Integration of metabolomic and transcriptomic analyses reveals novel regulatory functions of the ChREBP transcription factor in energy metabolism

Carbohydrate Response Element-Binding Protein (ChREBP) is a transcription factor that activates key genes involved in glucose, fructose, and lipid metabolism in response to carbohydrate feeding, but its other potential roles in metabolic homeostasis have not been as well studied. We used liver-selective GalNAc-siRNA technology to suppress expression of ChREBP in rats fed a high fat/high sucrose diet and characterized hepatic and systemic responses by integrating transcriptomic and metabolomic analyses. GalNAc-siChREBP-treated rats had lower levels of multiple short-chain acyl CoA metabolites compared to rats treated with GalNAc-siCtrl containing a non-targeting siRNA sequence. These changes were related to a sharp decrease in free CoA levels in GalNAc-siChREBP treated-rats, accompanied by lower expression of transcripts encoding enzymes and transporters involved in CoA biosynthesis. These activities of ChREBP likely contribute to its complex effects on hepatic lipid and energy metabolism. While core enzymes of fatty acid (FA) oxidation are induced by ChREBP knockdown, accumulation of liver acylcarnitines and circulating ketones indicate diversion of acetyl CoA to ketone production rather than complete oxidation in the TCA cycle. Despite strong suppression of pyruvate kinase and activation of pyruvate dehydrogenase, pyruvate levels were maintained, likely via increased expression of pyruvate transporters, and decreased expression of lactate dehydrogenase and alanine transaminase. GalNAc-siChREBP treatment increased hepatic citrate and isocitrate levels while decreasing levels of distal TCA cycle intermediates. The drop in free CoA levels, needed for the 2-ketoglutarate dehydrogenase reaction, as well as a decrease in transcripts encoding the anaplerotic enzymes pyruvate carboxylase, glutamate dehydrogenase, and aspartate transaminase likely contributed to these effects. GalNAc-siChREBP treatment caused striking increases in PRPP and ZMP/AICAR levels, and decreases in GMP, IMP, AMP, NaNM, NAD(P), and NAD(P)H levels, accompanied by reduced expression of enzymes that catalyze late steps in purine and NAD synthesis. ChREBP suppression also increased expression of a set of plasma membrane amino acid transporters, possibly as an attempt to replenish TCA cycle intermediates. In sum, combining transcriptomic and metabolomic analyses has revealed regulatory functions of ChREBP that go well beyond its canonical roles in control of carbohydrate and lipid metabolism to now include mitochondrial metabolism and cellular energy balance.碳水化合物反应元件结合蛋白（Carbohydrate Response Element-Binding Protein，ChREBP）是一种转录因子，可响应碳水化合物摄入，激活参与葡萄糖、果糖及脂质代谢的关键基因，但其在代谢稳态中的其他潜在作用尚未得到充分研究。我们采用肝脏选择性N-乙酰半乳糖胺修饰小干扰RNA（GalNAc-siRNA）技术，在高脂高蔗糖饮食喂养的大鼠中抑制ChREBP的表达，并通过整合转录组学与代谢组学分析，表征肝脏及全身的应答反应。经GalNAc-siChREBP处理的大鼠，其多种短链酰基辅酶A代谢物水平较经含非靶向siRNA序列的GalNAc-siCtrl处理的大鼠更低。这些变化与经GalNAc-siChREBP处理的大鼠体内游离辅酶A（free CoA）水平的急剧下降相关，同时伴随参与辅酶A生物合成的酶及转运蛋白的编码转录本表达水平降低。ChREBP的上述活性可能是其对肝脏脂质及能量代谢产生复杂影响的基础。尽管脂肪酸（FA）氧化的核心酶在ChREBP敲低后被诱导表达，但肝脏酰基肉碱及循环酮体的蓄积表明，乙酰辅酶A被转向生成酮体，而非在三羧酸循环（TCA循环）中完成氧化。尽管丙酮酸激酶受到显著抑制、丙酮酸脱氢酶被激活，但大鼠的丙酮酸水平得以维持，这可能是通过上调丙酮酸转运蛋白的表达，以及下调乳酸脱氢酶与丙氨酸转氨酶的表达实现的。经GalNAc-siChREBP处理后，大鼠肝脏内柠檬酸与异柠檬酸水平升高，而三羧酸循环远端中间产物的水平降低。2-酮戊二酸脱氢酶反应所需的游离辅酶A水平下降，以及参与回补反应的酶——丙酮酸羧化酶、谷氨酸脱氢酶和天冬氨酸转氨酶——的编码转录本水平降低，可能共同促成了上述变化。经GalNAc-siChREBP处理还可显著升高磷酸核糖焦磷酸（PRPP）与ZMP/AICAR的水平，并降低鸟苷单磷酸（GMP）、次黄嘌呤单磷酸（IMP）、腺苷单磷酸（AMP）、N-乙酰神经氨酸（NaNM）、烟酰胺腺嘌呤二核苷酸（及其磷酸化形式，NAD(P)）以及烟酰胺腺嘌呤二核苷酸磷酸（及其还原形式，NAD(P)H）的水平，同时伴随催化嘌呤及烟酰胺腺嘌呤二核苷酸合成晚期步骤的酶表达水平下调。ChREBP抑制还可上调一组质膜氨基酸转运蛋白的表达，这可能是为了补充三羧酸循环的中间产物。综上，通过整合转录组学与代谢组学分析，我们揭示了ChREBP的调控功能远不止其在碳水化合物与脂质代谢调控中的经典作用，其还可参与线粒体代谢及细胞能量平衡的调控。