Knockdown of Ketohexokinase Versus Inhibition of Its Kinase Activity Exert Divergent Effects on Fructose Metabolism

Excessive fructose intake is a risk factor for the development of obesity and its complications. Targeting ketohexokinase (KHK), the first enzyme of fructose metabolism, has been investigated for the management of MASLD. We compared the effects of systemic, small molecule inhibitor of KHK enzymatic activity to hepatocyte-specific, GalNAc-siRNA mediated knockdown of KHK in mice on a HFD. We measured KHK enzymatic activity, extensively quantified glycogen accumulation, performed RNAseq analysis, and enumerated hepatic metabolites using mass spectrometry. Both KHK siRNA and KHK inhibitor led to an improvement in liver steatosis, however, via substantially different mechanisms. KHK knockdown decreased the de novo lipogenesis pathway, whereas the inhibitor increased the fatty acid oxidation pathway. Moreover, KHK knockdown completely prevented hepatic fructolysis and improved glucose tolerance. Conversely, the KHK inhibitor only partially reduced fructolysis, but it also targeted triokinase, mediating the third step of fructolysis. This leads to the accumulation of fructose-1 phosphate, resulting in glycogen accumulation, hepatomegaly, and impaired glucose tolerance. Overexpression of wild-type, but not kinase-dead KHK in cultured hepatocytes increased hepatocyte injury and glycogen accumulation when treated with fructose. The differences between KHK inhibition and knockdown are, in part, explained by the kinase-dependent and independent effects of KHK on hepatic metabolism. Overall design: We had mice on normal diet or HFD. A subgroup of mice on HFD was treated with KHK inhibitor twice daily for 4 weeks, starting at 6 to 10 weeks on the diets or with KHK siRNA injected subcutaneously every two weeks starting from 6 to 10 weeks on the diet. The four groups of mice were sacrificed after 10 weeks. RNAseq was performed for RNA isolated from the liver.

过量摄入果糖是肥胖及其并发症发生的风险因素。靶向果糖代谢的首个酶——酮己糖激酶（ketohexokinase, KHK），已被研究用于代谢相关脂肪性肝病（MASLD）的管理。本研究对比了全身性小分子KHK酶活性抑制剂与肝细胞特异性、N-乙酰半乳糖胺修饰小干扰RNA（GalNAc-siRNA）介导的KHK敲低，在高脂饮食（HFD）小鼠中的作用效果。我们测定了KHK酶活性，全面定量了糖原蓄积情况，开展了RNA测序（RNA-seq）分析，并利用质谱分析法对肝脏代谢物进行了定量表征。KHK siRNA与KHK抑制剂均可改善肝脏脂肪变性，但二者的作用机制存在显著差异：KHK敲低会抑制从头脂肪生成通路，而KHK抑制剂则可激活脂肪酸氧化通路。此外，KHK敲低可完全阻断肝脏果糖分解，并改善糖耐量；与之相反，KHK抑制剂仅能部分降低果糖分解，且它还可靶向作用于介导果糖分解第三步的甘油醛激酶（triokinase），这会导致1-磷酸果糖积累，进而引发糖原蓄积、肝肿大以及糖耐量受损。在培养的肝细胞中，过表达野生型KHK（而非激酶失活型KHK）经果糖处理后，会加重肝细胞损伤并增加糖原蓄积。KHK抑制与敲低之间的差异，部分可通过KHK对肝脏代谢的激酶依赖型与非激酶依赖型效应来解释。总体实验设计：本研究设置正常饮食组与高脂饮食组小鼠。高脂饮食组小鼠进一步分为两个亚组：一组于造模6至10周后，每日两次给予KHK抑制剂，持续干预4周；另一组于同期开始每两周皮下注射一次KHK siRNA。四组小鼠均在造模10周后实施安乐死，采集肝脏组织分离RNA并开展RNA测序分析。