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. 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.

过量果糖摄入是肥胖及其并发症发生的风险因素。以果糖代谢的首步酶——己酮糖激酶（ketohexokinase, KHK）为靶点治疗代谢功能障碍相关脂肪性肝病（MASLD）的相关研究已被广泛开展。本研究在高脂饮食（High-Fat Diet, HFD）喂养的小鼠中，对比了全身性KHK酶活性小分子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基因敲低的干预效果。我们检测了KHK酶活性，全面定量分析了糖原积累情况，开展了RNA测序（RNA-seq）分析，并通过质谱分析法定量检测了肝脏代谢物。KHK基因敲低与KHK抑制剂均能改善肝脏脂肪变性，但二者的作用机制存在显著差异。KHK基因敲低可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基因敲低可完全阻断肝脏果糖分解过程，并改善葡萄糖耐量；与之相反，KHK抑制剂仅能部分降低果糖分解速率，同时还会靶向三碳糖激酶（triokinase），干扰果糖分解的第三步反应，进而导致1-磷酸果糖（fructose-1 phosphate）积累，引发糖原积累、肝肿大以及葡萄糖耐量受损。在体外培养的肝细胞中，过表达野生型（而非激酶失活型）KHK可在果糖处理条件下加重肝细胞损伤与糖原积累。KHK抑制与基因敲低之间的效应差异，部分可通过KHK在肝脏代谢中的激酶依赖与非依赖效应来解释。本研究共设置四组小鼠：正常饮食组、高脂饮食对照组、高脂饮食联合KHK抑制剂组以及高脂饮食联合KHK siRNA组。其中高脂饮食对照组与两个干预组均自造模6至10周起开始干预，正常饮食组不接受额外干预。高脂饮食联合KHK抑制剂组小鼠每日两次给药，持续4周；高脂饮食联合KHK siRNA组小鼠每两周皮下注射一次siRNA。所有小鼠均在造模10周后处死，采集肝脏组织分离RNA并进行RNA-seq分析。