Orosomucoid 2 maintains hepatic lipid homeostasis through suppression of de novo lipogenesis.

Non-alcoholic fatty liver disease (NAFLD) is caused by imbalance in lipid metabolism. In this study, we show that the hepatokine orosomucoid (ORM) 2 is a key regulator of de novo lipogenesis in the liver. Hepatic and plasma ORM2 levels are markedly decreased in obese murine models and patients with NAFLD. Through multiple loss- and gain-of function studies, we demonstrate that ORM2 is essential to maintain hepatic and systemic lipid homeostasis. At the mechanistic level, ORM2 binds to inositol 1, 4, 5-trisphosphate receptor type 2 to activate AMP-activated protein kinase signaling, thereby inhibiting sterol regulatory element binding protein 1c-mediated lipogenic gene program. Notably, intraperitoneal injections of recombinant ORM2 protein or stabilized ORM2-FC fusion protein markedly improved liver steatosis, steatohepatitis and atherosclerosis in preclinical mouse models, without adverse effects on body weight or food intake. Thus, these findings suggest that ORM2 may serve as a potential target for therapeutic intervention in NAFLD, non-alcoholic steatohepatitis and related lipid disorders.

ORM 1 as a biomarker of increased vascular invasion and decreased sorafenib sensitivity in hepatocellular carcinoma.

This study aimed to clarify the role of Orosomucoid 1 (ORM1) in the development and therapy resistance in hepatocellular carcinoma (HCC). The mRNA expression level of ORM1 was analyzed via integrative analysis of Gene Express Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. The protein expression level of ORM1 in our cohort was determined using immunohistochemistry. Correlation analysis was used to investigate the relationship between ORM1 expression and clinical parameters. The Cell Counting Kit-8 assay was used to clarify the role of ORM1 in HCC malignant behaviors, including cell growth and sorafenib sensitivity, in vitro. The results indicated that ORM1 was significantly downregulated in the hepatic cancer cells compared to that in the non-cancerous cells. However, it was upregulated in microvascular invasion samples, especially in the cancer embolus compared to that in the surrounding tumor cells. Though Kaplan-Meier analysis did not show an association of ORM1 expression with the overall survival rates of HCC patients, univariate analysis indicated that ORM1 expression was highly correlated with tumor grade and stage. An in vitro assay also revealed that downregulation of ORM1 led to the suppression of tumor growth and enhancement of sorafenib sensitivity without epithelial-to-mesenchymal transition (EMT) alteration, which was consistent with our bioinformatic analysis. Hence, ORM1 played a key role in HCC tumorigenesis and may serve as a potential target for the development of therapeutics against HCC in the future.

Human uridine-cytidine kinase phosphorylation of ribavirin: a convenient method for activation of ribavirin for conjugation to proteins.

Ribavirin is a synthetic nucleoside analog that is used for the treatment of hepatitis C virus (HCV) infection. Its primary toxicity is hemolytic anemia, which sometimes necessitates dose reduction or discontinuation of therapy. Selective delivery of ribavirin into liver cells would be desirable to enhance its antiviral activity and avoid systemic side effects. One approach to liver-specific targeting is conjugation of the ribavirin with asialoglycoprotein that is taken up specifically by liver cells. Human uridine-cytidine kinase-1 (UCK-1) was used for ribavirin phosphorylation to its monophosphate form. 1-Ethyl-3-diisopropylaminocarbodiimide (EDC) was used as a coupling agent. The best results were obtained using direct conjugation protocol with a molar ratio of 6.5 ribavirin monophosphate (RMP) molecules per one asialoorosomucoid (AsOR) molecule. Our findings show that ribavirin is a potential substrate of UCK-1, and RMP formed could be chemically coupled to AsOR to form a conjugate for liver specific targeting.

Protective effects of alpha1-acid glycoprotein and serum amyloid A on concanavalin A-induced liver failure via interleukin-6 induction by ME3738.

We examined whether the 22beta-methoxyolean-12-ene-3beta,24(4beta)-diol (ME3738)-mediated selective induction of interleukin-6 increased alpha1-acid glycoprotein and serum amyloid A expression, and whether these proteins protected against liver injury in vitro and in vivo. ME3738 treatment in male mice increased gene expression of alpha1-acid glycoprotein subtypes and serum amyloid A 2 genes, and plasma concentration of serum amyloid A. Treatment with alpha1-acid glycoprotein at 5 mg/animal or serum amyloid A at 0.03 and 0.1 mg/animal prior to concanavalin A administration reduced multifocal necrosis in the liver. Treatment with alpha1-acid glycoprotein and serum amyloid A, but not alpha1-antitrypsin, protected Hep G2 cells against cell injury. These results suggest that alpha1-acid glycoprotein and serum amyloid A, increased by ME3738-induced interleukin-6, might protect against concanavalin A-induced liver injury.

Preventive and therapeutic effects of alpha-acid glycoprotein in mice infected with B. anthracis.

We studied the effects of alpha1-acid glycoprotein preparations on the survival rate of BALB/c mice infected with the lethal dose of B. anthracis STI-1. Apart from native alpha1-acid glycoprotein from donor blood, we studied 3 glycoforms differing in the affinity for concanavalin A and structure of carbohydrate chains. The protective effect of alpha1-acid glycoprotein preparations did not depend on its dose and was observed 3 months after treatment (0.3 mg per mouse). The protective effect was revealed in mice receiving alpha1-acid glycoprotein preparations 2 h before infection and 24 h after inoculation of the bacterial culture. In the latter case the survival rate of animals was much higher compared to that observed in preventive administration of alpha1-acid glycoprotein. The protective effect practically did not depend on the time of treatment with glycoforms. Pretreatment with alpha1-acid glycoprotein preparations significantly decreased plasma interferon-gamma concentration. Administration of the test preparations 24 h after infection decreased the concentration of tumor necrosis factor-alpha.

Alpha1-acid-glycoprotein protects against trauma-hemorrhagic shock.

BACKGROUND: Recent studies have shown that the acute phase protein alpha(1)-acid-glycoprotein (AAG) directly modifies endothelial cell responsiveness and is a crucial factor for maintaining endothelial barrier function. We hypothesized that the addition of AAG to the resuscitation fluid will prevent edema formation, increases circulating blood volume, and reduces tissue inflammation following soft tissue trauma and hemorrhagic shock. MATERIALS AND METHODS: Male Sprague-Dawley rats (338 +/- 28 g) underwent a 5-cm midline laparotomy (i.e., induction of soft tissue trauma) and were bled to and maintained at a mean arterial pressure of 35 mm Hg for 90 min. The rats were then resuscitated with four times the shed blood volume with Ringer's lactate containing 200 mg/kg AAG or the same amount of albumin. At 6 h after resuscitation, organ wet-to-dry weight ratios and circulating blood volume (Evans blue dilution) were determined. Neutrophil accumulation (myeloperoxidase activity, MPO) and tissue lipid peroxidation (thiobarbituric acid reactive substances) were also measured in the lungs, liver, and intestine. RESULTS: Administration of AAG during the resuscitation significantly increased circulating blood volume and reduced edema formation, neutrophil accumulation, and lipid peroxidation. Interestingly, concomitant plasma IL-6 levels increased while TNF-alpha levels were not significantly affected. CONCLUSIONS: Since addition of AAG to the resuscitation fluid increased circulating blood volume, reduced edema formation, and neutrophil accumulation following trauma and hemorrhagic shock, supplementation of this acute phase protein appears to be a potential adjunct to prevent capillary leakage in patients undergoing major traumatic injury.

Effects of alpha 1-acid glycoprotein on acute pancreatitis and acute lung injury in rats.

alpha 1-Acid glycoprotein (AAG), a highly negatively charged glycoprotein, well known for its capillary stabilizing effect, was tested in rat models of acute edematous pancreatitis, acute hemorrhagic-necrotizing pancreatitis, and acute respiratory distress syndrome (ARDS). In cerulein-elicited edematous pancreatitis AAG improved histological alterations at 200 mg/kg i.v. and plasma amylase activity at 1800 or 4200 mg/kg i.v. All other parameters (edema, plasma lipase) were not affected in a biologically relevant manner. In glycodeoxycholic acid-induced hemorrhagic-necrotizing pancreatitis AAG was without effect on parameters measured (plasma amylase, plasma lipase activity, histological scores) at 1800 or 4200 mg/kg i.v. At the extremely high dose of 1500 mg/kg i.v. plasma amylase and lipase levels were decreased. In lipopolysaccharide-mediated ARDS, AAG was tested at 50, 200 or 600 mg/kg i.v. AAG, but also the placebo formulation decreased the myeloperoxidase content in the bronchoalveolar lavage fluid. Histological alterations were improved by AAG, however, not by the placebo formulation. Lung water content was not significantly influenced by AAG, whereas Evans blue extravasation was significantly diminished by all three doses of AAG. It is concluded that the edematous pancreatitis is the first in vivo condition with increased extravascular fluid accumulation, in which AAG is not effective. Based on data presented here and literature data, there is evidence for a beneficial effect of AAG in acute lung injury.

[Preclinical investigation of alpha 1-acid glycoprotein (orosomucoid)]. / Präklinische Untersuchung von alpha 1-saurem Glykoprotein (Orosomucoid).

The molecular properties of alpha 1-acid glycoprotein are briefly discussed. This molecule has been shown in in vitro experiments to have both a stabilizing effect on vascular permeability and antiinflammatory properties. We were able to demonstrate these two effects in vivo in guinea pigs (skin, Evan's Blue extravasation) and in rats (paw, carrageenan induced inflammation). Further experiments were performed in rats relating to possible therapeutic indications for alpha 1-acid glycoprotein: (1) inhibitory effect on brain edema formation after experimental stroke, (2) therapeutic effect in the puromycin aminonucleoside-induced minimal change nephrosis, (3) improvement of vital parameters in hemorrhagic-hypovolemic shock, (4) increase in survival rate in septic peritonitis, and (5) promising effects in burn-induced remote lung injury. The high content of sialic acid and the high negative charge of alpha 1-acid glycoprotein are believed to be major contributors to its stabilizing effect on vascular permeability. The protein is bound to the glycocalyx of the endothelial cells (and presumably to structures of the glomerular basement membrane), thereby hindering the passage of other polyanionic molecules through the vascular wall. The antiinflammatory/immunomodulatory effect of alpha 1-acid glycoprotein appears mainly due to suppression of polymorphonuclear neutrophils. This action is dependent on the glycan part of the molecule, which is highly variable (microheterogeneity). It is obvious that there are differences between the different glycan forms as far as the antiinflammatory property of the protein is concerned. Together with data in the literature, the results presented here suggest a variety of potential indications for therapeutic use of alpha 1-acid glycoprotein in humans.

Effects of alpha 1-acid glycoprotein in different rodent models of shock.

It was the aim of the present study to investigate the effects of the acute phase protein alpha 1-acid glycoprotein in different models of shock. The human plasma preparation used was without effect on mortality in lipopolysaccharide-injected mice when administered in two different doses (1 or 0.33 g/kg i.v.) and according to different treatment schedules. The same preparation significantly increased survival rate (48 h) in rats with septic peritonitis. This effect was seen when alpha 1-acid glycoprotein (200 mg/kg i.v.) was given 15 min prior to and 24 h after cecal puncture. All other dose regimes tested were without significant effect on survival rate. A hemorrhagic/hypovolemic shock model (including a defined trauma) in rats resuscitated with 200 mg/kg alpha 1-acid glycoprotein resulted in significantly higher values of mean arterial blood pressure, cardiac output and stroke volume when compared to corresponding values obtained after resuscitation with Ringer's solution or 200 mg/kg albumin i.v. (free of alpha 1-acid glycoprotein; placebo formulation). Taking all other possible mechanisms of alpha 1-acid glycoprotein into consideration, the partially protective effects of the preparation are explained by enhancing the capillary barrier function and thereby maintaining perfusion of vital organs.

Non-viral gene delivery: vehicle and delivery characterization.

The development of non-viral gene therapy has been hampered by an inability to reproducibly manufacture and characterize delivery system components and final formulations. Formation of interpolyelectrolyte complexes as the basis of various gene delivery methods has been approached as the first step towards development of synthetic viruses. We have found that preparation of interpolyelectrolyte complexes from disperse reagents gives a more homogeneous gene delivery vehicle than other methods. Methods which increase homogeneity also result in higher transfection efficiency in vivo. Expression levels of human growth hormone and other reporter proteins in mice confirm the potential of parenteral non-viral gene delivery for some therapeutic applications. Serum is demonstrated to inhibit transfection efficiency in vivo. Our results suggest that further development of methods to manufacture homogeneous disperse non-viral delivery vehicles with stealth characteristics may enhance both the potency and reproducibility of gene transfer in vivo.

[The effect of alpha-1-acidic glycoprotein on neutrophil chemiluminescence and lipid peroxidation in experimental thermal trauma]. / Vliianie alpha-1-kislogo glikoproteina na khemiliuminestsentsiiu neitrofilov i perekisnoe okislenie lipidov pri éksperimental'noi termicheskoi travme.

Induced chemoluminescence of neutrophils was distinctly inhibited both in intact mice and the animals with thermal injury after administration of acid alpha 1-glycoprotein. At the same time, the glycoprotein decreased the rate of thermal burn-induced activation of lipid peroxidation in rat blood, skin and liver tissue. Antioxidative and therapeutic effects of acid alpha 1-glycoprotein in burns appear to be related to regulation of neutrophil activity.

[Effect of alpha-acidic glycoprotein on oxyproline metabolism in experimental thermal trauma]. / Vliianie alpha-kislogo glikoproteina na obmen oksiprolina pri éksperimental'noi termicheskoi travme.

alpha 1-Acid glycoprotein, isolated from human blood plasma, was shown to accelerate healing of burn wounds in rats and to increase daily urine excretion. The glycoprotein restricted an elevation of the peptide-bound hydroxyproline in blood plasma, decreased the ratio between peptide-bound and free hydroxyproline within all the periods of experiment during 3 weeks as well as stimulated hydroxyproline excretion with urine at the step of active reparation of burn wounds. The glycoprotein appears to protect connective tissue metabolism in burns.

[Immunotropic activity of human alpha1-glucoprotein]. / Immunotropnaia aktivnost' alpha1-kislogo glikoproteina cheloveka.

Experiments were conducted to study the effect of alpha-acid glycoprotein on a fatal infection caused by Pseudomonas pyocyanea, growth of melanoma B-16, and transplantation of a skin graft from C57BL/6 mice to CBA mice. Injection of the agent significantly increased the anti-infection resistance in mice, suppressed growth of melanoma-16, and prolonged the survival of the skin grafts, which was evidence of marked glycoprotein immunomodulating activity.

[Acceleration of the healing of burn wounds in mice under the influence of alpha 1-acid glycoprotein]. / Uskorenie zazhivleniia ozhogovykh ran u myshei pod vliianiem al'fa 1-kislogo glikoproteida.

The effect of alpha 1-acid glycoprotein (alpha 1-AGP) was tested in experiments on rats with thermal burns. For comparison control mice with burns were given injections of albumin, physiological solution or did not receive injections. alpha 1-AGP was administered in the first 2 days after the burn to a total dose of 6 mg. Injection of physiological solution, alpha 1-AGP and albumin reduced animal lethality in the stage of shock. The burn disease took a more favourable course in patients given alpha 1-AGP than in those who received physiological solution and albumin. This was manifested by more rapid healing of the burn wounds and lower incidence of complications of burn disease. The possible mechanisms of the therapeutic action of alpha 1-AGP are discussed.

Control of malaria virulence by alpha 1-acid glycoprotein (orosomucoid), an acute-phase (inflammatory) reactant.

During malaria and other infections, the plasma concentration of alpha 1-acid glycoprotein (AGP) increases 3- to 4-fold, but the function of this glycoprotein has been unknown. This study demonstrates, by in vitro culture of the malaria parasite Plasmodium falciparum, that the AGP concentration achieved during malaria is sufficient to inhibit parasite multiplication by 80%. It was found that the inhibitory activity of AGP depends on and is a function of its sialic acid complement (12-16 mol/mol) and its higher-order structure. AGP acts by blocking parasite-erythrocyte interaction during the invasion process. These findings indicate a function for AGP with definite in vivo significance. Moreover, they reveal an important protective response to malaria and perhaps other infectious diseases.