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