Browsing by Author "Bittman, Robert"

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    DHA Alters Raft-like Membrane Domains as Revealed by Solid State 2H NMR Spectroscopy
    (Office of the Vice Chancellor for Research, 2015-04-17) Kinnun, Jacob J.; Williams, Justin A.; Stillwell, William; Bittman, Robert; Shaikh, Saame Raza; Wassall, Stephen R.
    Dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic acid (DHA, 22:6), are correlated with the prevention of neurological and autoimmune disorders in humans. These fatty acids must be obtained from the diet, such as oil fish or fish oil supplements, as they cannot be generated within the human body. The origin of the health benefits at the molecular level is still under question. A membrane-mediated mechanism in which n-3 PUFAs are incorporated into phospholipids and modulate molecular organization is one possibility. Cellular membranes are inhomogeneous where structurally diverse lipids can exist in separate domains. Regions rich in sphingomyelin (SM) and cholesterol, commonly called lipid rafts, contain important signaling proteins. In a recent solid-state 2H nuclear magnetic resonance (2H NMR) study of a model membrane composed of 1-[2H31] palmitoyl-2-docosahexaenoyl-phosphatidylcholine (PDPC-d31), a deuterated analog of a DHA-containing phospholipid, in mixtures with SM and cholesterol, we discovered that DHA could significantly enter raft-like domains. How DHA affects the molecular organization within the raft-like domains is addressed here by observing PSM-d31, an analog of SM with a perdeuterated N-palmitoyl chain. The 2H NMR spectra for PSM-d31, in mixtures with PDPC and cholesterol, exhibit two spectral components, a larger more ordered component that we attribute to raft-like domains and a smaller less ordered component that we attribute to non-raft-like domains. On average, the order of PSM-d31 is reduced and, thus, disordering of PSM-d31 by PDPC is indicated. Our observations confirm that DHA can infiltrate rafts and affect molecular organization, which has implications for the signaling of raft and non-raft proteins. Furthermore, these results are consistent with in vivo studies showing that DHA infiltrates rafts.
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    DHA and EPA Interaction with Raft Domains Observed With Solid-State 2H NMR Spectroscopy
    (Office of the Vice Chancellor for Research, 2013-04-05) Kinnun, Jacob J.; Williams, Justin A.; Stillwell, William H.; Bittman, Robert; Shaikh, Saame Raza; Wassall, Stephen R.
    Research continues to examine the health benefits of omega-3 polyunsaturated fatty acids (n-3 PUFA) found in fish oils. The major bioactive components are eicosapentaenoic acid (EPA, 20:5), with 20 carbons and 5 double bonds, and docosahexaenoic acid (DHA, 22:6), with 22 carbons and 6 double bonds. However, their molecular modes of action remain unclear. A suggested hypothesis is that these fatty acids are incorporated into membrane phospholipids and modify the structure and organization of lipid rafts, thus affecting cell signaling. We used solid-state 2H NMR spectroscopy to compare molecular organization in mixtures of 1-palmitoyl-2-eicosapentaenoylphosphatidylcholine (PEPC) and 1-palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC) with the raft-stabilizing molecules sphingomyelin (SM) and cholesterol. Our spectra for PEPC-d31 and PDPC-d31, analogs of PEPC and PDPC with a perdeuterated palmitoyl sn-1 chain, showed that DHA has a greater tendency than EPA to incorporate into raft-like domains enriched in SM and cholesterol. By using PSM-d31, an analog of SM with a perdeuterated N-palmitoyl chain, we now directly observe one of the raft-forming molecules and analyze the molecular order within the raft. These results will add to the growing information on how EPA and DHA differentially modify lipid domain organization in bilayers.
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    DHA Modifies the Size and Composition of Raftlike Domains: A Solid-State 2H NMR Study
    (Elsevier, 2018-01-23) Kinnun, Jacob J.; Bittman, Robert; Shaikh, Saame Raza; Wassall, Stephen R.; Physics, School of Science
    Docosahexaenoic acid is an omega-3 polyunsaturated fatty acid that relieves the symptoms of a wide variety of chronic inflammatory disorders. The structural mechanism is not yet completely understood. Our focus here is on the plasma membrane as a site of action. We examined the molecular organization of [2H31]-N-palmitoylsphingomyelin (PSM-d31) mixed with 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as a monounsaturated control, and cholesterol (chol) (1:1:1 mol) in a model membrane by solid-state 2H NMR. The spectra were analyzed in terms of segregation into ordered SM-rich/chol-rich (raftlike) and disordered PC-rich/chol-poor (nonraft) domains that are nanoscale in size. An increase in the size of domains is revealed when POPC was replaced by PDPC. Spectra that are single-component, attributed to fast exchange between domains (<45 nm), for PSM-d31 mixed with POPC and chol become two-component, attributed to slow exchange between domains (r > 30 nm), for PSM-d31 mixed with PDPC and chol. The resolution of separate signals from PSM-d31, and correspondingly from [3α-2H1]cholesterol (chol-d1) and 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d31), in raftlike and nonraft domains enabled us to determine the composition of the domains in the PDPC-containing membrane. Most of the lipid (28% SM, 29% chol, and 23% PDPC with respect to total lipid at 30°C) was found in the raftlike domain. Despite substantial infiltration of PDPC into raftlike domains, there appears to be minimal effect on the order of SM, implying the existence of internal structure that limits contact between SM and PDPC. Our results suggest a significant refinement to the model by which DHA regulates the architecture of ordered, sphingolipid-chol-enriched domains (rafts) in membranes.
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    Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures
    (American Physiological Society, 2015-07-15) Schweitzer, Kelly S.; Chen, Steven X.; Law, Sarah; Van Demark, Mary; Poirier, Christophe; Justice, Matthew J.; Hubbard, Walter C.; Kim, Elena S.; Lai, Xianyin; Wang, Mu; Kranz, William D.; Carroll, Clinton J.; Ray, Bruce D.; Bittman, Robert; Goodpaster, John V.; Petrache, Irina; Department of Biochemistry & Molecular Biology, IU School of Medicine
    The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.
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    Pharmacological sphingosine-1 phosphate receptor 1 targeting in cigarette smoke-induced emphysema in mice
    (American Physiological Society, 2022) Goel, Khushboo; Schweitzer, Kelly S.; Serban, Karina A.; Bittman, Robert; Petrache, Irina; Medicine, School of Medicine
    Primarily caused by chronic cigarette smoking (CS), emphysema is characterized by loss of alveolar cells comprising lung units involved in gas exchange and inflammation that culminate in airspace enlargement. Dysregulation of sphingolipid metabolism with increases of ceramide relative to sphingosine-1 phosphate (S1P) signaling has been shown to cause lung cell apoptosis and is emerging as a potential therapeutic target in emphysema. We sought to determine the impact of augmenting S1P signaling via S1P receptor 1 (S1P1) in a mouse model of CS-induced emphysema. DBA2 mice were exposed to CS for 4 or 6 mo and treated with pharmacological agonists of S1P1: phosphonated FTY720 (FTY720-1S and 2S analogs; 0.01–1.0 mg/kg) or GSK183303A (10 mg/kg). Pharmacological S1P1 agonists ameliorated CS-induced lung parenchymal apoptosis and airspace enlargement as well as loss of body weight. S1P1 agonists had modest inhibitory effects on CS-induced airspace inflammation and lung functional changes measured by Flexivent, improving lung tissue resistance. S1P1 abundance was reduced in chronic CS-conditions and remained decreased after CS-cessation or treatment with FTY720-1S. These results support an important role for S1P-S1P1 axis in maintaining the structural integrity of alveoli during chronic CS exposure and suggest that increasing both S1P1 signaling and abundance may be beneficial to counteract the effects of chronic CS exposure.
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    Raft Busters: A Molecular Role for DHA in Biological Membranes?
    (Office of the Vice Chancellor for Research, 2016-04-08) Kinnun, Jacob J.; Williams, Justin A.; Stillwell, William; Bittman, Robert; Shaikh, Saame Raza; Wassall, Stephen R.
    Dietary consumption of fish oils rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic acid (DHA, 22:6), has a wide variety of health benefits. However, a complete molecular mechanism is yet to be elucidated. One model that has emerged from biochemical and imaging studies of cells postulates that n-3 PUFAs are taken up into phospholipids in the plasma membrane of cells and, due to their high disorder and aversion for cholesterol, reorganize lipid rafts. Lipid rafts are ordered domains within biological membranes which contain high amounts of sphingomyelin (SM) and cholesterol. To investigate this model, we studied lipid bilayers composed of SM, PDPC (a DHA-containing phospholipid), and cholesterol (1:1:1 mol). The molecular organization of each lipid was investigated with solid-state 2H NMR using deuterated analogs of the lipids. Spectral components assigned to ordered raft-like domains and disordered non-raft domains were resolved, from which the composition of the domains and the order within them could be determined. Most of the SM (84%) and cholesterol (88%) was found in the raft-like domain, together with a substantial amount of PDPC (70%). Despite the infiltration of PDPC there appears to be minimal effect on the order of SM or cholesterol. We speculate that PDPC molecules sequester into small groups minimizing the contact of DHA chains with cholesterol, thereby interrupting the continuity of the raft-like environment.