Publications by authors named "David Orlicky"

126 Publications

SODB1 is essential for Leishmania major infection of macrophages and pathogenesis in mice.

PLoS Negl Trop Dis 2018 10 29;12(10):e0006921. Epub 2018 Oct 29.

Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, United States of America.

Leishmania species are sand fly-transmitted protozoan parasites that cause leishmaniasis, neglected tropical diseases that affect millions of people. Leishmania amastigotes must overcome a variety of host defenses, including reactive oxygen species (ROS) produced by the NADPH oxidase. Leishmania species encode three superoxide dismutases (SODs): the mitochondrial SODA and two glycosomal SODs (SODB1 and SODB2). SODs are metalloenzymes that function in antioxidant defense by converting superoxide to oxygen and hydrogen peroxide. Here, we investigated a role for SODB1 in Leishmania infection of macrophages and virulence in mice. We found that a single allele deletion of SODB1 (SODB1/Δsodb1) had minimal effects on the replication of axenically-grown L. major promastigotes or differentiation to infective metacyclic promastigotes. Disruption of a single SODB1 allele also did not affect L. donovani differentiation to amastigotes induced axenically, or the replication of axenically-grown L. donovani promastigotes and amastigotes. In contrast, the persistence of SODB1/Δsodb1 L. major in WT macrophages was impaired, and the development of cutaneous lesions in SODB1/Δsodb1 L. major-infected C57BL/6 and BALB/c mice was strongly reduced. The reduced disease severity in mice was associated with reduced burdens of SODB1/Δsodb1 L. major parasites in the foot at late, but not early times post-inoculation, as well as an impaired capacity to disseminate from the site of inoculation. Collectively, these data suggest that SODB1 is critical for L. major persistence in macrophages and virulence in mice.
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http://dx.doi.org/10.1371/journal.pntd.0006921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224164PMC
October 2018

Altered Cell-Cycle Control, Inflammation, and Adhesion in High-Risk Persistent Bronchial Dysplasia.

Cancer Res 2018 09 11;78(17):4971-4983. Epub 2018 Jul 11.

Department of Medicine/Division of Pulmonary Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell-cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G-M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention. Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell-cell adhesion that may underlie progression to invasive SCC. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-3822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6147150PMC
September 2018

Perilipin-2 deletion promotes carbohydrate-mediated browning of white adipose tissue at ambient temperature.

J Lipid Res 2018 08 4;59(8):1482-1500. Epub 2018 Jun 4.

Integrated Physiology Graduate Program, University of Colorado at Denver, Anschutz Medical Campus, Aurora, CO 80045

Mice lacking perilipin-2 (Plin2-null) are resistant to obesity, insulin resistance, and fatty liver induced by Western or high-fat diets. In the current study, we found that, compared with WT mice on Western diet, Plin2-null adipose tissue was more insulin sensitive and inguinal subcutaneous white adipose tissue (iWAT) exhibited profound browning and robust induction of thermogenic and carbohydrate-responsive genetic programs at room temperature. Surprisingly, these Plin2-null responses correlated with the content of simple carbohydrates, rather than fat, in the diet, and were independent of adipose Plin2 expression. To define Plin2 and sugar effects on adipose browning, WT and Plin2-null mice were placed on chow diets containing 20% sucrose in their drinking water for 6 weeks. Compared with WT mice, iWAT of Plin2-null mice exhibited pronounced browning and striking increases in the expression of thermogenic and insulin-responsive genes on this diet. Significantly, Plin2-null iWAT browning was associated with reduced sucrose intake and elevated serum fibroblast growth factor (FGF)21 levels, which correlated with greatly enhanced hepatic FGF21 production. These data identify Plin2 actions as novel mediators of sugar-induced adipose browning through indirect effects of hepatic FGF21 expression, and suggest that adipose browning mechanisms may contribute to Plin2-null resistance to obesity.
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http://dx.doi.org/10.1194/jlr.M086249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071773PMC
August 2018

Aberrant expression of redox regulatory proteins in patients with concomitant primary Sclerosing cholangitis/inflammatory bowel disease.

Exp Mol Pathol 2018 08 29;105(1):32-36. Epub 2018 May 29.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, United States. Electronic address:

Objective: Primary Sclerosing Cholangitis (PSC) is a severe cholestatic liver disease characterized by progressive peri-biliary tract inflammation, elevated oxidative stress and hepatocellular injury. A hallmark of PSC patients is the concurrent diagnosis of Inflammatory Bowel Disease occurring in approximately 70%-80% of PSC patients (PSC/IBD). We previously reported dysregulation of key anti-oxidant pathways in PSC/IBD. The objective of this study was to expand previous data by examining the abundance of thioredoxins (Trx) in PSC/IBD.

Methods: Using hepatic tissue and whole cell extracts isolated from age-matched healthy humans and patients diagnosed with end stage PSC/IBD, the protein abundance of thioredoxin, thioredoxin reductase (TrxR1), and their downstream substrates peroxiredoxins was assessed.

Results: Western blot analyses of thioredoxin and peroxiredoxin abundance revealed significant increases in abundance of Trx1 and TrxR1 whereas expression of thioredoxin-interacting protein was significantly decreased in PSC/IBD. Concurrently, abundance of cytosolic peroxiredoxins was not significantly impacted. The abundance of mitochondrial Trx2, along with peroxiredoxins 3, 5 and 6 were significantly decreased by concurrent PSC/IBD. Histological staining of Trx1/TrxR1 revealed elevated nuclear Trx1 and TrxR1 staining within cholangiocytes as well as an overall periportal increase in expression in PSC/IBD. An examination of additional anti-oxidant responses reveal suppression of gamma-glutamylcysteine synthetase and heme oxygenase (HO-1) whereas expression of the protein chaperone glucose regulated protein 78 increased suggesting elevated cellular stress in PSC/IBD.

Conclusions: Results herein suggest that in addition to severe dysregulation of anti-oxidant responses, cholestasis impacts both cytosolic/nuclear (Trx1) as well as mitochondrial (Trx2) redox signaling and control pathways.
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http://dx.doi.org/10.1016/j.yexmp.2018.05.012DOI Listing
August 2018

Elevated Nrf-2 responses are insufficient to mitigate protein carbonylation in hepatospecific PTEN deletion mice.

PLoS One 2018 25;13(5):e0198139. Epub 2018 May 25.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.

Objective: In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease (NASH) is oxidative stress, which leads to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine the impact of NASH on protein carbonylation and antioxidant responses in a murine model.

Methods: Liver-specific phosphatase and tensin homolog (PTEN)-deletion mice (PTENLKO) or control littermates were fed a standard chow diet for 45-55 weeks followed by analysis for liver injury, oxidative stress and inflammation.

Results: Histology and Picrosirius red-staining of collagen deposition within the extracellular matrix revealed extensive steatosis and fibrosis in the PTENLKO mice but no steatosis or fibrosis in controls. Increased steatosis and fibrosis corresponded with significant increases in inflammation. PTEN-deficient livers showed significantly increased cell-specific oxidative damage, as detected by 4-hydroxy-2-nonenal (4-HNE) and acrolein staining. Elevated staining correlated with an increase in nuclear DNA repair foci (γH2A.X) and cellular proliferation index (Ki67) within zones 1 and 3, indicating oxidative damage was zonally restricted and was associated with increased DNA damage and cell proliferation. Immunoblots showed that total levels of antioxidant response proteins induced by nuclear factor erythroid-2-like-2 (Nrf2), including GSTμ, GSTπ and CBR1/3, but not HO-1, were elevated in PTENLKO as compared to controls, and IHC showed this response also occurred only in zones 1 and 3. Furthermore, an analysis of autophagy markers revealed significant elevation of p62 and LC3II expression. Mass spectrometric (MS) analysis identified significantly more carbonylated proteins in whole cell extracts prepared from PTENLKO mice (966) as compared to controls (809). Pathway analyses of identified proteins did not uncover specific pathways that were preferentially carbonylated in PTENLKO livers but, did reveal specific strongly increased carbonylation of thioredoxin reductase and of glutathione-S-transferases (GST) M6, O1, and O2.

Conclusions: Results show that disruption of PTEN resulted in steatohepatitis, fibrosis and caused hepatic induction of the Nrf2-dependent antioxidant system at least in part due to elevation of p62. This response was both cell-type and zone specific. However, these responses were insufficient to mitigate the accumulation of products of lipid peroxidation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198139PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969769PMC
November 2018

Knockout of the Gsta4 Gene in Male Mice Leads to an Altered Pattern of Hepatic Protein Carbonylation and Enhanced Inflammation Following Chronic Consumption of an Ethanol Diet.

Alcohol Clin Exp Res 2018 07 30;42(7):1192-1205. Epub 2018 May 30.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Background: Glutathione S-transferase A4-4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE). In this study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease by examining the development of liver injury in male wild-type (WT) SV/J mice and SV/J mice lacking functional GSTA4 (GSTA4 mice).

Methods: Adult male WT and GSTA4 mice were fed chow (N = 10 to 12) or high-fat Lieber-DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18 to 20) for 116 days. At the end of the study, half of the EtOH-fed mice were acutely challenged with an EtOH binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4-HNE adduction and by comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) of purified carbonylated proteins.

Results: Chronic EtOH intake significantly increased hepatic 4-HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum alanine aminotransferase. Hepatic infiltration with B cells, T cells, and neutrophils and mRNA expression of pro-inflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)γ was modest in WT mice. However, an EtOH binge increased hepatic necrosis, hepatic cell proliferation, and expression of TNFα mRNA (p < 0.05). EtOH treatment of GSTA4 mice increased B-cell infiltration and increased mRNA expression of TNFα and IFNγ and of matrix remodeling markers MMP9, MMP13, and Col1A1 (p < 0.05). GSTA4 mice exhibited panlobular rather than periportal distribution of 4-HNE-adducted proteins and increased overall 4-HNE staining after EtOH binge. Comprehensive LC-MS of carbonylated proteins identified 1,022 proteins of which 189 were unique to the GSTA4 group.

Conclusions: These data suggest long-term adaptation to EtOH in WT mice does not occur in GSTA4 mice. Products of lipid peroxidation appear to play a role in inflammatory responses due to EtOH. And EtOH effects on B-cell infiltration and autoimmune responses may be secondary to formation of carbonyl adducts.
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http://dx.doi.org/10.1111/acer.13766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028296PMC
July 2018

Maternal obesity during lactation may protect offspring from high fat diet-induced metabolic dysfunction.

Nutr Diabetes 2018 04 25;8(1):18. Epub 2018 Apr 25.

Division of Reproductive Sciences, Department of Obstetrics & Gynecology, School of Medicine, University of Colorado Denver Anschutz Medical Campus, Aurora, CO, 80045, USA.

Background/objectives: The current obesity epidemic has spurred exploration of the developmental origin of adult heath and disease. A mother's dietary choices and health can affect both the early wellbeing and lifelong disease-risk of the offspring.

Subjects/methods: To determine if changes in the mother's diet and adiposity have long-term effects on the baby's metabolism, independently from a prenatal insult, we utilized a mouse model of diet-induced-obesity and cross-fostering. All pups were born to lean dams fed a low fat diet but were fostered onto lean or obese dams fed a high fat diet. This study design allowed us to discern the effects of a poor diet from those of mother's adiposity and metabolism. The weaned offspring were placed on a high fat diet to test their metabolic function.

Results: In this feeding challenge, all male (but not female) offspring developed metabolic dysfunction. We saw increased weight gain in the pups nursed on an obesity-resistant dam fed a high fat diet, and increased pathogenesis including liver steatosis and adipose tissue inflammation, when compared to pups nursed on either obesity-prone dams on a high fat diet or lean dams on a low fat diet.

Conclusion: Exposure to maternal over-nutrition, through the milk, is sufficient to shape offspring health outcomes in a sex- and organ-specific manner, and milk from a mother who is obesity-prone may partially protect the offspring from the insult of a poor diet.
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http://dx.doi.org/10.1038/s41387-018-0027-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916951PMC
April 2018

Cytokine-Producing B Cells Promote Immune-Mediated Bile Duct Injury in Murine Biliary Atresia.

Hepatology 2018 11 10;68(5):1890-1904. Epub 2018 Jul 10.

Department of Pediatrics, Section of Gastroenterology, Hepatology and Nutrition, Aurora, CO.

Biliary atresia (BA) is a neonatal T cell-mediated, inflammatory, sclerosing cholangiopathy. In the rhesus rotavirus (RRV)-induced neonatal mouse model of BA (murine BA), mice lacking B cells do not develop BA, and the lack of B cells is associated with loss of T-cell and macrophage activation. The aim of this study was to determine the mechanism of B cell-mediated immune activation (antigen presentation versus cytokine production) in murine BA. Normal neonatal B cells in the liver are predominantly at pro-B and pre-B cellular development. However, BA mice exhibit a significant increase in the number and activation status of mature liver B cells. Adoptively transferred B cells into RRV-infected, B cell-deficient mice were able to reinstate T-cell and macrophage infiltration and biliary injury. Nonetheless, neonatal liver B cells were incompetent at antigen presentation to T cells. Moreover, 3-83 immunoglobulin transgenic mice, in which B cells only present an irrelevant antigen, developed BA, indicating a B-cell antigen-independent mechanism. B cells from BA mice produced a variety of innate and adaptive immune cytokines associated with immune activation. In vitro trans-well studies revealed that BA B cells secreted cytokines that activated T cells based on increased expression of T-cell activation marker cluster of differentiation 69. Conclusion: Neonatal liver B cells are highly activated in murine BA and contribute to immune activation through production of numerous cytokines involved in innate and adaptive immunity; this work provides increased knowledge on the capacity of neonatal B cells to contribute to an inflammatory disease through cytokine-mediated mechanisms, and future studies should focus on targeting B cells as a therapeutic intervention in human BA.
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http://dx.doi.org/10.1002/hep.30051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195851PMC
November 2018

Supplemental Oxygen Improves In Vivo Mitochondrial Oxidative Phosphorylation Flux in Sedentary Obese Adults With Type 2 Diabetes.

Diabetes 2018 07 11;67(7):1369-1379. Epub 2018 Apr 11.

Center for Women's Health Research, Anschutz Medical Campus, Aurora, CO

Type 2 diabetes is associated with impaired exercise capacity. Alterations in both muscle perfusion and mitochondrial function can contribute to exercise impairment. We hypothesized that impaired muscle mitochondrial function in type 2 diabetes is mediated, in part, by decreased tissue oxygen delivery and would improve with oxygen supplementation. Ex vivo muscle mitochondrial content and respiration assessed from biopsy samples demonstrated expected differences in obese individuals with ( = 18) and without ( = 17) diabetes. Similarly, in vivo mitochondrial oxidative phosphorylation capacity measured in the gastrocnemius muscle via P-MRS indicated an impairment in the rate of ADP depletion with rest (27 ± 6 s [diabetes], 21 ± 7 s [control subjects]; = 0.008) and oxidative phosphorylation ( = 0.046) in type 2 diabetes after isometric calf exercise compared with control subjects. Importantly, the in vivo impairment in oxidative capacity resolved with oxygen supplementation in adults with diabetes (ADP depletion rate 5.0 s faster, = 0.012; oxidative phosphorylation 0.046 ± 0.079 mmol/L/s faster, = 0.027). Multiple in vivo mitochondrial measures related to HbA These data suggest that oxygen availability is rate limiting for in vivo mitochondrial oxidative exercise recovery measured with P-MRS in individuals with uncomplicated diabetes. Targeting muscle oxygenation could improve exercise function in type 2 diabetes.
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http://dx.doi.org/10.2337/db17-1124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463751PMC
July 2018

Ketohexokinase C blockade ameliorates fructose-induced metabolic dysfunction in fructose-sensitive mice.

J Clin Invest 2018 06 23;128(6):2226-2238. Epub 2018 Apr 23.

Department of Biology, Boston University, Boston, Massachusetts, USA.

Increasing evidence suggests a role for excessive intake of fructose in the Western diet as a contributor to the current epidemics of metabolic syndrome and obesity. Hereditary fructose intolerance (HFI) is a difficult and potentially lethal orphan disease associated with impaired fructose metabolism. In HFI, the deficiency of aldolase B results in the accumulation of intracellular phosphorylated fructose, leading to phosphate sequestration and depletion, increased adenosine triphosphate (ATP) turnover, and a plethora of conditions that lead to clinical manifestations such as fatty liver, hyperuricemia, Fanconi syndrome, and severe hypoglycemia. Unfortunately, there is currently no treatment for HFI, and avoiding sugar and fructose has become challenging in our society. In this report, through use of genetically modified mice and pharmacological inhibitors, we demonstrate that the absence or inhibition of ketohexokinase (Khk), an enzyme upstream of aldolase B, is sufficient to prevent hypoglycemia and liver and intestinal injury associated with HFI. Herein we provide evidence for the first time to our knowledge of a potential therapeutic approach for HFI. Mechanistically, our studies suggest that it is the inhibition of the Khk C isoform, not the A isoform, that protects animals from HFI.
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http://dx.doi.org/10.1172/JCI94427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983342PMC
June 2018

Pyrroloquinoline quinone prevents developmental programming of microbial dysbiosis and macrophage polarization to attenuate liver fibrosis in offspring of obese mice.

Hepatol Commun 2018 03 22;2(3):313-328. Epub 2018 Jan 22.

Department of Anesthesiology University of Colorado Anschutz Medical Campus Aurora CO.

Increasingly, evidence suggests that exposure to maternal obesity creates an inflammatory environment , exerting long-lasting postnatal signatures on the juvenile innate immune system and microbiome that may predispose offspring to development of fatty liver disease. We found that exposure to a maternal Western-style diet (WD) accelerated fibrogenesis in the liver of offspring and was associated with early recruitment of proinflammatory macrophages at 8-12 weeks and microbial dysbiosis as early as 3 weeks of age. We further demonstrated that bone marrow-derived macrophages (BMDMs) were polarized toward an inflammatory state at 8 weeks of age and that a potent antioxidant, pyrroloquinoline quinone (PQQ), reversed BMDM metabolic reprogramming from glycolytic toward oxidative metabolism by restoring trichloroacetic acid cycle function at isocitrate dehydrogenase. This resulted in reduced inflammation and inhibited collagen fibril formation in the liver at 20 weeks of age, even when PQQ was withdrawn at 3 weeks of age. Beginning at 3 weeks of age, WD-fed mice developed a decreased abundance of and , together with increased and decreased tight junction gene expression by 20 weeks, whereas microbiota of mice exposed to PQQ retained compositional stability with age, which was associated with improved liver health. : Exposure to a maternal WD induces early gut dysbiosis and disrupts intestinal tight junctions, resulting in BMDM polarization and induction of proinflammatory and profibrotic programs in the offspring that persist into adulthood. Disrupted macrophage and microbiota function can be attenuated by short-term maternal treatment with PQQ prior to weaning, suggesting that reshaping the early gut microbiota in combination with reprogramming macrophages during early weaning may alleviate the sustained proinflammatory environment, preventing the rapid progression of nonalcoholic fatty liver disease to nonalcoholic steatohepatitis in offspring of obese mothers. ( 2018;2:313-328).
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http://dx.doi.org/10.1002/hep4.1139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831029PMC
March 2018

High salt intake causes leptin resistance and obesity in mice by stimulating endogenous fructose production and metabolism.

Proc Natl Acad Sci U S A 2018 03 5;115(12):3138-3143. Epub 2018 Mar 5.

Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, Aurora, CO 80045.

Dietary guidelines for obesity typically focus on three food groups (carbohydrates, fat, and protein) and caloric restriction. Intake of noncaloric nutrients, such as salt, are rarely discussed. However, recently high salt intake has been reported to predict the development of obesity and insulin resistance. The mechanism for this effect is unknown. Here we show that high intake of salt activates the aldose reductase-fructokinase pathway in the liver and hypothalamus, leading to endogenous fructose production with the development of leptin resistance and hyperphagia that cause obesity, insulin resistance, and fatty liver. A high-salt diet was also found to predict the development of diabetes and nonalcoholic fatty liver disease in a healthy population. These studies provide insights into the pathogenesis of obesity and diabetes and raise the potential for reduction in salt intake as an additional interventional approach for reducing the risk for developing obesity and metabolic syndrome.
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http://dx.doi.org/10.1073/pnas.1713837115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866545PMC
March 2018

The Sodium-Glucose Cotransporter 2 Inhibitor Dapagliflozin Prevents Renal and Liver Disease in Western Diet Induced Obesity Mice.

Int J Mol Sci 2018 Jan 3;19(1). Epub 2018 Jan 3.

Department of Renal & Hypertension, Department of Pediatrics and Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, 12800 19th Ave., Aurora, CO 80045, USA.

Obesity and obesity related kidney and liver disease have become more prevalent over the past few decades, especially in the western world. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with promising effects on cardiovascular and renal function. Given SGLT2 inhibitors exert both anti-diabetic and anti-obesity effects by promoting urinary excretion of glucose and subsequent caloric loss, we investigated the effect of the highly selective renal SGLT2 inhibitor dapagliflozin in mice with Western diet (WD) induced obesity. Low fat (LF) diet or WD-fed male C57BL/6J mice were treated with dapagliflozin for 26 weeks. Dapagliflozin attenuated the WD-mediated increases in body weight, plasma glucose and plasma triglycerides. Treatment with dapagliflozin prevented podocyte injury, glomerular pathology and renal fibrosis determined by second harmonic generation (SHG), nephrin, synaptopodin, collagen IV, and fibronectin immunofluorescence microscopy. Oil Red O staining showed dapagliflozin also decreased renal lipid accumulation associated with decreased SREBP-1c mRNA abundance. Moreover, renal inflammation and oxidative stress were lower in the dapagliflozin-treated WD-fed mice than in the untreated WD-fed mice. In addition, dapagliflozin decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), hepatic lipid accumulation as determined by H&E and Oil Red O staining, and Coherent Anti-Stokes Raman Scattering (CARS) microscopy, and hepatic fibrosis as determined by picrosirius red (PSR) staining and TPE-SHG microscopy in WD-fed mice. Thus, our study demonstrated that the co-administration of the SGLT2 inhibitor dapagliflozin attenuates renal and liver disease during WD feeding of mice.
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http://dx.doi.org/10.3390/ijms19010137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796086PMC
January 2018

Dysregulation of antioxidant responses in patients diagnosed with concomitant Primary Sclerosing Cholangitis/Inflammatory Bowel Disease.

Exp Mol Pathol 2018 02 24;104(1):1-8. Epub 2017 Nov 24.

Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States.

Objective: Primary Sclerosing Cholangitis (PSC) is a chronic cholestatic liver disease that is characterized by severe peri-biliary tract inflammation and fibrosis, elevated oxidative stress and hepatocellular injury. A hallmark of PSC patients is the concurrent diagnosis of Inflammatory Bowel Disease occurring in approximately 70%-80% of PSC patients (PSC/IBD). The objective of this study was to determine the impact of end stage PSC/IBD on cellular antioxidant responses and the formation of protein carbonylation.

Methods: Using hepatic tissue and whole cell extracts isolated from age-matched healthy humans and patients diagnosed with end stage PSC/IBD, overall inflammation, oxidative stress, and protein carbonylation were assessed by Western blotting, and immunohistochemistry.

Results: Increased immunohistochemical staining for CD3+ (lymphocyte), CD68 (Kupffer cell) and myeloperoxidase (neutrophil) colocalized with the extensive Picrosirius red stained fibrosis confirming the inflammatory aspect of PSC. Importantly, the increased inflammation also colocalized with elevated periportal post-translational modification by the reactive aldehydes 4-HNE, MDA and acrolein. 4-HNE, MDA and acrolein IHC all displayed a significant component in hepatocytes adjacent to fibrotic regions. Furthermore, acrolein was also elevated within the nuclei of periportal inflammatory cells whereas MDA staining was increased in hepatocytes across the lobule. Prussian Blue staining, when compared to the positive controls (ALD, NASH), did not display any evidence of iron accumulation in PSC/IBD livers. Western analysis of PSC/IBD anti-oxidant responses revealed elevated expression of SOD2, GSTπ as well as upregulation of Akt Ser473 phosphorylation. In contrast, expression of GSTμ, GSTA4, catalase, Gpx1 and Hsp70 were suppressed. These data were further supported by a significant decrease in measured GST activity. Dysregulation of anti-oxidant responses in the periportal region of the liver was supported by elevated SOD2 and GSTπ IHC signals in periportal hepatocytes and cholangiocytes. Expression of the Nrf2-regulated proteins HO-1, NAD(P)H quinone reductase (NQO1) and Gpx1 was primarily localized to macrophages. In contrast, catalase staining decreased within periportal hepatocytes and was not evident within cholangiocytes.

Conclusions: Results herein provide additional evidence that cholestasis induces significant increases in periportal oxidative stress and suggest that there are significant differences in the cellular and subcellular generation of reactive aldehydes formed during cholestatic liver injury. Furthermore, these data suggest that anti-oxidant responses are dysregulated during end-stage PSC/IBD supporting pathological data. This work was funded by NIH5R37AA009300-22 D.R.P.
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http://dx.doi.org/10.1016/j.yexmp.2017.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801122PMC
February 2018

FXR/TGR5 Dual Agonist Prevents Progression of Nephropathy in Diabetes and Obesity.

J Am Soc Nephrol 2018 01 31;29(1):118-137. Epub 2017 Oct 31.

Departments of Medicine and

Bile acids are ligands for the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5. We have shown that FXR and TGR5 have renoprotective roles in diabetes- and obesity-related kidney disease. Here, we determined whether these effects are mediated through differential or synergistic signaling pathways. We administered the FXR/TGR5 dual agonist INT-767 to DBA/2J mice with streptozotocin-induced diabetes, db/db mice with type 2 diabetes, and C57BL/6J mice with high-fat diet-induced obesity. We also examined the individual effects of the selective FXR agonist obeticholic acid (OCA) and the TGR5 agonist INT-777 in diabetic mice. The FXR agonist OCA and the TGR5 agonist INT-777 modulated distinct renal signaling pathways involved in the pathogenesis and treatment of diabetic nephropathy. Treatment of diabetic DBA/2J and db/db mice with the dual FXR/TGR5 agonist INT-767 improved proteinuria and prevented podocyte injury, mesangial expansion, and tubulointerstitial fibrosis. INT-767 exerted coordinated effects on multiple pathways, including stimulation of a signaling cascade involving AMP-activated protein kinase, sirtuin 1, PGC-1, sirtuin 3, estrogen-related receptor-, and Nrf-1; inhibition of endoplasmic reticulum stress; and inhibition of enhanced renal fatty acid and cholesterol metabolism. Additionally, in mice with diet-induced obesity, INT-767 prevented mitochondrial dysfunction and oxidative stress determined by fluorescence lifetime imaging of NADH and kidney fibrosis determined by second harmonic imaging microscopy. These results identify the renal signaling pathways regulated by FXR and TGR5, which may be promising targets for the treatment of nephropathy in diabetes and obesity.
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http://dx.doi.org/10.1681/ASN.2017020222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748904PMC
January 2018

Differential carbonylation of proteins in end-stage human fatty and nonfatty NASH.

Free Radic Biol Med 2017 12 6;113:280-290. Epub 2017 Oct 6.

Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States.

Objective: In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease is oxidative stress leading to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine if significant differences were evident when evaluating carbonylation in human end-stage fatty nonalcoholic steatohepatitis (fNASH) compared to end-stage nonfatty NASH (nfNASH).

Methods: Using hepatic tissue obtained from healthy humans and patients diagnosed with end stage nfNASH or fNASH, overall carbonylation was assessed by immunohistochemistry (IHC) and LC-MS/MS followed by bioinformatics.

Results: Picrosirius red staining revealed extensive fibrosis in both fNASH and nfNASH which corresponded with increased reactive aldehyde staining. Although significantly elevated when compared to normal hepatic tissue, no significant differences in overall carbonylation and fibrosis were evident when comparing fNASH with nfNASH. Examining proteins that are critical for anti-oxidant defense revealed elevated expression of thioredoxin, thioredoxin interacting protein, glutathione S-transferase p1 and mitochondrial superoxide dismutase in human NASH. As important, using immunohistochemistry, significant colocalization of the aforementioned proteins occurred in cytokeratin 7 positive cells indicating that they are part of the ductular reaction. Expression of catalase and Hsp70 decreased in both groups when compared to normal human liver. Mass spectrometric analysis revealed a total of 778 carbonylated proteins. Of these, 194 were common to all groups, 124 unique to tissue prepared from healthy individuals, 357 proteins exclusive to NASH, 124 proteins distinct to samples from patients with fNASH and 178 unique to nfNASH. Using functional enrichment analysis of hepatic carbonylated proteins revealed a propensity for increased carbonylation of proteins regulating cholesterol and Huntington's disease related pathways occurred in nfNASH. Examining fNASH, increased carbonylation was evident in proteins regulating Rho cytoskeletal pathways, nicotinic acetylcholine receptor signaling and chemokine/cytokine inflammatory pathways. Using LC-MS/MS analysis and trypsin digests, sites of carbonylation were identified on peptides isolated from vimentin, endoplasmin and serum albumin in nfNASH and fNASH respectively.

Conclusions: These results indicate that cellular factors regulating mechanisms of protein carbonylation may be different depending on pathological diagnosis of NASH. Furthermore these studies are the first to use LC-MS/MS analysis of carbonylated proteins in human NAFLD and explore possible mechanistic links with end stage cirrhosis due to fatty liver disease and the generation of reactive aldehydes.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704928PMC
December 2017

Serelaxin improves cardiac and renal function in DOCA-salt hypertensive rats.

Sci Rep 2017 08 29;7(1):9793. Epub 2017 Aug 29.

Renal Diseases and Hypertension, School of Medicine, University of Colorado ANSCHUTZ MEDICAL CAMPUS, Aurora, Colorado, 80045, USA.

Serelaxin, a recombinant form of the naturally occurring peptide hormone relaxin-2, is a pleiotropic vasodilating hormone that has been studied in patients with acute heart failure. In this study, the effects of serelaxin on cardiac and renal function, fibrosis, inflammation and lipid accumulation were studied in DOCA-salt treated rats. Uninephrectomized rats were assigned to two groups: controls provided with normal drinking water and DOCA provided with DOCA pellets and sodium chloride drinking water. After 4 weeks, the DOCA-salt rats were randomly selected and implanted with osmotic minipumps delivering vehicle or serelaxin for another 4 weeks. Treatment with serelaxin prevented cardiac and renal dysfunction in DOCA-salt rats. Serelaxin prevented cardiac and renal fibrosis, as determined by Picrosirius Red staining and Second Harmonic Generation (SHG) Microscopy. Treatment of DOCA-salt rats with serelaxin decreased renal inflammation, including the expression of TGF-β, NFκB, MCP-1, IL-1, IL-6, ICAM-1, VCAM-1 and CD68 macrophages. Serelaxin also decreased lipid accumulation in kidney in part by decreasing SREBP-1c, SREBP-2, ChREBP, FATP1, HMGCoAR, and LDL receptor, and increasing Acox1 and ABCA1. In summary, serelaxin reversed DOCA-salt induced cardiac and renal dysfunction.
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http://dx.doi.org/10.1038/s41598-017-09470-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574886PMC
August 2017

Chronic Ethanol Metabolism Inhibits Hepatic Mitochondrial Superoxide Dismutase via Lysine Acetylation.

Alcohol Clin Exp Res 2017 Oct 14;41(10):1705-1714. Epub 2017 Sep 14.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Aurora, Colorado.

Background: Chronic ethanol (EtOH) consumption is a major cause of liver disease worldwide. Oxidative stress is a known consequence of EtOH metabolism and is thought to contribute significantly to alcoholic liver disease (ALD). Therefore, elucidating pathways leading to sustained oxidative stress and downstream redox imbalances may reveal how EtOH consumption leads to ALD. Recent studies suggest that EtOH metabolism impacts mitochondrial antioxidant processes through a number of proteomic alterations, including hyperacetylation of key antioxidant proteins.

Methods: To elucidate mechanisms of EtOH-induced hepatic oxidative stress, we investigate a role for protein hyperacetylation in modulating mitochondrial superoxide dismutase (SOD2) structure and function in a 6-week Lieber-DeCarli murine model of EtOH consumption. Our experimental approach includes immunoblotting immunohistochemistry (IHC), activity assays, mass spectrometry, and in silico modeling.

Results: We found that EtOH metabolism significantly increased the acetylation of SOD2 at 2 functionally relevant lysine sites, K68 and K122, resulting in a 40% decrease in enzyme activity while overall SOD2 abundance was unchanged. In vitro studies also reveal which lysine residues are more susceptible to acetylation. IHC analysis demonstrates that SOD2 hyperacetylation occurs near zone 3 within the liver, which is the main EtOH-metabolizing region of the liver.

Conclusions: Overall, the findings presented in this study support a role for EtOH-induced lysine acetylation as an adverse posttranslational modification within the mitochondria that directly impacts SOD2 charge state and activity. Last, the data presented here indicate that protein hyperacetylation may be a major factor contributing to an imbalance in hepatic redox homeostasis due to chronic EtOH metabolism.
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http://dx.doi.org/10.1111/acer.13473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626652PMC
October 2017

Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase.

Cell Rep 2017 06;19(13):2771-2781

Microbiology & Immunology, Montana State University, Bozeman, MT 59718, USA. Electronic address:

Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1) disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr), respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null) causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.
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http://dx.doi.org/10.1016/j.celrep.2017.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730093PMC
June 2017

Cystathionine beta-synthase deficiency alters hepatic phospholipid and choline metabolism: Post-translational repression of phosphatidylethanolamine N-methyltransferase is a consequence rather than a cause of liver injury in homocystinuria.

Mol Genet Metab 2017 04 2;120(4):325-336. Epub 2017 Mar 2.

Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

Classical homocystinuria (HCU) due to inactivating mutation of cystathionine β-synthase (CBS) is a poorly understood life-threatening inborn error of sulfur metabolism. A previously described cbs-/- mouse model exhibits a semi-lethal phenotype due to neonatal liver failure. The transgenic HO mouse model of HCU exhibits only mild liver injury and recapitulates multiple aspects of the disease as it occurs in humans. Disruption of the methionine cycle in HCU has the potential to impact multiple aspect of phospholipid (PL) metabolism by disruption of both the Kennedy pathway and phosphatidylethanolamine N-methyltransferase (PEMT) mediated synthesis of phosphatidylcholine (PC). Comparative metabolomic analysis of HO mouse liver revealed decreased levels of choline, and choline phosphate indicating disruption of the Kennedy pathway. Alterations in the relative levels of multiple species of PL included significant increases in PL degradation products consistent with enhanced membrane PL turnover. A significant decrease in PC containing 20:4n6 which primarily formed by the methylation of phosphatidylethanolamine to PC was consistent with decreased flux through PEMT. Hepatic expression of PEMT in both the cbs-/- and HO models is post-translationally repressed with decreased levels of PEMT protein and activity that inversely-correlates with the scale of liver injury. Failure to induce further repression of PEMT in HO mice by increased homocysteine, methionine and S-adenosylhomocysteine or depletion of glutathione combined with examination of multiple homocysteine-independent models of liver injury indicated that repression of PEMT in HCU is a consequence rather than a cause of liver injury. Collectively, our data show significant alteration of a broad range of hepatic PL and choline metabolism in HCU with the potential to contribute to multiple aspects of pathogenesis in this disease.
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http://dx.doi.org/10.1016/j.ymgme.2017.02.010DOI Listing
April 2017

Transcriptomic analysis and plasma metabolomics in Aldh16a1-null mice reveals a potential role of ALDH16A1 in renal function.

Chem Biol Interact 2017 Oct 28;276:15-22. Epub 2017 Feb 28.

Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT 06520, United States. Electronic address:

ALDH16A1 is a novel member of the ALDH superfamily that is enzymatically-inactive and highly expressed in the kidney. Recent studies identified an association between a rare missense single nucleotide variant (SNV) in the ALDH16A1 gene and elevated serum uric acid levels and gout. The present study explores the mechanisms by which ALDH16A1 influences uric acid homeostasis in the kidney. We generated and validated a mouse line with global disruption of the Aldh16a1 gene through gene targeting and performed RNA-seq analyses in the kidney of wild-type (WT) and Aldh16a1 knockout (KO) mice, along with plasma metabolomics. We found that ALDH16A1 is expressed in proximal and distal convoluted tubule cells in the cortex of the kidney and in zone 3 hepatocytes. RNA-seq and gene ontology enrichment analyses showed that cellular lipid and lipid metabolic processes are up-regulated. Three transporters localized in the apical membrane of the proximal convoluted tubule of the kidney known to influence urate/uric acid homeostasis were found to be up-regulated (Abcc4, Slc16a9) or down-regulated (Slc17a3). An initial metabolomics analysis in plasma revealed an altered lipid profile in KO mice that is in agreement with our RNA-seq analysis. This is the first study demonstrating a functional role of ALDH16A1 in the kidney.
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http://dx.doi.org/10.1016/j.cbi.2017.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725231PMC
October 2017

Cutaneous exposure to vesicant phosgene oxime: Acute effects on the skin and systemic toxicity.

Toxicol Appl Pharmacol 2017 02 11;317:25-32. Epub 2017 Jan 11.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.

Phosgene Oxime (CX), an urticant or nettle agent categorized as a vesicant, is a potential chemical warfare and terrorist weapon. Its exposure can result in widespread and devastating effects including high mortality due to its fast penetration and ability to cause immediate severe cutaneous injury. It is one of the least studied chemical warfare agents with no effective therapy available. Thus, our goal was to examine the acute effects of CX following its cutaneous exposure in SKH-1 hairless mice to help establish a relevant injury model. Results from our study show that topical cutaneous exposure to CX vapor causes blanching of exposed skin with an erythematous ring, necrosis, edema, mild urticaria and erythema within minutes after exposure out to 8h post-exposure. These clinical skin manifestations were accompanied with increases in skin thickness, apoptotic cell death, mast cell degranulation, myeloperoxidase activity indicating neutrophil infiltration, p53 phosphorylation and accumulation, and an increase in COX-2 and TNFα levels. Topical CX-exposure also resulted in the dilatation of the peripheral vessels with a robust increase in RBCs in vessels of the liver, spleen, kidney, lungs and heart tissues. These events could cause a drop in blood pressure leading to shock, hypoxia and death. Together, this is the first report on effects of CX cutaneous exposure, which could help design further comprehensive studies evaluating the acute and chronic skin injuries from CX topical exposure and elucidate the related mechanism of action to aid in the identification of therapeutic targets and mitigation of injury.
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http://dx.doi.org/10.1016/j.taap.2017.01.003DOI Listing
February 2017

Cytochrome bd-Dependent Bioenergetics and Antinitrosative Defenses in Salmonella Pathogenesis.

mBio 2016 12 20;7(6). Epub 2016 Dec 20.

Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA

In the course of an infection, Salmonella enterica occupies diverse anatomical sites with various concentrations of oxygen (O) and nitric oxide (NO). These diatomic gases compete for binding to catalytic metal groups of quinol oxidases. Enterobacteriaceae express two evolutionarily distinct classes of quinol oxidases that differ in affinity for O and NO as well as stoichiometry of H translocated across the cytoplasmic membrane. The investigations presented here show that the dual function of bacterial cytochrome bd in bioenergetics and antinitrosative defense enhances Salmonella virulence. The high affinity of cytochrome bd for O optimizes respiratory rates in hypoxic cultures, and thus, this quinol oxidase maximizes bacterial growth under O-limiting conditions. Our investigations also indicate that cytochrome bd, rather than cytochrome bo, is an intrinsic component of the adaptive antinitrosative toolbox of Salmonella Accordingly, induction of cytochrome bd helps Salmonella grow and respire in the presence of inhibitory NO. The combined antinitrosative defenses of cytochrome bd and the flavohemoglobin Hmp account for a great part of the adaptations that help Salmonella recover from the antimicrobial activity of NO. Moreover, the antinitrosative defenses of cytochrome bd and flavohemoglobin Hmp synergize to promote Salmonella growth in systemic tissues. Collectively, our investigations indicate that cytochrome bd is a critical means by which Salmonella resists the nitrosative stress that is engendered in the innate response of mammalian hosts while it concomitantly allows for proper O utilization in tissue hypoxia.

Importance: It is becoming quite apparent that metabolism is critically important to the virulence potential of pathogenic microorganisms. Bacterial cells use a variety of terminal electron acceptors to power electron transport chains and metabolic processes. Of all the electron acceptors available to bacteria, utilization of O yields the most energy while diversifying the type of substrates that a pathogen can use. Recent investigations have demonstrated important roles for bd-type quinol oxidases with high affinity for O in bacterial pathogenesis. The investigations presented here have revealed that cytochrome bd potentiates virulence of a clinically relevant bacterial pathogen by fueling bioenergetics of prokaryotic cells while protecting the respiratory chain against NO toxicity. The adaptive antinitrosative defenses afforded by cytochrome bd synergize with other NO-detoxifying systems to preserve cellular bioenergetics, thereby promoting bacterial virulence in tissue hypoxia.
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http://dx.doi.org/10.1128/mBio.02052-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5181779PMC
December 2016

Catalase deletion promotes prediabetic phenotype in mice.

Free Radic Biol Med 2017 02 8;103:48-56. Epub 2016 Dec 8.

Department of Environmental Health Services, Yale School of Public Health, Yale University, 60 College St, New Haven CT 06520-8034, USA. Electronic address:

Hydrogen peroxide is produced endogenously and can be toxic to living organisms by inducing oxidative stress and cell damage. However, it has also been identified as a signal transduction molecule. By metabolizing hydrogen peroxide, catalase protects cells and tissues against oxidative damage and may also influence signal transduction mechanisms. Studies suggest that acatalasemic individuals (i.e., those with very low catalase activity) have a higher risk for the development of diabetes. We now report catalase knockout (Cat) mice, when fed a normal (6.5% lipid) chow, exhibit an obese phenotype that manifests as an increase in body weight that becomes more pronounced with age. The mice demonstrate altered hepatic and muscle lipid deposition, as well as increases in serum and hepatic triglycerides (TGs), and increased hepatic transcription and protein expression of PPARγ. Liver morphology revealed steatosis with inflammation. Cat mice also exhibited pancreatic morphological changes that correlated with impaired glucose tolerance and increased fasting serum insulin levels, conditions consistent with pre-diabetic status. RNA-seq analyses revealed a differential expression of pathways and genes in Cat mice, many of which are related to metabolic syndrome, diabetes, and obesity, such as Pparg and Cidec. In conclusion, the results of the present study show mice devoid of catalase develop an obese, pre-diabetic phenotype and provide compelling evidence for catalase (or its products) being integral in metabolic regulation.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513671PMC
February 2017

Role of p53 in silibinin-mediated inhibition of ultraviolet B radiation-induced DNA damage, inflammation and skin carcinogenesis.

Carcinogenesis 2017 01 11;38(1):40-50. Epub 2016 Oct 11.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences,

Non-melanoma skin cancers (NMSC) are a growing problem given that solar ultraviolet B (UVB) radiation exposure is increasing most likely due to depletion of the atmospheric ozone layer and lack of adequate sun protection. Better preventive methods are urgently required to reduce UV-caused photodamage and NMSC incidence. Earlier, we have reported that silibinin treatment activates p53 and reduces photodamage and NMSC, both in vitro and in vivo; but whether silibinin exerts its protective effects primarily through p53 remains unknown. To address this question, we generated p53 heterozygous (p53) and p53 knockout (p53) mice on SKH-1 hairless mouse background, and assessed silibinin efficacy in both short- and long-term UVB exposure experiments. In the chronic UVB-exposed skin tumorigenesis study, compared to p53 mice, p53 mice developed skin tumors earlier and had higher tumor number, multiplicity and volume. Silibinin topical treatment significantly reduced the tumor number, multiplicity and volume in p53 mice but silibinin' protective efficacy was significantly compromised in p53 mice. Additionally, silibinin treatment failed to inhibit precursor skin cancer lesions in p53 mice but improved the survival of the mice. In short-term studies, silibinin application accelerated the removal of UVB-induced DNA damage in p53 mice while its efficacy was partially compromised in p53 mice. Interestingly, silibinin treatment also inhibited the UVB-induced inflammatory markers in skin tissue. These results further confirmed that absence of the p53 allele predisposes mice to photodamage and photocarcinogenesis, and established that silibinin mediates its protection against UVB-induced photodamage, inflammation and photocarcinogenesis partly through p53 activation.
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http://dx.doi.org/10.1093/carcin/bgw106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5219048PMC
January 2017

Sevelamer Improves Steatohepatitis, Inhibits Liver and Intestinal Farnesoid X Receptor (FXR), and Reverses Innate Immune Dysregulation in a Mouse Model of Non-alcoholic Fatty Liver Disease.

J Biol Chem 2016 10 7;291(44):23058-23067. Epub 2016 Sep 7.

From the Departments of Gastroenterology and Hepatology,

Bile acid sequestrants are synthetic polymers that bind bile acids in the gut and are used to treat dyslipidemia and hyperphosphatemia. Recently, these agents have been reported to lower blood glucose and increase insulin sensitivity by altering bile acid signaling pathways. In this study, we assessed the efficacy of sevelamer in treating mice with non-alcoholic fatty liver disease (NAFLD). We also analyzed how sevelamer alters inflammation and bile acid signaling in NAFLD livers. Mice were fed a low-fat or Western diet for 12 weeks followed by a diet-plus-sevelamer regimen for 2 or 12 weeks. At the end of treatment, disease severity was assessed, hepatic leukocyte populations were examined, and expression of genes involved in farnesoid X receptor (FXR) signaling in the liver and intestine was analyzed. Sevelamer treatment significantly reduced liver steatosis and lobular inflammation. Sevelamer-treated NAFLD livers had notably fewer pro-inflammatory infiltrating macrophages and a significantly greater fraction of alternatively activated Kupffer cells compared with controls. Expression of genes involved in FXR signaling in the liver and intestine was significantly altered in mice with NAFLD as well as in those treated with sevelamer. In a mouse model of NAFLD, sevelamer improved disease and counteracted innate immune cell dysregulation in the liver. This study also revealed a dysregulation of FXR signaling in the liver and intestine of NAFLD mice that was counteracted by sevelamer treatment.
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http://dx.doi.org/10.1074/jbc.M116.731042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087726PMC
October 2016

Prolonged feeding with guanidinoacetate, a methyl group consumer, exacerbates ethanol-induced liver injury.

World J Gastroenterol 2016 Oct;22(38):8497-8508

Natalia A Osna, Dan Feng, Murali Ganesan, Dean J Tuma, Kusum K Kharbanda, Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, NE 68105, United States.

Aim: To investigate the hypothesis that exposure to guanidinoacetate (GAA, a potent methyl-group consumer) either alone or combined with ethanol intake for a prolonged period of time would cause more advanced liver pathology thus identifying methylation defects as the initiator and stimulator for progressive liver damage.

Methods: Adult male Wistar rats were fed the control or ethanol Lieber DeCarli diet in the absence or presence of GAA supplementation. At the end of 6 wk of the feeding regimen, various biochemical and histological analyses were conducted.

Results: Contrary to our expectations, we observed that GAA treatment alone resulted in a histologically normal liver without evidence of hepatosteatosis despite persistence of some abnormal biochemical parameters. This protection could result from the generation of creatine from the ingested GAA. Ethanol treatment for 6 wk exhibited changes in liver methionine metabolism and persistence of histological and biochemical defects as reported before. Further, when the rats were fed the GAA-supplemented ethanol diet, similar histological and biochemical changes as observed after 2 wk of combined treatment, including inflammation, macro- and micro-vesicular steatosis and a marked decrease in the methylation index were noted. In addition, rats on the combined treatment exhibited increased liver toxicity and even early fibrotic changes in a subset of animals in this group. The worsening liver pathology could be related to the profound reduction in the hepatic methylation index, an increased accumulation of GAA and the inability of creatine generated to exert its hepato-protective effects in the setting of ethanol.

Conclusion: To conclude, prolonged exposure to a methyl consumer superimposed on chronic ethanol consumption causes persistent and pronounced liver damage.
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http://dx.doi.org/10.3748/wjg.v22.i38.8497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064031PMC
October 2016

Perilipin-2 Deletion Impairs Hepatic Lipid Accumulation by Interfering with Sterol Regulatory Element-binding Protein (SREBP) Activation and Altering the Hepatic Lipidome.

J Biol Chem 2016 Nov 27;291(46):24231-24246. Epub 2016 Sep 27.

From the Integrated Physiology Graduate Program,

Perilipin-2 (PLIN2) is a constitutively associated cytoplasmic lipid droplet coat protein that has been implicated in fatty liver formation in non-alcoholic fatty liver disease. Mice with or without whole-body deletion of perilipin-2 (Plin2-null) were fed either Western or control diets for 30 weeks. Perilipin-2 deletion prevents obesity and insulin resistance in Western diet-fed mice and dramatically reduces hepatic triglyceride and cholesterol levels in mice fed Western or control diets. Gene and protein expression studies reveal that PLIN2 deletion suppressed SREBP-1 and SREBP-2 target genes involved in de novo lipogenesis and cholesterol biosynthetic pathways in livers of mice on either diet. GC-MS lipidomics demonstrate that this reduction correlated with profound alterations in the hepatic lipidome with significant reductions in both desaturation and elongation of hepatic neutral lipid species. To examine the possibility that lipidomic actions of PLIN2 deletion contribute to suppression of SREBP activation, we isolated endoplasmic reticulum membrane fractions from long-term Western diet-fed wild type (WT) and Plin2-null mice. Lipidomic analyses reveal that endoplasmic reticulum membranes from Plin2-null mice are markedly enriched in ω-3 and ω-6 long-chain polyunsaturated fatty acids, which others have shown inhibit SREBP activation and de novo lipogenesis. Our results identify PLIN2 as a determinant of global changes in the hepatic lipidome and suggest the hypothesis that these actions contribute to SREBP-regulated de novo lipogenesis involved in non-alcoholic fatty liver disease.
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http://dx.doi.org/10.1074/jbc.M116.759795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104945PMC
November 2016

Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis.

Kidney Int 2016 11 21;90(5):1123-1128. Epub 2016 Aug 21.

Department of Medicine, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA.

All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. Here we develop a fast and operator-independent method to measure fibrosis utilizing the murine unilateral ureteral obstruction model which manifests a time-dependent fibrotic increase in obstructed kidneys while the contralateral kidneys are used as controls. After ureteral obstruction, kidneys were analyzed at 7, 14, and 21 days. Fibrosis was quantified using fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) in a Deep Imaging via Enhanced photon Recovery deep tissue imaging microscope. This microscope was developed for deep tissue along with second and third harmonic generation imaging and has extraordinary sensitivity toward harmonic generation. SHG data suggest the presence of more fibrillar collagen in the obstructed kidneys. The combination of short-wavelength FLIM and SHG analysis results in a robust assessment procedure independent of observer interpretation and let us create criteria to quantify the extent of fibrosis directly from the image. Thus, the FLIM-SHG technique shows remarkable improvement in quantification of renal fibrosis compared to standard histological techniques.
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http://dx.doi.org/10.1016/j.kint.2016.06.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473685PMC
November 2016

Xanthine oxidoreductase mediates membrane docking of milk-fat droplets but is not essential for apocrine lipid secretion.

J Physiol 2016 10 3;594(20):5899-5921. Epub 2016 Aug 3.

Division of Reproductive Sciences, Department of Obstetrics and Gynecology, School of Medicine, University of Colorado Anschutz Medical Center, Aurora, CO, 80045, USA.

Key Points: Xanthine oxidoreductase (XOR) modulates milk lipid secretion and lactation initiation. XOR is required for butyrophilin1a1 clustering in the membrane during milk lipid secretion. XOR mediates apical membrane reorganization during milk lipid secretion. Loss of XOR delays milk fat globule secretion. XOR loss alters the proteome of milk fat globules.

Abstract: Apocrine secretion is utilized by epithelial cells of exocrine glands. These cells bud off membrane-bound particles into the lumen of the gland, losing a portion of the cytoplasm in the secretion product. The lactating mammary gland secretes milk lipid by this mechanism, and xanthine oxidoreductase (XOR) has long been thought to be functionally important. We generated mammary-specific XOR knockout (MGKO) mice, expecting lactation to fail. Histology of the knockout glands showed very large lipid droplets enclosed in the mammary alveolar cells, but milk analysis showed that these large globules were secreted. Butyrophilin, a membrane protein known to bind to XOR, was clustered at the point of contact of the cytoplasmic lipid droplet with the apical plasma membrane, in the wild-type gland but not in the knockout, suggesting that XOR mediates 'docking' to this membrane. Secreted milk fat globules were isolated from mouse milk of wild-type and XOR MGKO dams, and subjected to LC-MS/MS for analysis of protein component. Proteomic results showed that loss of XOR leads to an increase in cytoplasmic, cytoskeletal, Golgi apparatus and lipid metabolism proteins associated with the secreted milk fat globule. Association of XOR with the lipid droplet results in membrane docking and more efficient retention of cytoplasmic components by the secretory cell. Loss of XOR then results in a reversion to a more rudimentary, less efficient, apocrine secretion mechanism, but does not prevent milk fat globule secretion.
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http://dx.doi.org/10.1113/JP272390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063925PMC
October 2016