Analysis of hepatic transcriptome modulation exerted by γ-conglutin from lupins in a streptozotocin-induced diabetes model. Analysis of hepatic transcriptome modulation exerted by γ-conglutin from lupins in a streptozotocin-induced diabetes model

Lupinus albus γ-conglutin is proposed to positively affect glucose metabolism through inhibition of hepatic glucose production and insulin-mimetic activity; however, the action mechanism is not entirely known. Besides, most studies had focused on its effect on molecular targets directly related to glucose metabolism, and few studies have investigated how γ-conglutin may affect the liver gene expression or if it plays a role in other metabolic processes. Therefore, we investigated the influence of γ-conglutin on the liver transcriptome of streptozotocin-induced diabetic rats using DNA microarrays, ontological analyses, and quantitative PCR. Of the 22,000 genes evaluated, 803 and 173 were downregulated and upregulated, respectively. The ontological analyses of the differentially expressed genes revealed that among others, the mitochondria, microtubules, cytoskeleton, and oxidoreductase activity terms were enriched, implying a possible role of γ-conglutin on autophagy. To corroborate the microarray results, we selected and quantified, by PCR, the expression of two genes associated with autophagy (Atg7 and Snx18) and found their expression augmented two and threefold, respectively; indicating a higher autophagy activity in animals treated with γ-conglutin. Although complementary studies are required, our findings indicate for the first time that the hypoglycaemic effects of γ-conglutin may involve an autophagy induction mechanism, a pivotal process for the preservation of cell physiology and glucose homeostasis. Overall design: A two-color microarrays experiment was performed. We separately pooled equal amounts of high-quality RNA from five diabetic rats of the treated (with γ-conglutin, n = 5) and non-treated (without γ-conglutin, n = 5) groups before carrying out the cDNA synthesis and hybridization. We also replicated the cDNA hybridization using the dye-swap replication technique in two separate two-color microarray slides. Each slide contained samples from both the control and treatment groups differentially stained.

已有研究表明，白羽扇豆γ-伴球蛋白（Lupinus albus γ-conglutin）可通过抑制肝脏葡萄糖生成、发挥胰岛素样活性来正向调控葡萄糖代谢，但其具体作用机制尚未完全阐明。此外，现有研究大多聚焦于其对葡萄糖代谢直接相关分子靶点的调控作用，极少有研究探讨γ-伴球蛋白如何影响肝脏基因表达，或其在其他代谢过程中的潜在功能。因此，本研究采用DNA微阵列（DNA microarrays）、基因本体分析（ontological analyses）及定量聚合酶链反应（quantitative PCR），探究了γ-伴球蛋白对链脲佐菌素诱导的糖尿病大鼠肝脏转录组的影响。在本次检测的22000个基因中，分别有803个基因表达下调、173个基因表达上调。对差异表达基因的基因本体分析显示，线粒体、微管、细胞骨架以及氧化还原酶活性等条目显著富集，提示γ-伴球蛋白可能参与细胞自噬调控。为验证微阵列实验结果，我们通过定量PCR筛选并定量了两个与细胞自噬相关的基因（Atg7与Snx18）的表达水平，发现二者的表达量分别上调了2倍和3倍，表明经γ-伴球蛋白处理的动物体内细胞自噬活性升高。尽管仍需开展补充验证实验，但本研究首次证实，γ-伴球蛋白的降糖效应可能通过诱导细胞自噬实现——细胞自噬是维持细胞生理稳态与葡萄糖稳态的关键过程。

实验整体设计：本研究采用双色微阵列（two-color microarrays）实验方案。我们分别从5只经γ-伴球蛋白处理的糖尿病大鼠（处理组，n=5）与5只未处理的糖尿病大鼠（对照组，n=5）中提取高质量RNA，将等量RNA混合后进行互补DNA（cDNA）合成与杂交实验。同时，我们采用染料互换重复技术（dye-swap replication）在两张独立的双色微阵列玻片上重复进行cDNA杂交实验。每张玻片均包含经不同荧光染料标记的对照组与处理组样本。