Cγ and lupanine enhances in vivo the antidiabetic effect and involves modulation of the liver gene expression profile

Previous studies have individually shown the antidiabetic potential of gamma conglutin (Cγ) and lupanine from lupins. Until now, the influence of combines both compounds and the effective dose of the combination is not assessed. Moreover, the resulting gene profile from this novel combination remains to be explored. Therefore, we proposed to study different dose combinations of Cγ and lupanine by the oral glucose tolerance test (OGTT) to identify the greater antidiabetic effect on the T2D rat model. Later, we administered the selected dose combination for a week. Finally, we evaluated the gene expression profile using DNA microarrays and its bioinformatic analysis. We found that the combination of 28 mg/kg BW Cγ + 20 mg/kg BW lupanine positively influences the expression of Pdk4, Ppargc1a, Foxo1, and Foxo3 genes. The reestablishment of Pdk4 gene expression is involved in several biological processes associated with regulation, metabolism, and homeostasis of glucose and fatty acids. For the first time, we reported the beneficial effect of the combination of two functional lupin compounds in a T2D rat model. Nevertheless, further studies are needed to investigate the pharmacokinetics and pharmacodynamics of the Cγ + lupanine combination. Gene expression analysis of RNA samples from liver tissue of healthy and diabetic-induced rats. Three biological replicates of liver total RNA pools were prepared for each experimental group: healthy rat group, type 2 diabetic control group, and type 2 diabetic rats treated with 28 mg/kg BW Cγ + 20 mg/kg BW lupanine. Each pool consisted of the mixture of RNA isolated from three different animals. A total of three technical replicates (derived from the RNA of nine animals per group) were processed for hybridization into the Affymetrix´s Rat Transcriptome Array 1.0 (Affymetrix, Santa Clara, CA, USA) at the Microarray Unit of the National Institute of Genomic Medicine (INMEGEN), Mexico. The procedures of hybridization and scanning of the microarray were performed according to GeneChipTM Whole Transcript (WT) Expression Arrays User Guide of Affymetrix Corporation. The gene expression files were processed and analyzed with the Applied Biosystem-sTM Transcriptome Analysis Console 4.0.2 (TAC, ThermoFisher, CA, USA). Gene expression data were RMA normalized, and differential expression determined with the following parameters: RMA+DABG (Rattus norvegicus) and expression analysis settings: Fold Change < -2 or > 2, p-value < 0.05, and ANOVA method: ebayes. For functional enrichment analysis, STRING software was used to analyze differentially expressed targets interactions. Gene Ontology (GO), KEGG pathways, and WikiPathways analysis were performed.

过往研究已分别证实，来源于羽扇豆的γ-伴球蛋白（gamma conglutin, Cγ）与羽扇豆碱（lupanine）均具备抗糖尿病活性。截至目前，尚未有研究评估二者联合使用的影响及其联合给药的有效剂量，且该新型组合所诱导的基因表达谱仍有待探索。为此，本研究通过口服葡萄糖耐量试验（oral glucose tolerance test, OGTT），针对2型糖尿病（type 2 diabetes, T2D）大鼠模型，探究Cγ与羽扇豆碱的不同剂量组合的抗糖尿病活性，以筛选最优给药方案；随后对筛选得到的联合给药组进行为期一周的干预；最终通过DNA微阵列（DNA microarray）技术及生物信息学分析，对基因表达谱进行评估。研究发现，28 mg/kg BW的Cγ联合20 mg/kg BW的羽扇豆碱组合，可正向调控Pdk4、Ppargc1a、Foxo1及Foxo3基因的表达。Pdk4基因表达的恢复，参与葡萄糖与脂肪酸的调控、代谢及稳态维持相关的多种生物学过程。本研究首次在T2D大鼠模型中，报道了两种羽扇豆功能化合物联合使用的有益调控作用。不过，仍需开展进一步研究以阐明Cγ与羽扇豆碱联合给药的药代动力学与药效动力学特征。本研究针对健康大鼠及糖尿病造模大鼠的肝脏组织RNA样本开展基因表达分析。每个实验组均制备3份生物学重复的肝脏总RNA混合样本，实验组包括：健康大鼠组、2型糖尿病对照组，以及经28 mg/kg BW Cγ联合20 mg/kg BW 羽扇豆碱处理的2型糖尿病给药组；每份混合样本均来自3只不同个体的分离RNA。每组共制备3份技术重复样本（每组样本来自9只个体的RNA），于墨西哥国家基因组医学研究所（INMEGEN）微阵列平台，将样本与Affymetrix大鼠转录组芯片1.0（Affymetrix，美国加利福尼亚州圣克拉拉）进行杂交。微阵列的杂交与扫描流程严格按照Affymetrix公司的GeneChip™ 全转录组（Whole Transcript, WT）表达阵列用户指南执行。基因表达数据文件通过应用生物系统（Applied Biosystems）转录组分析控制台4.0.2（TAC，赛默飞世尔，美国加利福尼亚州）进行处理与分析。基因表达数据采用RMA法进行标准化，差异表达分析采用如下参数：RMA+DABG（褐家鼠，Rattus norvegicus），表达分析阈值设置为：折叠变化（Fold Change）<-2或>2，P值<0.05，统计方法采用经验贝叶斯（ebayes）法。功能富集分析方面，本研究采用STRING软件分析差异表达基因的靶标相互作用，同时开展了基因本体（Gene Ontology, GO）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路及WikiPathways分析。