Dual oxidase 1 induced by Th2 cytokines promotes STAT6 phosphorylation via oxidative inactivation of protein tyrosine phosphatase 1B in human epidermal keratinocytes

Although hydrogen peroxide (H(2)O(2)) is better known for its cytotoxic effects, in recent years it has been shown to play a crucial role in eukaryotic signal transduction. In respiratory tract epithelial cells, the dual oxidase (DUOX) proteins 1 and 2 has been identified as the cellular source of H(2)O(2). However, the expression of DUOX1 or DUOX2 has not yet been examined in keratinocytes. In this study, using a DNA microarray, we demonstrated that, of the seven NOX/DUOX family members in normal human epidermal keratinocytes (NHEK), IL-4/IL-13 treatment augments the expression of only DUOX1 mRNA. We next confirmed the IL-4/IL-13 induction of DUOX1 in NHEK at the mRNA and protein level using quantitative real-time PCR and Western blotting, respectively. In addition, we demonstrated that this augmented DUOX1 expression was accompanied by increased H(2)O(2) production, which was significantly suppressed both by diphenyleneiodonium, an inhibitor of NADPH oxidase, and by small interfering RNA against DUOX1. Finally, we demonstrated that the increased expression of DUOX1 in IL-4/IL-13-treated NHEK augments STAT6 phosphorylation via oxidative inactivation of protein tyrosine phosphatase 1B. These results revealed a novel role of IL-4/IL-13-induced DUOX1 expression in making a positive feedback loop for IL-4/IL-13 signaling in keratinocytes.     

Mice deficient in dual oxidase maturation factors are severely hypothyroid

Dual oxidases (DUOX1 and DUOX2) are evolutionary conserved reduced nicotinamide adenine dinucleotide phosphate oxidases responsible for regulated hydrogen peroxide (H(2)O(2)) release of epithelial cells. Specific maturation factors (DUOXA1 and DUOXA2) are required for targeting of functional DUOX enzymes to the cell surface. Mutations in the single-copy Duox and Duoxa genes of invertebrates cause developmental defects with reduced survival, whereas knockdown in later life impairs intestinal epithelial immune homeostasis. In humans, mutations in both DUOX2 and DUOXA2 can cause congenital hypothyroidism with partial iodide organification defects compatible with a role of DUOX2-generated H(2)O(2) in driving thyroid peroxidase activity. The DUOX1/DUOXA1 system may account for residual iodide organification in patients with loss of DUOX2, but its physiological function is less clear. To provide a murine model recapitulating complete DUOX deficiency, we simultaneously targeted both Duoxa genes by homologous recombination. Knockout of Duoxa genes (Duoxa(-/-) mice) led to a maturation defect of DUOX proteins lacking Golgi processing of N-glycans and to loss of H(2)O(2) release from thyroid tissue. Postnatally, Duoxa(-/-) mice developed severe goitreous congenital hypothyroidism with undetectable serum T4 and maximally disinhibited TSH levels. Heterozygous mice had normal thyroid function parameters. (125)I uptake and discharge studies and probing of iodinated TG epitopes corroborated the iodide organification defect in Duoxa(-/-) mice. Duoxa(-/-) mice on continuous T4 replacement from P6 showed normal growth without an overt phenotype. Our results confirm in vivo the requirement of DUOXA for functional expression of DUOX-based reduced nicotinamide adenine dinucleotide phosphate oxidases and the role of DUOX isoenzymes as sole source of hormonogenic H(2)O(2).     

The type of DUOX-dependent ROS production is dictated by defined sequences in DUOXA

A deliberate generation of ROS is now recognized to be achieved by specific NADPH oxidases (NOX). Dual oxidases (DUOXs) are Ca(2+)-activated NOXs and operate as H(2)O(2)-generators in various tissues. A tight regulation is however required to avoid ROS overproduction that can rapidly be harmful to biological systems. DUOX activator (DUOXA) proteins act as organizing elements for surface expression and activity of the DUOX enzymes. To study DUOX activation by the maturation factors, chimeric DUOXA proteins were generated by replacing particular domains between DUOXA1 and DUOXA2. Their impact on DUOX function and membrane expression were explored in a reconstituted heterologous cell system composed of COS-7 cells. We have shown that the COOH-terminal end of DUOXA1 is responsible for DUOX1-dependent H(2)O(2) generation. The NH(2)-terminal tail of DUOXA2 is critical to specify the type of ROS released by DUOX2, hydrogen peroxide or superoxide. Native DUOXA2 would constrain DUOX2 to produce H(2)O(2). However, alterations of the DUOXA2 NH(2)-terminal domain modify DUOX2 activity triggering superoxide leaking. Our results demonstrate that specific domains of the DUOX maturation factors promote the activation of DUOXs as well as the type of ROS generated by the oxidases.     

Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity

Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.     

Developmental regulation of claudin localization by fetal alveolar epithelial cells

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.     

Identification of the maturation factor for dual oxidase. Evolution of an eukaryotic operon equivalent

Dual oxidase 2 (DUOX2), an NADPH:O(2) oxidoreductase flavoprotein, is a component of the thyroid H(2)O(2) generator crucial for hormone synthesis at the apical membrane. Mutations in DUOX2 produce congenital hypothyroidism in humans. However, no functional DUOX-based NADPH oxidase has ever been reconstituted at the plasma membrane of transfected cells. It has been proposed that DUOX retention in the endoplasmatic reticulum (ER) of heterologous systems is due to the lack of an unidentified component required for functional maturation of the enzyme. By data mining of a massively parallel signature sequencing tissue expression data base, we identified an uncharacterized gene named DUOX maturation factor (DUOXA2) arranged head-to-head to and co-expressed with DUOX2. A paralog (DUOXA1) was similarly linked to DUOX1. The genomic rearrangement leading to linkage of ancient DUOX and DUOXA genes could be traced back before the divergence of echinoderms. We demonstrate that co-expression of DUOXA2, an ER-resident transmembrane protein, allows ER-to-Golgi transition, maturation, and translocation to the plasma membrane of functional DUOX2 in a heterologous system. The identification of DUOXA genes has important implications for studies of the molecular mechanisms controlling DUOX expression and the molecular genetics of congenital hypothyroidism.     

Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production

NADPH oxidases are major sources of superoxide (O2*-) and hydrogen peroxide (H2O2) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation and differentiation, while ROS overproduction has been implicated in hypertension and atherosclerosis. It is known that the heme-containing catalytic subunits Nox1 and Nox4 are responsible for oxygen reduction in vascular smooth muscle cells from large arteries. However, the exact mechanism of ROS production by NADPH oxidases is not completely understood. We hypothesized that Nox1 and Nox4 play distinct roles in basal and angiotensin II (AngII)-stimulated production of O2*- and H2O2. Nox1 and Nox4 expression in rat aortic smooth muscle cells (RASMCs) was selectively reduced by treatment with siNox4 or antisense Nox1 adenovirus. Production of O2*- and H2O2 in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was measured by NADPH-dependent O2*- and H2O2 production using electron spin resonance (ESR) and 1-hydroxy-3-carboxypyrrolidine (CPH) in the membrane fraction in the absence of cytosolic superoxide dismutase. It was found that production of O2*- by quiescent RASMC NADPH oxidases was five times less than H2O2 production. Stimulation of cells with AngII led to a 2-fold increase of O2*- production by NADPH oxidases, with a small 15 to 30% increase in H2O2 formation. Depletion of Nox4 in RASMCs led to diminished basal H2O2 production, but did not affect O2*- or H2O2 production stimulated by AngII. In contrast, depletion of Nox1 in RASMCs inhibited production of O2*- and AngII-stimulated H2O2 in the membrane fraction and intact cells. Our data suggest that Nox4 produces mainly H2O2, while Nox1 generates mostly O2*- that is later converted to H2O2. Therefore, Nox4 is responsible for basal H2O2 production, while O2*- production in nonstimulated and AngII-stimulated cells depends on Nox1. The difference in the products generated by Nox1 and Nox4 may help to explain the distinct roles of these NADPH oxidases in cell signaling. These findings also provide important insight into the origin of H2O2 in vascular cells, and may partially account for the limited pharmacological effect of antioxidant treatments with O2*- scavengers that do not affect H2O2.     

NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H(2)O(2)) increases endothelial nitric oxide (NO(.)) production. The current study is designed to determine whether endogenous levels of H(2)O(2) are ever sufficient to stimulate NO(.) production in intact endothelial cells. NO(.) production was detected by a NO(.)-specific microelectrode or by an electron spin resonance spectroscopy using Fe(2+)-(DETC)(2) as a NO(.)-specific spin trap. The addition of H(2)O(2) to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO(.) release. Incubation with angiotensin II (10(-7) mol) elevated intracellular H(2)O(2) levels, which were attenuated with PEG-catalase. Angiotensin II increased NO(.) production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H(2)O(2) in this response(.) In contrast, NO(.) production evoked by either bradykinin or calcium ionophore was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO(.) production in aortic endothelial cells from C57BL/6 mice but had no effect on NO(.) production in endothelial cells from p47(phox-/-) mice. In contrast, stimulated NO(.) production to a similar extent in endothelial cells from wild-type and p47(phox-/-) mice. In summary, the present study provides direct evidence that endogenous H(2)O(2), derived from the NAD(P)H oxidase, mediates endothelial NO(.) production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O(2)() production from the NAD(P)H oxidase, the H(2)O(2) stimulation of NO(.) may facilitate peroxynitrite formation in response to this octapeptide.     

Alveolar epithelial changes in rabbits after a 21-day exposure to 60% O2

This study characterizes the biochemical and physiological effects of prolonged exposure of rabbits to sublethal (60%) O2 concentrations. After 3 wk in 60% O2, rabbits had arterial PO2 values of 69 +/- 2 vs. 79 +/- 3 Torr for control animals (means +/- SE; P less than 0.05) and a small but significant rise in pulmonary wet weight-to-dry weight ratios (5.6 +/- 0.3 vs. 4.1 +/- 0.3; P less than 0.05). Alveolar permeability to solute, lung compliance, total lung capacity, and alveolar protein levels were unchanged from control, but the amount of lavagable alveolar phospholipid was 90% higher in the O2-exposed rabbits. The lipid biosynthetic ability of isolated alveolar type II pneumocytes, measured by radiolabeled precursor [3H]choline incorporation, indicated that type II cells isolated from hyperoxic animals synthesized phosphatidylcholine at a rate 110% higher than those from control animals. Laser flow cytometric analyses of isolated type II cells showed a significant increase in type II cell diameter, based on time-of-flight measurements, and an average 60% increase in lipid content per cell, based on phosphine-3R fluorescence intensity. These findings indicate that exposure to 60% O2 for 21 days results in a decrease in arterial PO2 and induces several important biochemical and morphological changes in alveolar type II pneumocytes.     

Missense mutations of dual oxidase 2 (DUOX2) implicated in congenital hypothyroidism have impaired trafficking in cells reconstituted with DUOX2 maturation factor

Dual oxidase 2 (DUOX2), a reduced NAD phosphate:O2 oxidoreductase flavoprotein, is a component of the thyrocyte H2O2 generator required for hormone synthesis at the apical plasma membrane. We recently identified a specific DUOX2 maturation factor (DUOXA2) that is necessary and sufficient for expression of functional DUOX2 in mammalian cell lines. We have now used a DUOXA2 reconstituted system to provide the first characterization of natural DUOX2 missense variants (Q36H, R376W, D506N) at the molecular level, analyzing their impact on H2O2 generation, trafficking, stability, folding, and DUOXA2 interaction. The Q36H and R376W mutations completely prevent routing of DUOX2 to the cell surface. The mutant proteins are predominantly present as core N-glycosylated, thiol-reduced folding intermediates, which are retained by the quality control system within the endoplasmic reticulum (ER) as indicated by increased complexation with the lectin calnexin. D506N displays a partial deficiency phenotype with reduced surface expression of a mutant protein with normal intrinsic activity in generating H2O2. D506N N-glycan moieties are not subject to normal modification in the Golgi apparatus, suggesting that nonnative protein can escape the quality control in the ER. Oxidative folding of DUOX2 in the ER appears to be the rate-limiting step in the maturation of DUOX2, but is not facilitated by DUOXA2. Rather, DUOXA2 allows rapid ER exit of folded DUOX2 or enhanced degradation of mutant DUOX2 proteins not competent for ER exit. DUOXA2 may thus be part of a secondary quality control system specific for DUOX2.     

Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development

Apoptosis plays a central role in the cellular remodeling of the developing lung. We determined the spatiotemporal patterns of the cell death regulators Fas and Fas ligand (FasL) during rabbit lung development and correlated their expression with pulmonary and type II cell apoptosis. Fetal rabbit lungs (25-31 days gestation) were assayed for apoptotic activity by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and DNA size analysis. Fas and FasL expression were analyzed by RT-PCR, immunoblot, and immunohistochemistry. Type II cell apoptosis increased significantly on gestational day 28; the type II cell apoptotic index increased from 0.54 +/- 0.34% on gestational day 27 to 3.34 +/- 1.24% on day 28, P < 0.01 (ANOVA). This corresponded with the transition from the canalicular to the terminal sac stage of development. The day 28 rise in epithelial apoptosis was synchronous with a robust if transient 20-fold increase in FasL mRNA and a threefold increase in FasL protein levels. In contrast, Fas mRNA levels remained constant, suggestive of constitutive expression. Fas and FasL proteins were immunolocalized to alveolar type II cells and bronchiolar Clara cells. The correlation of this highly specific pattern of FasL expression with alveolar epithelial apoptosis and remodeling implicates the Fas/FasL system as a potentially important regulatory pathway in the control of postcanalicular alveolar cytodifferentiation.     

Reactivity of Hg(II) with superoxide: evidence for the catalytic dismutation of superoxide by Hg(II).

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O2-) and hydrogen peroxide (H2O2) production by rat kidney mitochondria, Hg(II) (1-6 microM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H2O2 production but an apparent decrease in mitochondrial O2- production. Hg(II) also inhibited O(2-)-dependent cytochrome c reduction (IC50 approximately 2-3 microM) when O2- was produced from xanthine oxidase. In contrast, Hg(I) did not react with O2- in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O2- by Hg(II). Hg(II) also inhibited the reactions of KO2 (i.e., O2-) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO2 in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO2. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O2- dismutation, leading to increased production of H2O2 by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.     

Concerted generation of Ig isotype diversity in human fetal bone marrow

The human fetal bone marrow B cell compartment of 14- to 21-wk gestational age was examined phenotypically and with respect to Ig H chain commitment and diversity. A dramatic expansion of fetal marrow B cell pools at 16- to 18-wk gestational age characterizes a rapid and concerted chain of differentiation events. Transiently up to 1/4 of nucleated marrow cells are CD20+/CD21+ cells which begin to express surface Ig other than IgM. Limiting dilution analysis of EBV-infected marrow cells delineated a virtually exclusive commitment to IgM production until 15 wk and the absolute and relative number of these cells were small (approximately 5% of comparable adult values). In parallel to the rapid increase in total B cell pools size, cells committed and able to secrete any of the five Ig isotypes are generated by 16-wk gestational age and by 18 wk the frequencies of these cells rapidly reach levels typical for adult peripheral tissue such as blood or lymph node. Fetal L chain diversity always anticipated that observed in adult serum. In addition to rising pool sizes and diverse IgH expression, EBV transformability is a major variable during this period of B cell development with up to 2/3 of B lineage cells transformable, about half of which are pre-B cells. By 21-wk gestational age transformable pre-B cells have disappeared and (as in adult tissue) approximately 10 to 20% of CD20+ cells are transformable. The rapid, concerted expression of full H chain diversity during a narrow period in fetal development is unique to marrow and implies a lymphopoietic process in a privileged site rather than an immunologic differentiation event. During this event, the relative proportions between the different IgH classes expressed, resembled that found in adult tissue, perhaps suggesting that B cell inherent programming rather than only antigenic forces determine heavy chain choice. The staggered expression, early in postnatal life, of IgH regions 3' of the C mu locus may reflect regulatory functions rather than inherent immaturity of the B lineage.     

cAMP-response element-binding protein mediates acid-induced NADPH oxidase NOX5-S expression in Barrett esophageal adenocarcinoma cells

Gastroesophageal reflux disease complicated by Barrett esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not known. We found that NOX1 and NOX5-S were the major isoforms of NADPH oxidase in SEG1-EA cells. The expression of NOX5-S mRNA was significantly higher in these cells than in esophageal squamous epithelial cells. NOX5 mRNA was also significantly higher in Barrett tissues with high grade dysplasia than without dysplasia. Pulsed acid treatment significantly increased H(2)O(2) production in both SEG1-EA cells and BE mucosa, which was blocked by the NADPH oxidase inhibitor apocynin. In SEG1 cells, acid treatment increased mRNA expression of NOX5-S, but not NOX1, and knockdown of NOX5 by NOX5 small interfering RNA abolished acid-induced H(2)O(2) production. In addition, acid treatment increased intracellular Ca(2+) and phosphorylation of cAMP-response element-binding protein (CREB). Acid-induced NOX5-S expression and H(2)O(2) production were significantly inhibited by removal of extracellular Ca(2+) and by knockdown of CREB using CREB small interfering RNA. Two novel CREB-binding elements TGACGAGA and TGACGCTG were identified in the NOX5-S gene promoter. Overexpression of CREB significantly increased NOX5-S promoter activity. Knockdown of NOX5 significantly decreased [(3)H]thymidine incorporation, which was restored by 10(-13) M H(2)O(2). Knockdown of NOX5 also significantly decreased retinoblastoma protein phosphorylation and increased cell apoptosis and caspase-9 expression. In conclusion, in SEG1 EA cells NOX5-S is overexpressed and mediates acid-induced H(2)O(2) production. Acid-induced NOX5-S expression depends on an increase in intracellular Ca(2+) and activation of CREB. NOX5-S contributes to increased cell proliferation and decreased apoptosis.     

High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates

Despite the considerable interest in superoxide as a potential cause of pathology, the mechanisms of its deleterious production by mitochondria remain poorly understood. Previous studies in purified mitochondria have found that the highest rates of superoxide production are observed with succinate-driven reverse-electron transfer through complex I, although the physiological importance of this pathway is disputed because it necessitates high concentrations of succinate and is thought not to occur when NAD is in the reduced state. However, very few studies have examined the rates of superoxide production with mitochondria respiring on both NADH-linked (e.g. glutamate) and complex II-linked substrates. In the present study, we find that the rates of superoxide production (measured indirectly as H2O2) with glutamate+succinate (approximately 1100 pmol of H2O2 x min(-1) x mg(-1)) were unexpectedly much higher than with succinate (approximately 400 pmol of H2O2 x min(-1) x mg(-1)) or glutamate (approximately 80 pmol of H2O2 x min(-1) x mg(-1)) alone. Superoxide production with glutamate+succinate remained high even at low substrate concentrations (<1 mM), was decreased by rotenone and was completely eliminated by FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), indicating that it must in large part originate from reverse-electron transfer through complex I. Similar results were obtained when glutamate was replaced with pyruvate, alpha-ketoglutarate or palmitoyl carnitine. In contrast, superoxide production was consistently lowered by the addition of malate (malate+succinate approximately 30 pmol of H2O2 x min(-1) x mg(-1)). We propose that the inhibitory action of malate on superoxide production can be explained by oxaloacetate inhibition of complex II. In summary, the present results indicate that reverse-electron transfer-mediated superoxide production can occur under physiologically realistic substrate conditions and suggest that oxaloacetate inhibition of complex II may be an adaptive mechanism to minimize this.     

Alveolar macrophage function in rats with severe protein calorie malnutrition. Arachidonic acid metabolism, cytokine release, and antimicrobial activity

To investigate the effects of protein calorie malnutrition (PCM) on alveolar macrophage function, we measured antimicrobial activity, IL-1 and TNF production, and arachidonic acid metabolism in alveolar macrophages of infant rats with moderate and severe PCM. Groups of weanling male rats were fed a diet containing 0.8% protein (PCM) or 24% protein (control). A third group (pair fed) was fed limited amounts of the control diet that matched the mean daily dietary intake of the PCM group. After 4 wk on the diets, alveolar macrophages from all three groups functioned similarly with respect to surface adherence, phagocytosis and killing of Listeria monocytogenes, release of hydrogen peroxide and superoxide anion, and production of IL-1 and TNF. In contrast, Listeria-stimulated alveolar macrophages from the PCM group exhibited a marked shift in arachidonic acid metabolism, with impaired production of leukotriene B4 and enhanced release of thromboxane B2 and PGE2. The membrane arachidonic acid content and the uptake of [3H]arachidonate by alveolar macrophages did not differ among the three groups. The shift toward the cyclooxygenase pathway was not seen after 2 wk of dietary restriction and was reversed if PCM animals were fed the control diet for 1 wk. Thus, PCM does not affect the antimicrobial activity or cytokine production of alveolar macrophages, but causes alterations in arachidonic acid metabolism that may interfere with the modulatory functions of alveolar macrophages.     

Adipose tissue triglyceride turnover, de novo lipogenesis, and cell proliferation in humans measured with 2H2O

The turnover of adipose tissue components (lipids and cells) and the pathways of adipose lipid deposition have been difficult to measure in humans. We apply here a (2)H(2)O long-term labeling technique for concurrent measurement of adipose-triglyceride (TG) turnover, cell (DNA) proliferation, and de novo lipogenesis (DNL). Healthy subjects drank (2)H(2)O (70 ml/day) for 5-9 wk. Subcutaneous adipose tissue aspirates were taken (gluteal, thigh, and flank depots). Deuterium incorporation into TG glycerol (representing all-source TG synthesis), TG palmitate (representing DNL, by mass isotopomer distribution analysis), and DNA (representing cell proliferation) was measured by gas chromatography-mass spectrometry. Subjects tolerated the protocol well, and body (2)H(2)O enrichments were stable. Mean TG-glycerol fractional synthesis was 0.12 (i.e., 12%) with a range of 0.03-0.32 after 5 wk and 0.20 (range 0.08-0.49) after 9 wk (TG half-life 200-270 days). Label decay measurements 5-8 mo after discontinuing (2)H(2)O gave similar turnover estimates. Net lipolysis (TG turnover) was 50-60 g/day. DNL contribution to adipose-TG was 0.04 after 9 wk, representing approximately 20% of newly deposited TG. Cell proliferation was 0.10-0.17 after 9 wk (half-life 240-425 days). In summary, long-term (2)H(2)O administration to human subjects allows measurement of the dynamics of adipose tissue components. Turnover of all elements is slow, and DNL contributes approximately 20% of new TG.