝氨酸（serine，缩写SER）、丙氨酸（alanine，缩写ALA）与谷氨酸（glutamate，缩写GLU）的质量同位素异构体分布（mass isotopologue distribution），以此计算氨基酸的¹³C富集率以及各氨基酸间的比值。本研究假设氨基酸库与三羧酸循环中间产物库之间存在自由碳交换，因此以各氨基酸作为三羧酸循环代谢物的替代指标。具体而言，天冬氨酸富集率（eASP）可作为草酰乙酸（oxaloacetate，缩写OAA）的替代指标，丝氨酸富集率（eSER）可作为3-磷酸甘油酸（3-phosphoglycerate）的替代指标，丙氨酸富集率（eALA）可作为丙酮酸（pyruvate）的替代指标，谷氨酸富集率（eGLU）可作为α-酮戊二酸（α-ketoglutarate，缩写aKG）的替代指标。

补充表S1展示了每头奶牛在对应处理组下各氨基酸的摩尔富集率（molar enrichment），以及各氨基酸间的比值。氨基酸的代谢通量比值通过以下方式计算：将单种氨基酸的M+1至M+3同位素异构体信号求和，再除以对照氨基酸的同位素异构体总和（isotopomer sum）。eSER:eASP比值可反映碳从草酰乙酸流向葡萄糖的通量；eALA:eASP比值可反映经磷酸烯醇式丙酮酸羧激酶（phosphoenolpyruvate carboxykinase）的碳流，其反向比值则可反映经丙酮酸羧化酶（pyruvate carboxylase）反应的碳流；而eALA/eGLU比值可反映三羧酸循环通量。此外，本数据集还展示了丙酸盐对草酰乙酸、丙酮酸与α-酮戊二酸的贡献占比，计算方式为将作为草酰乙酸、丙酮酸及α-酮戊二酸替代指标的氨基酸的M+3同位素异构体信号，除以丙酸盐的富集率（99.5%）。本研究的数据来自4头对照组奶牛与4头相位偏移组奶牛。整体而言，相位偏移组奶牛的eSER/eASP比值（0.75）较对照组（0.81）显著降低（P<0.05），表明昼夜节律扰乱暴露会降低糖异生水平。

补充表S2列出了与eSER/eASP比值存在显著关联（P<0.05，|r|≥0.70）的蛋白质编码基因，这些基因的RNA测序（RNA-seq）标准化读长计数的以2为底对数与eSER/eASP比值相关。经线性相关分析（linear correlation analysis），共有781个基因的标准化读长计数与eSER:eASP比值存在关联，其中366个基因呈负相关，415个基因呈正相关。

补充表S3为注释、可视化与集成发现数据库（Database for Annotation, Visualization and Integrated Discovery, DAVID）对与eSER:eASP比值呈负相关的基因所输出的功能注释（functional annotation）结果。

补充表S4为DAVID数据库对与eSER:eASP比值呈正相关的基因所输出的功能注释结果。

样本唯一标识符说明：22PS-BEC-LIVER2中，22代表奶牛编号；PS为相位偏移组、C为对照组；BEC代表预计产犊前21天，即样本采集时间点；LIVER代表肝脏活检样本。