George Marek

Hyperuricemia as a Mediator of the Proinflammatory Endocrine Imbalance in the Adipose Tissue in a Murine Model of the Metabolic Syndrome

OBJECTIVE Hyperuricemia is strongly associated with obesity and metabolic syndrome and can predict visceral obesity and insulin resistance. Previously, we showed that soluble uric acid directly stimulated the redox-dependent proinflammatory signaling in adipocytes. In this study we demonstrate the role of hyperuricemia in the production of key adipokines. RESEARCH DESIGN AND METHODS We used mouse 3T3-L1 adipocytes, human primary adipocytes, and a mouse model of metabolic syndrome and hyperuricemia. RESULTS Uric acid induced in vitro an increase in the production (mRNA and secreted protein) of monocyte chemotactic protein-1 (MCP-1), an adipokine playing an essential role in inducing the proinflammatory state in adipocytes in obesity. In addition, uric acid caused a decrease in the production of adiponectin, an adipocyte-specific insulin sensitizer and anti-inflammatory agent. Uric acid–induced increase in MCP-1 production was blocked by scavenging superoxide or by inhibiting NADPH oxidase and by stimulating peroxisome-proliferator–activated receptor-γ with rosiglitazone. Downregulation of the adiponectin production was prevented by rosiglitazone but not by antioxidants. In obese mice with metabolic syndrome, we observed hyperuricemia. Lowering uric acid in these mice by inhibiting xanthine oxidoreductase with allopurinol could improve the proinflammatory endocrine imbalance in the adipose tissue by reducing production of MCP-1 and increasing production of adiponectin. In addition, lowering uric acid in obese mice decreased macrophage infiltration in the adipose tissue and reduced insulin resistance. CONCLUSIONS Hyperuricemia might be partially responsible for the proinflammatory endocrine imbalance in the adipose tissue, which is an underlying mechanism of the low-grade inflammation and insulin resistance in subjects with the metabolic syndrome.

Uric Acid Induces Hepatic Steatosis by Generation of Mitochondrial Oxidative Stress POTENTIAL ROLE IN FRUCTOSE-DEPENDENT AND -INDEPENDENT FATTY LIVER

Background: Uric acid is an independent risk factor in fructose-induced fatty liver, but whether it is a marker or a cause remains unknown. Results: Hepatocytes exposed to uric acid developed mitochondrial dysfunction and increased de novo lipogenesis, and its blockade prevented fructose-induced lipogenesis. Conclusion: Rather than a consequence, uric acid induces fatty liver Significance: Hyperuricemic people are more prone to develop fructose-induced fatty liver. Metabolic syndrome represents a collection of abnormalities that includes fatty liver, and it currently affects one-third of the United States population and has become a major health concern worldwide. Fructose intake, primarily from added sugars in soft drinks, can induce fatty liver in animals and is epidemiologically associated with nonalcoholic fatty liver disease in humans. Fructose is considered lipogenic due to its ability to generate triglycerides as a direct consequence of the metabolism of the fructose molecule. Here, we show that fructose also stimulates triglyceride synthesis via a purine-degrading pathway that is triggered from the rapid phosphorylation of fructose by fructokinase. Generated AMP enters into the purine degradation pathway through the activation of AMP deaminase resulting in uric acid production and the generation of mitochondrial oxidants. Mitochondrial oxidative stress results in the inhibition of aconitase in the Krebs cycle, resulting in the accumulation of citrate and the stimulation of ATP citrate lyase and fatty-acid synthase leading to de novo lipogeneis. These studies provide new insights into the pathogenesis of hepatic fat accumulation under normal and diseased states.

Adiponectin Resistance and Proinflammatory Changes in the Visceral Adipose Tissue Induced by Fructose Consumption via Ketohexokinase-Dependent Pathway

An epidemic of obesity and type 2 diabetes is linked with the increase in consumption of fructose-containing sugars, such as sucrose and high-fructose corn syrup. In mammalian cells, fructose is metabolized predominantly via phosphorylation to fructose-1 phosphate by ketohexokinase (KHK) or by alternative pathways. Here we demonstrate that a KHK-dependent pathway mediates insulin resistance and inflammatory changes in the visceral fat in response to high fructose. We used mice (males, C57BL/6 background) including littermate wild-type control and mice lacking both isoforms of KHK (KHK-null). Fructose diet induced metabolic syndrome, including visceral obesity, insulin resistance, proinflammatory changes in the visceral fat (production of proinflammatory adipokines and macrophage infiltration), the endoplasmic reticulum stress signaling, and decrease of the high–molecular weight adiponectin followed by decrease in the downstream signaling. KHK-KO mice consuming the same high-fructose diet remained lean, with normal insulin sensitivity and healthy visceral adipose tissue with normal adiponectin function not distinguishable from the control by any of the tested parameters. This study demonstrates that blocking KHK and redirecting fructose metabolism to alternative pathways is an effective way to prevent visceral obesity and insulin resistance induced by high fructose, a widespread component of Western diets.

A high-throughput method for isolation of salicylic acid metabolic mutants

BackgroundSalicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor Acinetobacter sp. ADPWH_lux-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.ResultsOn the basis of the previously described biosensor-based method, we simplified the tissue collection and the SA extraction procedure. Leaf discs were collected and boiled in Luria-Bertani (LB), and then the released SA was measured with the biosensor. The time-consuming steps of weighing samples, grinding tissues and centrifugation were avoided. The direct boiling protocol detected similar differences in SA levels among pathogen-infected wild-type, npr1 (nonexpressor of pathogenesis-related genes), and sid2 (SA induction-deficient) plants as did the previously described biosensor-based method and an HPLC-based approach, demonstrating the efficacy of the protocol presented here. We adapted this protocol to a high-throughput format and identified six npr1 suppressors that accumulated lower levels of SA than npr1 upon pathogen infection. Two of the suppressors were found to be allelic to the previously identified eds5 mutant. The other four are more susceptible than npr1 to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 and their identity merits further investigation.ConclusionsThe rapid SA extraction method by direct boiling of leaf discs further reduced the cost and time required for the biosensor Acinetobacter sp. ADPWH_lux-based SA estimation, and allowed the screening for npr1 suppressors that accumulated less SA than npr1 after pathogen infection in a high-throughput manner. The highly efficacious SA estimation protocol can be applied in genetic screen for SA metabolic mutants and characterization of enzymes involved in SA metabolism. The mutants isolated in this study may help identify new components in the SA-related signaling pathways.

Myeloid-derived Suppressor Cells Are Necessary for Development of Pulmonary Hypertension

&NA; Pulmonary hypertension (PH) complicates the care of patients with chronic lung disease, such as idiopathic pulmonary fibrosis (IPF), resulting in a significant increase in morbidity and mortality. Disease pathogenesis is orchestrated by unidentified myeloid‐derived cells. We used murine models of PH and pulmonary fibrosis to study the role of circulating myeloid cells in disease pathogenesis and prevention. We administered clodronate liposomes to bleomycin‐treated wild‐type mice to induce pulmonary fibrosis and PH with a resulting increase in circulating bone marrow‐derived cells. We discovered that a population of C‐X‐C motif chemokine receptor (CXCR) 2+ myeloid‐derived suppressor cells (MDSCs), granulocytic subset (G‐MDSC), is associated with severe PH in mice. Pulmonary pressures worsened despite improvement in bleomycin‐induced pulmonary fibrosis. PH was attenuated by CXCR2 inhibition, with antagonist SB 225002, through decreasing G‐MDSC recruitment to the lung. Molecular and cellular analysis of clinical patient samples confirmed a role for elevated MDSCs in IPF and IPF with PH. These data show that MDSCs play a key role in PH pathogenesis and that G‐MDSC trafficking to the lung, through chemokine receptor CXCR2, increases development of PH in multiple murine models. Furthermore, we demonstrate pathology similar to the preclinical models in IPF with lung and blood samples from patients with PH, suggesting a potential role for CXCR2 inhibitor use in this patient population. These findings are significant, as there are currently no approved disease‐specific therapies for patients with PH complicating IPF.

Erdj3 Has an Essential Role for Z Variant Alpha-1-Antitrypsin Degradation

Alpha‐1‐antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at amino acid 342 in the mature protein, resulting in the Z mutation of the alpha‐1‐antitrypsin gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes and monocytes, causing a toxic gain of function. Retained ZAAT is eliminated by ER‐associated degradation and autophagy. We hypothesized that alpha‐1‐antitrypsin (AAT)‐interacting proteins play critical roles in quality control of human AAT. Using co‐immunoprecipitation, we identified ERdj3, an ER‐resident Hsp40 family member, as a part of the AAT trafficking network. Depleting ERdj3 increased the rate of ZAAT degradation in hepatocytes by redirecting ZAAT to the ER calreticulin‐EDEM1 pathway, followed by autophagosome formation. In the Huh7.5 cell line, ZAAT ER clearance resulted from enhancing ERdj3‐mediated ZAAT degradation by silencing ERdj3 while simultaneously enhancing autophagy. In this context, ERdj3 suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD‐related liver disease. J. Cell. Biochem. 118: 3090–3101, 2017.

Alpha-1 Antitrypsin-Deficient Macrophages Have Increased Matriptase-Mediated Proteolytic Activity

&NA; Alpha‐1 antitrypsin (AAT) deficiency‐associated emphysema is largely attributed to insufficient inhibition of neutrophil elastase released from neutrophils. Correcting AAT levels using augmentation therapy only slows disease progression, and that suggests a more complex process of lung destruction. Because alveolar macrophages (M&phis;) express AAT, we propose that the expression and intracellular accumulation of mutated Z‐AAT (the most common mutation) compromises M&phis; function and contributes to emphysema development. Extracellular matrix (ECM) degradation is a hallmark of emphysema pathology. In this study, M&phis; from individuals with Z‐AAT (Z‐M&phis;) have greater proteolytic activity on ECM than do normal M&phis;. This abnormal Z‐M&phis; activity is not abrogated by supplementation with exogenous AAT and is likely the result of cellular dysfunction induced by intracellular accumulation of Z‐AAT. Using pharmacologic inhibitors, we show that several classes of proteases are involved in matrix degradation by Z‐M&phis;. Importantly, compared with normal M&phis;, the membrane‐bound serine protease, matriptase, is present in Z‐M&phis; at higher levels and contributes to their proteolytic activity on ECM. In addition, we identified matrix metalloproteinase (MMP)‐14, a membrane‐anchored metalloproteinase, as a novel substrate for matriptase, and showed that matriptase regulates the levels of MMP‐14 on the cell surface. Thus, high levels of matriptase may contribute to increased ECM degradation by Z‐M&phis;, both directly and through MMP‐14 activation. In summary, the expression of Z‐AAT in M&phis; confers increased proteolytic activity on ECM. This proteolytic activity is not rescued by exogenous AAT supplementation and could thus contribute to augmentation resistance in AAT deficiency‐associated emphysema.

SVIP regulates Z variant alpha-1 antitrypsin retro-translocation by inhibiting ubiquitin ligase gp78

Alpha-1 antitrypsin deficiency (AATD) is an inherited disorder characterized by early-onset emphysema and liver disease. The most common disease-causing mutation is a single amino acid substitution (Glu/Lys) at amino acid 342 of the mature protein, resulting in disruption of the 290–342 salt bridge (an electrophoretic abnormality defining the mutation [Z allele, or ZAAT]), protein misfolding, polymerization, and accumulation in the endoplasmic reticulum of hepatocytes and monocytes. The Z allele causes a toxic gain of function, and the E3 ubiquitin ligase gp78 promotes degradation and increased solubility of endogenous ZAAT. We hypothesized that the accumulation of ZAAT is influenced by modulation of gp78 E3 ligase and SVIP (small VCP-interacting protein) interaction with p97/VCP in ZAAT-expressing hepatocytes. We showed that the SVIP inhibitory effect on ERAD due to overexpression causes the accumulation of ZAAT in a human Z hepatocyte–like cell line (AT01). Overexpression of gp78, as well as SVIP suppression, induces gp78-VCP/p97 interaction in AT01 cells. This interaction leads to retro-translocation of ZAAT and reduction of the SVIP inhibitory role in ERAD. In this context, overexpression of gp78 or SVIP suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD.

98. Bortezomib Enhances AAV Vector Mediated Transduction But Inhibits the Secretion of Transgene Product

Adeno-associated virus (rAAV) mediated gene therapy has shown promise for the treatment of alpha1 antitrypsin deficiency (AATD). Sustained alpha 1 antitrypsin (AAT) expression has been achieved in clinical trails, but the serum AAT concentrations do not reach therapeutic levels. Enhancing transduction efficiency is critical to developing a successful treatment. Since rAAV transduction may be limited by the proteasome, which degrade AAV capsids, we tested the effect of proteasome inhibitors on rAAV mediated AAT production. We treated HeLa and 293 cells with proteasome inhibitors including bortezomib and infected HeLa cells with rAAV2-GFP, rAAV2-AAT or AAV1-AAT and we Infected 293 cells with rAAV2-AAT. We measured AAT in the medium and the lysates of cells. Consistent with previous reports, bortezomib significantly enhanced AAV2 mediated GFP transduction in Hela cells. In rAAV-AAT infected cells, bortezomib treatment resulted in 9.7-13.1 fold increase in of intracellular AAT, while only modest (1.16-1.6 fold) increases of secreted AAT compared to that in cells without bortezomib. Similarly, intramuscular injection of rAAV1-AAT to C57BL6 mice treated with bortezomib also resulted in a small (~1 fold) increases in serum AAT compared with no treatment. These results indicate that bortezomib inhibited AAT protein secretion. To test the role unfolded protein response (UPR) in AAT accumulation, we measured UPR-related genes and showed that bortezomib treatment increased the mRNA levels of the protein chaperone and UPR sensor GRP78 (BiP) indicating an activation of ER stress pathways. Together, our results indicate that bortezomib can enhance rAAV transduction, but it can also inhibit the secretion of transgene product, which will limit the application of this drug for enhancing the efficiency of certain gene therapy, such as alpha 1 antitrypsin gene therapy.