NorUDCA promotes degradation of α1-antitrypsin mutant Z protein by inducing autophagy through AMPK/ULK1 pathway.

Alpha-1 Antitrypsin (α1AT) Deficiency is a genetic disease in which accumulation of α1AT mutant Z (α1ATZ) protein in the ER of hepatocytes causes chronic liver injury, liver fibrosis, and hepatocellular carcinoma. No effective medical therapy is currently available for the disease. We previously found that norUDCA improves the α1AT deficiency associated liver disease by promoting autophagic degradation of α1ATZ protein in liver in a mouse model of the disease. The current study unravels the novel underlying cellular mechanism by which norUDCA modulates autophagy. HTOZ cells, modified from HeLa Tet-Off cells by transfection with the resulting pTRE1-ATZ plasmid and expressing mutant Z proteins, were studied in these experiments. The role of norUDCA in inducing autophagy, autophagy-mediated degradation of α1ATZ and the role of AMPK in norUDCA-induced autophagy were examined in the current report. NorUDCA promoted disposal of α1ATZ via autophagy-mediated degradation of α1ATZ in HTOZ cells. Activation of AMPK was required for norUDCA-induced autophagy and α1ATZ degradation. Moreover, mTOR/ULK1 was involved in norUDCA-induced AMPK activation and autophagy in HTOZ cells. Our results provide novel mechanistic insights into the therapeutic action of norUDCA in promoting the clearance of α1ATZ in vitro and suggest a novel therapeutic approach for the treatment of α1ATZ deficiency disease and its associated liver diseases.

Pathophysiology of Alpha-1 Antitrypsin Deficiency Liver Disease.

Classical alpha-1 antitrypsin (a1AT) deficiency is an autosomal recessive disease associated with an increased risk of liver disease in adults and children, and with lung disease in adults (Teckman and Jain, Curr Gastroenterol Rep 16(1):367, 2014). The vast majority of the liver disease is associated with homozygosity for the Z mutant allele, the so-called PIZZ. These homozygous individuals synthesize large quantities of a1AT mutant Z protein in the liver, but the mutant protein folds improperly during biogenesis and approximately 85% of the molecules are retained within the hepatocytes rather than appropriately secreted. The resulting low, or "deficient," serum level leaves the lungs vulnerable to inflammatory injury from uninhibited neutrophil proteases. Most of the mutant Z protein molecules retained within hepatocytes are directed into intracellular proteolysis pathways, but some molecules remain in the endoplasmic reticulum for long periods of time. Some of these molecules adopt an unusual aggregated or "polymerized" conformation (Duvoix et al., Rev Mal Respir 31(10):992-1002, 2014). It is thought that these intracellular polymers trigger a cascade of intracellular injury which can lead to end-organ liver injury including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (Lindblad et al., Hepatology 46(4):1228-1235, 2007). The hepatocytes with the largest accumulations of mutant Z polymers undergo apoptotic death and possibly other death mechanisms. This intracellular death cascade appears to involve ER stress, mitochondrial depolarization, and caspase cleavage, and is possibly linked to autophagy and redox injury. Cells with lesser burdens of mutant Z protein proliferate to maintain the liver cell mass. This chronic cycle of cell death and regeneration activates hepatic stellate cells and initiates the process of hepatic fibrosis. Cirrhosis and hepatocellular carcinoma then result in some patients. Since not all patients with the same homozygous PIZZ genotype develop end-stage disease, it is hypothesized that there is likely to be a strong influence of genetic and environmental modifiers of the injury cascade and of the fibrotic response.

Semiquantitation of Monomer and Polymer Alpha-1 Antitrypsin by Centrifugal Separation and Assay by Western Blot of Soluble and Insoluble Components.

Alpha-1 antitrypsin (a1AT) deficiency, in its classical form, is an autosomal recessive disease associated with an increased risk of liver disease in adults and children, and with lung disease in adults. The vast majority of liver disease is associated with homozygosity for the Z mutant allele, also called PiZZ. This homozygous allele synthesizes large quantities of a1AT mutant Z protein in the liver, but the mutant protein also folds improperly during biogenesis. As a result, approximately 85% of the molecules are retained within the hepatocytes instead of being appropriately secreted. The resulting low, or "deficient," serum level leaves the lungs vulnerable to inflammatory injury from uninhibited neutrophil proteases. Most of the mutant Z protein retained within hepatocytes is directed into intracellular proteolysis pathways, but some molecules remain in the endoplasmic reticulum for long periods of time and others adopt an unusual aggregated or "polymerized" conformation. It is thought that these intracellular polymers trigger a cascade of intracellular injury which can lead to end organ liver injury including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. It is widely accepted that the disease causing factor in mutant Z-alpha-1 antitrypsin deficiency (AATD-Z) is the toxic build-up of the mutant Z protein. Since misfolding of some but not all of the Z protein during its maturation leads to homopolymerization, an assay to assess the amount of normally folded ATZ and accumulated polymeric ATZ would be very useful. Here we describe a method to semiquantitatively assess these two fractions in a tissue or cell culture source.

Oleanolic Acid Improves Gut Atrophy Induced by Parenteral Nutrition.

BACKGROUND: Nutrition support with parenteral nutrition (PN) is associated with gut atrophy. Prior studies have shown improvement with enteral chenodeoxycholic acid, a dual agonist for the farnesoid X receptor (FXR) and bile acid receptor TGR5. We hypothesized that gut growth is induced by TGR5 activation, and gut atrophy during PN administration could be prevented with the TGR5-specific agonist oleanolic acid (OA). METHODS: Neonatal pigs were implanted with duodenal and jugular vein catheters. Animals were provided equi-nutritious PN or enteral swine milk. A PN subgroup received enteral OA at 50 mg/kg/d. RESULTS: PN caused marked gut atrophy compared with enterally fed (EN) control animals. OA treatment led to preservation of gut mass demonstrated grossly and histologically. The mean ± SD gut weight as a percentage of body weight was 4.30 ± 0.26 for EN, 1.92 ± 0.06 for PN (P < .05, EN vs PN), and 3.39 ± 0.79 for PN+OA (P < .05, PN+OA vs PN). Mean ± SD gut density (g/cm) was 0.31 ± 0.03 for EN, 0.18 ± 0.03 for PN (P < .05 EN vs PN), and 0.27 ± 0.01 for PN+OA (P < .05 PN+OA vs PN). Histologically, a markedly decreased villous to crypt ratio was noted with PN, and OA significantly prevented this decrease. The mean ± SD v/c ratio was 3.51 ± 0.59 for EN, 1.69 ± 0.10 for PN (P < .05, EN vs PN), and 2.90 ± 0.23 for PN+OA (P < .05, PN+OA vs PN). Gut TGR5 messenger RNA expression was significantly elevated with OA treatment compared with both PN and EN. CONCLUSION: The bile acid-activated G protein-coupled receptor TGR5 agonist OA prevented gut atrophy associated with PN.

Validating hyperbilirubinemia and gut mucosal atrophy with a novel ultramobile ambulatory total parenteral nutrition piglet model.

Total parenteral nutrition (TPN) provides all nutrition intravenously. Although TPN therapy has grown enormously, it causes significant complications, including gut and hepatic dysfunction. Current models use animal tethering which is unlike ambulatory human TPN delivery and is cost prohibitive. We hypothesize that using ultramobile infusion pumps, TPN can be delivered cost-effectively, resulting in classical gut and hepatic injury, and we thus aim to establish a new model system. Neonatal pigs (n=8) were implanted with jugular vein and duodenal catheters. Animals were fitted in dual-pocket jackets. An ultramobile ambulatory pump was placed in one pocket and connected to the jugular vein or duodenal catheter. Isocaloric TPN or swine formula was placed in the other pocket. Rigorous Wifi-based video and scheduled monitoring was performed. After 14days, the animals were euthanized. The mean (±SD) daily weight gain (in grams) for enteral-fed control (EN) vs TPN animals was 102.4±10.8 and 91.03±12.1 respectively (P<.05). Total parenteral nutrition resulted in significant conjugated bilirubin elevation and hepatomegaly. Mean (±SD) serum conjugated bilirubin (in µmol/L) was 1.5±0.7 for EN and 6.3±2.8 for TPN (P<.05). Marked gut atrophy was noted with TPN. The mean (±SD) gut weight as a percent of body weight was 4.30±0.26 for EN and 2.62±0.48 for TPN (P<.05). Surgical sites healed well. All animals remained completely mobile. We thus established that TPN can be successfully delivered using ultramobile pumps and believe that this remains the first such description of an ambulatory piglet TPN model system. In addition to cholestasis and gut atrophy, classical TPN-induced injury was documented.

In vivo post-transcriptional gene silencing of alpha-1 antitrypsin by adeno-associated virus vectors expressing siRNA.

alpha-1 Antitrypsin (AAT) deficiency is one of the most common genetic diseases in North America, with a carrier frequency of approximately 4% in the US population. Homozygosity for the most common mutation (Glu342Lys, PI(*)Z) leads to the synthesis of a mutant protein, which accumulates and polymerizes within hepatocytes rather than being efficiently secreted. This lack of secretion causes severe serum deficiency predisposing to chronic lung disease. Twelve to fifteen percent of patients with PI(*)ZZ also develop liver disease, which can be severe, even in infancy. This is thought to be due to toxic effects of the accumulated mutant Z-AAT within the hepatocyte. Thus, an approach to reduce AAT-deficient liver disease will likely require some mechanism to decrease the amount of Z-AAT within hepatocytes. In this report, we describe studies of small-interfering RNAs (siRNAs) designed to downregulate endogenous AAT within hepatocytes. Three different siRNA sequences were identified and cloned into a recombinant adeno-associated virus (rAAV) backbone, either singly or as a trifunctional (3X) construct. Each had activity independently, but the levels of AAT expression in cell culture models showed the greatest decrease with the 3X construct, resulting in levels that were five-fold lower than controls. The rAAV-3X-siRNA was then packaged into AAV8 capsids and used in vivo to transduce the livers of human Z-AAT overexpressing transgenic mice. Those studies showed a decrease in total human AAT, a clearing of Z-AAT accumulation by immunohistochemistry, and a decrease in monomer Z-AAT within the liver within 3 weeks after vector injection. The rAAV8-3X-siRNA vector may hold promise as a potential therapy for patients with AAT liver disease.