Targeting autophagy for therapy of hyperammonemia

ABSTRACT

Ammonia is a highly neurotoxic metabolite that is efficiently converted into urea or glutamine. During liver failure due to hepatocellular dysfunction or in inherited deficiencies of urea cycle enzymes, ammonia clearance is impaired resulting in systemic hyperammonemia and hepatic encephalopathy that can rapidly progress into coma and death if left untreated. Because available therapeutic options are often unsatisfactory, the development of effective therapies for hyperammonemia is highly needed. Here, we review our recent findings on the role of hepatic macroautophagy/autophagy in ammonia detoxification. We found that during hyperammonemia, ammonia-induced depletion of liver alpha-ketoglutarate and its consequent inhibition of the mechanistic target of rapamycin kinase complex 1 results in autophagy induction. Metabolite recycling induced by enhanced hepatic autophagy increases the efficiency of ammonia detoxification by furnishing key urea cycle intermediates and ATP, and stimulating ureagenesis. Moreover, autophagy enhancement by liver-directed gene transfer of the master regulator of autophagy TFEB (transcription factor EB) or treatments with the autophagy enhancers rapamycin and Tat-beclin 1 improve ammonia detoxification during hyperammonemia occurring as a consequence of either acquired or inherited diseases.

KEYWORDS:

• Alpha-ketoglutarate
• autophagy
• hyperammonemia
• mTORC1
• ornithine transcarbamylase deficiency
• rapamycin
• Tat-beclin 1
• TFEB
• ureagenesis

Ammonia is a highly neurotoxic metabolite produced during breakdown of proteins and other nitrogen-containing molecules. The liver plays a major role in regulating the levels of ammonia via 2 detoxification systems: i) the urea cycle that generates urea from ammonia and is made by enzymes that altogether are only expressed in the liver, and ii) the GLUL/glutamine synthetase that incorporates ammonia into glutamine (Figure 1). During liver failure or in inherited deficiency of urea cycle enzymes, ammonia is poorly removed from the blood resulting in hyperammonemia and hepatic encephalopathy that can rapidly progress into coma and death if not treated. Despite treatments, hyperammonemia remains an extremely challenging medical condition with high risks of mortality and irreversible brain damage. Therefore, the development of more effective therapies for hyperammonemia is highly needed.

Figure 1. Liver autophagy and hyperammonemia. In hepatocytes under normal conditions (left): i) MTORC1, involved in a major signaling pathway controlling autophagy, is regulated by various signals including growth factors, energy and nutrients such as amino acids (AA) and alpha-ketoglutarate (a-KG); and ii) ammonia (NH +) is cleared by synthesis of urea through the urea cycle and glutamine (Gln) by GLUL/glutamine synthetase. During hyperammonemia (right), ammonia-induced depletion of a-KG and its consequent inhibition of MTORC1 induce autophagy. Metabolite recycling induced by increased autophagy makes ammonia removal more efficient because it stimulates ureagenesis by furnishing key urea cycle intermediates (aspartate [Asp] and glutamate [Glu]), acetyl-coA, and ATP. Autophagy enhancement by gene transfer of Tfeb or treatments with the autophagy enhancers rapamycin (Rapa) and Tat-beclin 1 (TB-1), increases ureagenesis and can be exploited to reduce hyperammonemia.

Ammonia has a dual effect on autophagy: i) at higher concentrations it impairs lysosome function by increasing lysosomal pH, thus inhibiting autophagic cargo degradation, and ii) at lower concentrations it stimulates autophagy. Interestingly, research in the cancer field revealed that tumor cells are protected from chemotherapeutic drugs by ammonia-induced autophagy. Moreover, in vivo hyperammonemia inhibits mechanistic target of rapamycin kinase complex 1 (MTORC1) signaling and activates autophagy in skeletal muscle. These latter findings provided a potential mechanism underlying muscle wasting (i.e., sarcopenia) in patients with liver cirrhosis. However, the role of ammonia-induced autophagy in liver has not been evaluated so far. In our recent study, we investigated hepatic autophagy during hyperammonemia and we found that it plays an important role in ammonia detoxification with broad implications for both physiological and disease state conditions. Moreover, we found that autophagy enhancement has potential for therapy of both primary and secondary causes of hyperammonemia.

We investigated liver autophagy in well-established mouse models of acute and chronic hyperammonemia in which wild-type mice received intraperitoneal injections of ammonium chloride or were fed with an ammonium supplemented diet, respectively. We found that hyperammonemia activates liver autophagy through inhibition of MTORC1 signaling as a consequence of reduced hepatic alpha-ketoglutarate (a-KG), a tricarboxylic acid cycle intermediate (Figure 1). The reduced hepatic levels of a-KG during hyperammonemia are consistent with its previously reported function as a nitrogen scavenger in which ammonia is incorporated into glutamate by reductive amination of a-KG. At molecular mechanistic level, a-KG is an essential electron donor for EGLNs/prolyl hydroxylases that have been proposed to be responsible for MTORC1 recruitment to, and activation at, the lysosome surface. Nevertheless, whether EGLNs/prolyl hydroxylases are involved in the activation of autophagy during hyperammonemia requires further investigation.

To investigate the physiological relevance of ammonia-induced hepatic autophagy, ammonia detoxification was evaluated in mice with either increased or deficient autophagy. Ammonia clearance was impaired in mice with defective autophagy induced by knockdown of hepatic ATG7, a component of the autophagic machinery. Similarly, we observed gher concentrations of serum ammonia following ammonia challenge in mice with hepatic-specific deletion of Tfeb (transcription factor EB), a master regulator of autophagy, lysosomal biogenesis and exocytosis. Taken together, these data suggest that hepatic cells require a functional autophagic pathway for efficient ammonia detoxification. Conversely, autophagy enhancement by viral vector-mediated hepatic gene transfer of TFEB or treatments with 2 autophagy inducers, namely rapamycin and Tat-beclin 1, protect against acute loads of ammonium chloride by increasing ammonia clearance (Figure 1). Enhancement of liver autophagy is associated with increased ureagenesis as shown by greater incorpo- ration of 15N-labeled ammonium into urea. Notably, despite no detected changes in glutamine levels, the contribution of hepatic glutamine synthesis to ammonia detox- ification cannot be ruled out.

To investigate how autophagy induction potentiates ureagenesis during hyperammonemia, we focused on selected liver metabolites important for functioning of the urea cycle. We found that liver contents of ATP, aspartate, glutamate, and acetyl-coA were all increased by enhanced autophagy during hyperammonemia (Figure 1). When the hepatic ratio ammonia:aspartate is greater than 1:1, intra-hepatic proteolysis is stimulated to provide aspartate that fuels the urea cycle. This enhancement of protein catabolism has been interpreted as the metabolic price that must be paid for effective ammonia detoxification when the liver is facing high ammonia levels. Our data support the concept that autophagy enhancement results in increased liver protein degradation that generates more aspartate available for ureagenesis and more efficient ammonia detoxification. In addition to aspartate, enhancement of auto- phagic recycling also provides the urea cycle with key substrates for production of N-acetylglutamate, the allosteric activator of CPS1 (carbamoyl-phosphate synthetase 1) that increases the flux through the cycle, and ATP.

To investigate how autophagy induction potentiates ureagenesis during hyperammonemia, we focused on selected liver metabolites important for functioning of the urea cycle. We found that liver contents of ATP, aspartate, glutamate, and acetyl-coA were all increased by enhanced autophagy during hyperammonemia (Figure 1). When the hepatic ratio ammonia:aspartate is greater than 1:1, intra-hepatic proteolysis is stimulated to provide aspartate that fuels the urea cycle. This enhancement of protein catabolism has been interpreted as the metabolic price that must be paid for effective ammonia detoxification when the liver is facing high ammonia levels. Our data support the concept that autophagy enhancement results in increased liver protein degradation that generates more aspartate available for ureagenesis and more efficient ammonia detoxification. In addition to aspartate, enhancement of autophagic recycling also provides the urea cycle with key substrates for production of N-acetylglutamate, the allosteric activator of CPS1 (carbamoyl-phosphate synthetase 1) that increases the flux through the cycle, and ATP.

Finally, pharmacological enhancement of autophagy in mouse models of both acquired and inherited hyperammonemia including thioacetamide-induced acute liver failure and OTC (ornithine transcarbamylase) deficiency, the most frequent urea cycle disorder, result in reduced serum ammonia levels and normalization of blood orotic acid, a clinically relevant end-point in OTC deficiency.

Therapeutic targeting of autophagy is currently an available option to treat several pathological conditions ranging from cancer to neurodegenerative, inflammatory, infectious, and metabolic diseases. Our findings suggest that drugs stimulating autophagy might be effective in both primary and secondary hyperammonemias. Food and Drug Administration-approved drugs or nutritional supplements known as autophagy inducers are available for use in humans. However, the choice of the optimal autophagy activator for hyperammonemia must take into consideration potential pitfalls related to target specificity and undesired side effects. For example, sodium valproate and metformin are known autophagy inducers but they are also associated with reduced N-acetylglutamate production and lactic acidosis, respectively, 2 undesirable effects in inborn errors of metabolism. Conversely, pharmacological inhibition of autophagy by chloroquine or hydroxychloroquine, either alone or in combination with other drugs, are used as an anti-cancer therapy and might affect ammonia detoxification.

In summary, our study suggests the novel concept that hepatic autophagy potentiates ureagenesis by providing key urea cycle intermediates and ATP under both acute and chronic hyperammonemia. The enhancement of hepatic autophagy has potential for therapy of both inherited and acquired causes of hyperammonemia.

Disclosure of potential conflicts of interest

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the European Research Council (IEMTx) (N.B.-P.) and Fondazione Telethon Italy Grants TCBP37TELC and TCBMT3TELD (to N.B.-P.). L.R.S. was supported by the Dulbecco Telethon Institute International Mobility for Postdoctoral Research Training (DTI-IMPORT) Marie Sk»odowska-Curie COFUND program.