Quantitative isolation of alphalAT mutant Z protein polymers from human and mouse livers and the effect of heat.

Alpha-1-antitrypsin (alpha1AT) deficiency in its most common form is caused by homozygosity for the alpha1AT mutant Z gene. This gene encodes a mutant Z secretory protein, primarily synthesized in the liver, that assumes an abnormal conformation and accumulates within hepatocytes causing liver cell injury. Studies have shown that mutant alpha1ATZ protein molecules form unique protein polymers. These Z protein polymers have been hypothesized to play a critical role in the pathophysiology of liver injury in this disease, although a lack of quantitative methods to isolate the polymers from whole liver has hampered further analysis. In this study, we demonstrate a quantitative alpha1ATZ polymer isolation technique from whole liver and show that the hepatocellular periodic acid-Schiff-positive globular inclusions that are the histopathological hallmark of this disease are composed almost entirely of the polymerized alpha1ATZ protein. Furthermore, we examine the previously proposed but untested hypothesis that induction of alpha1ATZ polymerization by the heat of physiological fever is part of the mechanism of hepatic alpha1ATZ protein accumulation. The results, however, show that fever-range temperature elevations have no detectable effect on steady-state levels of intrahepatic Z protein polymer in a model in vivo system. In conclusion, methods to separate insoluble protein aggregates from liver can be used for quantitative isolation of alpha1ATZ protein polymers, and the effect of heat from physiological fever may be different in vivo compared with in vitro systems.

Analyses of hepatocellular proliferation in a mouse model of alpha-1-antitrypsin deficiency.

alpha-1-Antitrypsin (alpha1-AT) deficiency is the most common cause of metabolic pediatric liver disease. Hepatocellular injury is caused by toxicity of the mutant alpha-1-antitrypsin Z (alpha1-ATZ) molecule retained within hepatocytes. In these studies, we used the PiZ transgenic mouse model of alpha1-AT deficiency to examine hepatocellular proliferation in response to chronic liver injury resulting from this metabolic disease. The results showed increased hepatocellular proliferation and caspase 9 activation in male PiZ mice compared with female PiZ and wild-type mice. Hepatic alpha1-AT mRNA and protein expression also were increased in male PiZ mice, suggesting that greater hepatocellular proliferation and caspase activation in males results from increased hepatotoxicity associated with greater intracellular alpha1-ATZ accumulation. Testosterone treatment of female PiZ mice increased alpha1-ATZ expression and hepatocellular proliferation to a level comparable with that in males. In PiZ mice, hepatocytes devoid of intracellular alpha1-AT globules had a proliferative advantage compared with globule-containing hepatocytes. However, this advantage is relative because both globule-containing and globule-devoid hepatocytes exhibited comparable proliferation after partial hepatectomy. In conclusion, these data indicate that intracellular retention of mutant alpha1-ATZ is associated with a regenerative stimulus leading to increased hepatocellular proliferation, that gender-specific signals influence the degree of alpha1-AT expression and associated hepatic injury, and that hepatocytes devoid of alpha1-ATZ have a proliferative advantage over cells that accumulate the mutant protein. This selective proliferation suggests that hepatocellular transplantation may be applicable for treatment of this and other slowly progressive metabolic liver diseases.

Mitochondrial autophagy and injury in the liver in alpha 1-antitrypsin deficiency.

Homozygous, PIZZ alpha(1)-antitrypsin (alpha(1)-AT) deficiency is associated with chronic liver disease and hepatocellular carcinoma resulting from the toxic effects of mutant alpha(1)-anti-trypsin Z (alpha(1)-ATZ) protein retained in the endoplasmic reticulum (ER) of hepatocytes. However, the exact mechanism(s) by which retention of this aggregated mutant protein leads to cellular injury are still unknown. Previous studies have shown that retention of mutant alpha(1)-ATZ in the ER induces an intense autophagic response in hepatocytes. In this study, we present evidence that the autophagic response induced by ER retention of alpha(1)-ATZ also involves the mitochondria, with specific patterns of both mitochondrial autophagy and mitochondrial injury seen in cell culture models of alpha(1)-AT deficiency, in PiZ transgenic mouse liver, and in liver from alpha(1)-AT-deficient patients. Evidence for a unique pattern of caspase activation was also detected. Administration of cyclosporin A, an inhibitor of mitochondrial permeability transition, to PiZ mice was associated with a reduction in mitochondrial autophagy and injury and reduced mortality during experimental stress. These results provide evidence for the novel concept that mitochondrial damage and caspase activation play a role in the mechanism of liver cell injury in alpha(1)-AT deficiency and suggest the possibility of mechanism-based therapeutic interventions.

Fasting in alpha1-antitrypsin deficient liver: constitutive [correction of consultative] activation of autophagy.

Alpha1-antitrypsin (alpha1-AT) deficiency causes severe liver injury in a subgroup of patients. Liver injury is thought to be caused by retention of a polymerized mutant alpha1-ATZ molecule in the endoplasmic reticulum (ER) of hepatocytes and is associated with an intense autophagic response. However, there is limited information about what physiologic stressors might influence liver injury. In this study, we examined the effect of fasting in the PiZ mouse model of alpha1-AT deficiency, because fasting is a well-characterized physiological stressor and a known stimulus for autophagy. Results show that there is a marked increase in fat accumulation and in alpha1-AT-containing globules in the liver of the PiZ mouse induced by fasting. Although fasting induced a marked autophagic response in wild-type mice, the autophagic response was already activated in PiZ mice and did not further increase with fasting. PiZ mice also had a significantly decreased tolerance for prolonged fasting compared with wild-type mice (PiZ mice 0% survival of 72-h fast; wild-type 100% survivial). These results demonstrate an altered response to stress in the alpha1-AT-deficient liver, including inability to further increase an activated autophagic response, a developmental state-specific increase in alpha1-AT-containing globules, and increased mortality.