Chronic liver disease and impaired hepatic glycogen metabolism in argininosuccinate lyase deficiency

Abstract

BACKGROUNDLiver disease in urea cycle disorders (UCDs) ranges from hepatomegaly and chronic hepatocellular injury to cirrhosis and end-stage liver disease. However, the prevalence and underlying mechanisms are unclear.METHODSWe estimated the prevalence of chronic hepatocellular injury in UCDs using data from a multicenter, longitudinal, natural history study. We also used ultrasound with shear wave elastography and FibroTest to evaluate liver stiffness and markers of fibrosis in individuals with argininosuccinate lyase deficiency (ASLD), a disorder with high prevalence of elevated serum alanine aminotransferase (ALT). To understand the human observations, we evaluated the hepatic phenotype of the AslNeo/Neo mouse model of ASLD.RESULTSWe demonstrate a high prevalence of elevated ALT in ASLD (37%). Hyperammonemia and use of nitrogen-scavenging agents, 2 markers of disease severity, were significantly (P < 0.001 and P = 0.001, respectively) associated with elevated ALT in ASLD. In addition, ultrasound with shear wave elastography and FibroTest revealed increased echogenicity and liver stiffness, even in individuals with ASLD and normal aminotransferases. The AslNeo/Neo mice mimic the human disorder with hepatomegaly, elevated aminotransferases, and excessive hepatic glycogen noted before death (3-5 weeks of age). This excessive hepatic glycogen is associated with impaired hepatic glycogenolysis and decreased glycogen phosphorylase and is rescued with helper-dependent adenovirus expressing Asl using a liver-specific (ApoE) promoter.CONCLUSIONOur results link urea cycle dysfunction and impaired hepatic glucose metabolism and identify a mouse model of liver disease in the setting of urea cycle dysfunction.TRIAL REGISTRATIONThis study has been registered at ClinicalTrials.gov (NCT03721367, NCT00237315).FUNDINGFunding was provided by NIH, Burroughs Wellcome Fund, NUCDF, Genzyme/ACMG Foundation, and CPRIT.

Keywords: Amino acid metabolism; Glucose metabolism; Hepatology; Metabolism; Mouse models.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Increased aminotransferase levels in individuals with UCDs.

(A) The percentage of participants with each disorder who had 2 or more ALT levels above the indicated threshold are shown. (B) The percentage of participants with each disorder who had 2 or more AST levels above the indicated threshold are shown. ALT, alanine aminotransferase; AST, aspartate aminotransferase; OTCD, ornithine transcarbamylase deficiency; ASS1D, argininosuccinate synthetase deficiency; ASLD, argininosuccinate lyase deficiency; ARG1D, arginase deficiency; OTHER, other urea cycle disorders; M, male, F, female.

Figure 2. Hepatic fibrosis in ASLD.

(A) H&E stain of section of liver explant from a 20-month-old male with ASLD shows periportal fibrosis (stage 1; original magnification, 100×). Hepatocytes are swollen. (B) Trichrome stain of section of liver explant from a 2-year, 7-month-old female with ASLD shows diffuse bridging fibrosis (stage 3; original magnification, 10×). Hepatocytes are swollen. (C) PAS stain (without diastase) of section of liver explant from 5-year, 11-month-old female with ASLD and stage 4 fibrosis (original magnification, 400×). (D) PAS stain with diastase of section of liver explant from the same individual as in C demonstrates absence of PAS stain, which indicates that the PAS-stained material, in the absence of diastase, is glycogen (original magnification, 400×).

Figure 3. Hepatocellular injury and hepatic glycogen accumulation in AslNeo/Neo mice after 4-hour fast.

(A) Elevated ALT was observed in AslNeo/Neo mice. (B) Elevated AST was observed in AslNeo/Neo mice. (C) Hepatomegaly was observed in AslNeo/Neo mice. (D) Electron microscopy demonstrates increased glycogen deposition within hepatocytes of AslNeo/Neo mice, causing displacement of organelles to the cell membrane. Scale bars: 10 μm. (E) Quantification of hepatic glycogen in AslNeo/Neo mice versus WT mice confirms increased hepatic glycogen accumulation in AslNeo/Neo mice. For statistical comparisons, Student’s independent 2-sample t test was used.

Figure 4. Impaired hepatic glycogenolysis in AslNeo/Neo mice.

(A) Hepatic glycogen was quantified in WT and AslNeo/Neo mice that were not-fasted (fed) or mice that were fasted for 3, 12, or 24 hours. A 2-way ANOVA followed by Sidak’s post hoc test was used to make pair-wise comparisons between WT versus AslNeo/Neo mice at each time point. (B) Hepatic glycogen phosphorylase enzyme activity is reduced in AslNeo/Neo mice. Student’s independent 2-sample t test was used. (C) Gene expression in WT and AslNeo/Neo mice is similar. n = 8 per genotype. (D) PYGL protein levels are reduced in AslNeo/Neo mice versus WT mice. Immunoblotting was performed with tissue from 8 WT and 8 AslNeo/Neo mice with technical replicates (representative samples shown).

Figure 5. Plasma glucagon levels in AslNeo/Neo mice and WT mice.

(A) Plasma glucose levels after a 12-hour fast (anesthetized mice used in glucose bolus study) are not significantly different between mutant and WT mice. (B) Plasma glucagon levels after a 3- and 24-hour fast are similar in AslNeo/Neo mice and WT mice. (C) Hepatic glycogen after a glucose bolus appears to rise similarly in AslNeo/Neo mice versus WT mice. For plasma glucagon and glucose levels, Student’s independent 2-sample t test was used. For glycogen study, a 1-way ANOVA with Sidak’s post hoc test was used to compare the results from the 2 genotypes before glucose bolus and in the groups after glucose bolus. Pre, after 12-hour fast but before glucose bolus; Post, 2 hours after glucose bolus.

Figure 6. Helper-dependent adenovirus gene therapy (liver) corrects hepatic phenotypes in AslNeo/Neo mice.

(A) There is a trend suggesting improvement in ALT (3-hour fast, P = 0.11) and AST (3-hour fast, P = 0.13) after treatment with helper-dependent adenovirus expressing Asl using the ApoE promoter with liver-specific enhancer. Liver/body weight ratio was significantly improved after virus treatment. (B) Hepatic glycogen accumulation is improved and hepatic glycogen phosphorylase enzyme activity is increased after a 12-hour fast in AslNeo/Neo mice treated with virus. AslNeo/Neo mice treated with empty virus died before the end of the experiment and, thus, data from historical controls at 3–4 weeks of age are shown and used for comparison. Student’s independent 2-sample t test was used.