Novel sequencing technologies have led to the identification of multiple candidate genes for human diseases. Gene targeting and mutagenesis using stem cell technologies are powerful tools to elucidate essential functions of genes in normal physiology and the pathogenesis of disease. Using gene-targeted mice, haploid genetics in stem cells, and fly genetics, my group tries to genetically dissect disease mechanisms.
More than one billion people are malnourished in poor countries. In fact, malnutrition is a major problem even in rich countries. The consequences of malnutrition are often severe, making it a leading cause of death in the world. For more than a hundred years now, doctors have known that a diet deficient in protein or amino acids - the building blocks of proteins - may cause disorders of the immune system, diarrhea and intestinal inflammation, which weaken the body and are potentially life threatening. However, the molecular mechanism that would explain how malnutrition causes such severe symptoms has remained largely unexplored.
The renin-angiotensin system (RAS) has been studied for more than a century now. RAS is regulated by the opposing actions of two key carboxypeptidases known as angiotensin-converting enzyme (ACE) and ACE2. We were the first to demonstrate that ACE2 is a potent negative regulator of RAS, counterbalancing the multiple functions of ACE via catalytic cleavage of angiotensin II (Crackower et al. Nature 2002). ACE2 also functions as a key receptor for the SARS Coronavirus involved in acute lung failure (Imai et al. Nature 2005; Kuba et al. Nature Med. 2005). ACE2 is a chimeric protein that has emerged from the duplication and fusion of two genes: homology with ACE at the catalytic domain and homology with Collectrin (Tmem27) in the membrane proximal domain.
We showed that the inactivation of Collectrin in mice results in nearly complete downregulation of apical amino acid transporters such as the he neutral amino acid transporter B0AT1 in the kidney, thus regulating renal amino acid re-absorption (Danilczyk et al. Nature 2006). Variants in the B0AT1 (SLC6A19) gene have been identified as a cause of Hartnup's disease, an autosomal recessive disorder associated with pellagra-like symptoms including diarrhea, which is manifested in the presence of malnutrition. However, the in vivo function of ACE2 in gut epithelium remained to be investigated.
In addition to its catalytic activity ACE2 associates with B0AT1 on the luminal surface of intestinal epithelial cells, where ACE2 is required for expression of this transporter on the luminal surface of intestinal epithelial cells (Fig. 1a). Genetic inactivation of ACE2 results in impaired uptake of dietary amino acids, particularly the essential amino acid tryptophan, in the intestine. As enterocytes also express di- and tripeptide transporters, we provided tryptophan in the form of a dipeptide. Dietary dipeptidic tryptophan restored serum tryptophan levels (Fig. 1b). Thus, ACE2 exhibits a novel RAS-independent function, regulating intestinal amino acid homeostasis.
Intriguingly, when we challenged ACE2-deficient mice with dextran sodium sulfate (DSS), a chemical irritant that disrupts the intestinal epithelial barrier and results in the induction of colitis, we observed a distinctly stronger inflammatory reaction compared to wild-type littermates (Fig. 2). Thus, genetic inactivation of the key RAS enzyme ACE2 results in severe colitis after intestinal injury. Moreover, protein malnutrition may directly affect the severity of DSS-induced colitis. Mechanistically ACE2 regulates the expression of innate antimicrobial peptides and the ecology of the gut microbiome. ACE2-dependent changes in epithelial immunity and the gut microbiome can be directly regulated by the dietary amino acid tryptophan and its metabolic product nicotinamide (Fig. 3a). Tryptophan supplementation as well as treatment with the tryptophan metabolite nicotinamide almost completely alleviated severe colitis and diarrhea to the level seen in control mice. Finally, transplantation of the altered microbiome from ACE2-mutant mice into wild-type germfree hosts was able to transmit the increased propensity to develop severe colitis (Fig. 3b).
Conclusions. Malnutrition is a major global health burden, affecting approximately a billion people in the world. Some of its consequences, such as colitis and diarrhea, can be fatal1,2. Chronic inflammatory conditions of the intestine, cachexia, anorexia nervosa, or amino acid mal-absorption are severe clinical problems30. Our data provide novel mechanistic insights into how protein malnutrition leads to colitis and diarrhea. Our data also revealed molecular crosstalk between the RAS system and intestinal amino acid homeostasis via ACE2, and demonstrated a direct link between dietary amino acid metabolism and innate immunity, the composition of the intestinal microbiota, and susceptibility to colitis (Hashimoto, Perlot et al. Nature 2012).