Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.

Effects of supplementation on food intake, body weight and hepatic metabolites in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mouse model of human citrin deficiency.

The C57BL/6:Slc23a13(-/-);Gpd2(-/-) double-knockout (a.k.a., citrin/mitochondrial glycerol 3-phosphate dehydrogenase double knockout or Ctrn/mGPD-KO) mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2), making it a suitable model of human citrin deficiency. In the present study, we show that when mature Ctrn/mGPD-KO mice are switched from a standard chow diet (CE-2) to a purified maintenance diet (AIN-93M), this resulted in a significant loss of body weight as a result of reduced food intake compared to littermate mGPD-KO mice. However, supplementation of the purified maintenance diet with additional protein (from 14% to 22%; and concomitant reduction or corn starch), or with specific supplementation with alanine, sodium glutamate, sodium pyruvate or medium-chain triglycerides (MCT), led to increased food intake and body weight gain near or back to that on chow diet. No such effect was observed when supplementing the diet with other sources of fat that contain long-chain fatty acids. Furthermore, when these supplements were added to a sucrose solution administered enterally to the mice, which has been shown previously to lead to elevated blood ammonia as well as altered hepatic metabolite levels in Ctrn/mGPP-KO mice, this led to metabolic correction. The elevated hepatic glycerol 3-phosphate and citrulline levels after sucrose administration were suppressed by the administration of sodium pyruvate, alanine, sodium glutamate and MCT, although the effect of MCT was relatively small. Low hepatic citrate and increased lysine levels were only found to be corrected by sodium pyruvate, while alanine and sodium glutamate both corrected hepatic glutamate and aspartate levels. Overall, these results suggest that dietary factors including increased protein content, supplementation of specific amino acids like alanine and sodium glutamate, as well as sodium pyruvate and MCT all show beneficial effects on citrin deficiency by increasing the carbohydrate tolerance of Ctrn/mGPD-KO mice, as observed through increased food intake and maintenance of body weight.

Pivotal role of inter-organ aspartate metabolism for treatment of mitochondrial aspartate-glutamate carrier 2 (citrin) deficiency, based on the mouse model.

Previous studies using citrin/mitochondrial glycerol-3-phosphate (G3P) dehydrogenase (mGPD) double-knockout mice have demonstrated that increased dietary protein reduces the extent of carbohydrate-induced hyperammonemia observed in these mice. This study aimed to further elucidate the mechanisms of this effect. Specific amino acids were initially found to decrease hepatic G3P, or increase aspartate or citrulline levels, in mGPD-knockout mice administered ethanol. Unexpectedly, oral glycine increased ammonia in addition to lowering G3P and increasing citrulline. Subsequently, simultaneous glycine-plus-sucrose (Gly + Suc) administration led to a more severe hyperammonemic state in double-KO mice compared to sucrose alone. Oral arginine, ornithine, aspartate, alanine, glutamate and medium-chain triglycerides all lowered blood ammonia following Gly + Suc administration, with combinations of ornithine-plus-aspartate (Orn + Asp) or ornithine-plus-alanine (Orn + Ala) suppressing levels similar to wild-type. Liver perfusion and portal vein-arterial amino acid differences suggest that oral aspartate, similar to alanine, likely activated ureagenesis from ammonia and lowered the cytosolic NADH/NAD+ ratio through conversion to alanine in the small intestine. In conclusion, Gly + Suc administration induces a more severe hyperammonemic state in double-KO mice that Orn + Asp or Orn + Ala both effectively suppress. Aspartate-to-alanine conversion in the small intestine allows for effective oral administration of either, demonstrating a pivotal role of inter-organ aspartate metabolism for the treatment of citrin deficiency.