Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform increases endoplasmic reticulum (ER) stress in a dose dependent fashion, so when fructose is coupled with a HFD intake it leads to unresolved ER stress. Conversely, a liver-specific knockdown of KHK in C57BL/6J male mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in genetically obesity ob/ob, db/db and lipodystrophic FIRKO male mice, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

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Diets high in sugar and fat promote the development of obesity and metabolic complications, while high-sugar, low-fat or high-fat, sugar-free (ketogenic) diets do not. We assessed the importance of sugar in a commonly used 60% high-fat diet (HFD), which contains 6.7% of calories from sucrose. The sucrose content was replaced either with an equal amount of fructose (HFD+RF) or glucose (HFD+RG) and all other components of the diet were identical. After 12 weeks on the diets, the mice on HFD and HFD+RF diets gained more weight, developed hepatic steatosis, glucose intolerance and insulin resistance, compared to the mice on normal chow or HFD+RG diets. A relatively small (6.7%) amount of sucrose or fructose, but not glucose, in a HFD was sufficient to increase the fructolysis (KHK-C, ALDO B and TKFC) and de novo lipogenesis (ME1, ACLY, ACC1, FASN, SCD1) pathways by WB. Metabolomics analysis revealed higher hepatic diacylglycerol (DAG) content in HFD and HFD+RF groups, while HFD+RG group had elevated hepatic triacylglycerol content. Proteomics analysis quantified 3058 unique proteins, 736 of which were significantly different between the mice on chow and HFD. Replacing sucrose with fructose altered only 2 proteins, while replacing sucrose with fructose significantly changed 196 proteins in the liver. Reactome pathway analysis showed that the most altered processes mediate lipid biosynthesis and metabolism. In conclusion, HFDs containing sucrose or fructose but not glucose increase the fructolysis pathway and lead to hepatic DAG accumulation and insulin resistance. Thus, a relatively small amount of fructose, regardless of its source, is sufficient to alter metabolic outcomes in mice consuming a HFD. Disclosure S.Park: None. T.I.Fadhul: None. C.Kahn: None. S.Softic: Research Support; Alnylam Pharmaceuticals, Inc.

Consumption of diets high in sugar and fat are well-established risk factors for the development of obesity and its metabolic complications, including non-alcoholic fatty liver disease. Metabolic dysfunction associated with sugar intake is dependent on fructose metabolism via ketohexokinase (KHK). Here, we compared the effects of systemic, small molecule inhibition of KHK enzymatic activity to hepatocyte-specific, GalNAc-siRNA mediated knockdown of KHK in mice on a HFD. Both modalities led to an improvement in liver steatosis, however, via substantially different mechanisms. KHK knockdown profoundly decreased lipogenesis, while the inhibitor increased the fatty acid oxidation pathway. Moreover, hepatocyte-specific KHK knockdown completely prevented hepatic fructose metabolism and improved glucose tolerance. Conversely, KHK inhibitor only partially reduced fructose metabolism, but it also decreased downstream triokinase. This led to the accumulation of fructose-1 phosphate, resulting in glycogen accumulation, hepatomegaly, and impaired glucose tolerance. In summary, KHK profoundly impacts hepatic metabolism, likely via both kinase-dependent and independent mechanisms.