Night eating syndrome and nocturnal snacking: association with obesity, binge eating and psychological distress.

OBJECTIVE: Night eating syndrome (NES) is characterized by a time-delayed pattern of eating relative to sleep, where most food is consumed in the evening and night. This study aimed to investigate the clinical significance of NES and nocturnal snacking by exploring the relationship between NES and (1) obesity, (2) binge eating disorder (BED) and (3) psychological distress. SUBJECTS: One hundred and eighty bariatric surgery candidates, 93 members of a non-surgical weight loss support group and 158 general community respondents (81 males/350 females, mean age: 45.8+/-13.3 years, mean body mass index (BMI): 34.8+/-10.8 and BMI range: 17.7-66.7). METHODS: NES diagnosis required within the previous 3 months: (1) no appetite for breakfast, (2) consumption of > or =50% of daily energy after 1900 hours and (3) sleep difficulties > or =3 nights/week. Nocturnal snacking (awakening to eat) was recorded. Validated questionnaires assessed BED, symptoms of depression, appearance dissatisfaction (AD) and mental health-related quality of life (MHQoL). NES and binge eating (BE) (> or =1 episode/week) were confirmed by interview. RESULTS: NES criteria were met by 11.1% of the total cohort. Across all groups, BE (P=0.001), BMI (P=0.003) and male gender (P=0.013) explained 10% of NES variance. Individuals with co-morbid NES and BE reported similarly elevated psychological distress as other binge eaters. NES alone was not associated with psychological distress. Those with NES who consumed nocturnal snacks reported poorer MHQoL (P=0.007) and greater depressive symptoms (P=0.039) and hunger (P=0.013) than others with NES. Low MHQoL (P=0.007) and male gender (P=0.022) explained 27% of the variance in the nocturnal snacking group. DISCUSSION: In this study, NES was positively associated with BMI, BE and male gender. Elevated psychological distress was only apparent in those who consumed nocturnal snacks. Further characterization and understanding of the clinical significance of NES and nocturnal snacking is required.

Oxidized LDL-induced injury and apoptosis in atherosclerosis. Potential roles for oxysterols.

The cell injury caused by oxidized lipoproteins was among the first findings that led to the theory that it is the oxidation of low-density lipoprotein (LDL), not just LDL concentration, that leads to arterial disease. Voluminous studies have now revealed that oxidized lipoproteins and their constituents can induce numerous effects on cells that can be construed to be atherogenic. Cell injury is but one of these, and it is these injurious effects that are the focus of this brief review. Cell injury and death appear to play multiple roles in lesion development and the toxic lipid constituents of oxidized lipoproteins, including a variety of oxysterols, are candidates for the in vivo effectors of this cytotoxicity. Recent studies have focused on the mechanisms of oxidized lipoprotein-induced cell death, whether the cells die by apoptosis or necrosis, and the identities of the toxins that induce injury. Understanding the roles of these agents in lesion development could lead to therapies that modulate cell death and inhibit lesion formation.

Lysophosphatidylcholine-induced cellular injury in cultured fibroblasts involves oxidative events.

Lysophosphatidylcholine (lysoPC), formed during LDL oxidation and located within atherosclerotic plaques, induces numerous cellular responses, but via unknown mechanisms. Cellular events involved in sublethal lysoPC-induced injury were examined because these are relevant to mechanisms by which lysoPC alters cell behavior. LysoPC evoked transient membrane permeabilization in fibroblasts within 10 min. Cells underwent reversible rounding within 2 h, returning 3 h later to grossly normal appearance and a normal response to growth stimulation. We asked whether this sublethal permeabilization resulted from physical perturbation of the plasma membrane or if it required cellular events. LysoPC induced leakage of fluorescent dye from unilamellar phospholipid vesicles, suggesting physical membrane perturbation was a significant contributor. To characterize this further we increased the cholesterol content of cells and vesicles to stabilize membranes, and found decreased lysoPC-induced permeabilization in both cell and cell-free systems as cholesterol levels increased. Interestingly, vitamin E, a known antioxidant, blunted lysoPC-induced permeabilization and morphological changes in cells. Thus, lysoPC appeared to cause an unexpected oxidant stress-dependent enhancement of cell injury. To confirm this, several structurally distinct antioxidants, including N, N'-diphenyl-1,4-phenylenediamine, Desferal, Tiron, and 4-hydroxy TEMPO, were applied and these also were inhibitory. Oxidant stress was observed by a lysoPC-induced increase in fluorescence of 5- and 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, an intracellular marker of reactive oxygen species. Lysophosphatidylethanolamine (lysoPE) caused qualitatively similar morphological changes to cells and induced permeabilization, but injury by lysoPE was not inhibited by antioxidants. These data suggest that generation of intracellular reactive oxygen species follows lysoPC-induced plasma membrane destabilization and that this lysoPC-specific oxidant stress enhances cell injury. This intracellular oxidant stress in response to lysoPC may be an integral part of the multiple influences lysoPC has on gene expression and cell function.

Leukocytes utilize myeloperoxidase-generated nitrating intermediates as physiological catalysts for the generation of biologically active oxidized lipids and sterols in serum.

The initiation of lipid peroxidation and the concomitant formation of biologically active oxidized lipids and sterols is believed to play a central role in the pathogenesis of inflammatory and vascular disorders. Here we explore the role of neutrophil- and myeloperoxidase (MPO)-generated nitrating intermediates as a physiological catalyst for the initiation of lipid peroxidation and the formation of biologically active oxidized lipids and sterols. Activation of human neutrophils in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide (nitrogen monoxide, NO) metabolism, generated an oxidant capable of initiating peroxidation of lipids. Formation of hydroxy- and hydroperoxyoctadecadienoic acids [H(P)ODEs], hydroxy- and hydroperoxyeicosatetraenoic acids [H(P)ETEs], F(2)-isoprostanes, and a variety of oxysterols was confirmed using on-line reverse phase HPLC tandem mass spectrometry (LC/MS/MS). Lipid oxidation by neutrophils required cell activation and NO(2)(-), occurred in the presence of metal chelators and superoxide dismutase, and was inhibited by catalase, heme poisons, and free radical scavengers. LC/MS/MS studies demonstrated formation of additional biologically active lipid and sterol oxidation products known to be enriched in vascular lesions, such as 1-hexadecanoyl-2-oxovalaryl-sn-glycero-3-phosphocholine, which induces upregulation of endothelial cell adhesion and chemoattractant proteins, and 5-cholesten-3beta-ol 7beta-hydroperoxide, a potent cytotoxic oxysterol. In contrast to the oxidant formed during free metal ion-catalyzed reactions, the oxidant formed during MPO-catalyzed oxidation of NO(2)(-) readily promoted lipid peroxidation in the presence of serum constituents. Collectively, these results suggest that phagocytes may employ MPO-generated reactive nitrogen intermediates as a physiological pathway for initiating lipid peroxidation and forming biologically active lipid and sterol oxidation products in vivo.

Isolation and characterization of two distinct forms of liver fatty acid binding protein from the rat.

Liver fatty acid binding protein (L-FABP) appears to contain several different forms that may result from post-translational modification or bound ligand. To further assess this possibility, L-FABP was purified from rat liver homogenate and two putative isoforms separated using a sulfonyl column, a strong cation exchange resin. Fraction I eluted at 0.2 M NaCl, had a pI of 7.59, and following a final size exclusion step contained > 98% L-FABP. Fraction II eluted at 1.0 M NaCl, had a pI of 7.59, and following a final size exclusion step contained > 99% L-FABP. Both fractions contained approx. 0.15 moles of endogenous bound fatty acid per mole of protein, while L-FABP not subjected to the cation exchange step contained 0.75 moles of fatty acid per mole of protein. Fractions I and II had a greater proportion of saturated and monounsaturated fatty acids with a large reduction in polyunsaturated fatty acids compared to L-FABP not fractionated by cation exchange. Mass spectral analysis indicated the molecular mass of Fraction I was 14,315.02 +/- 0.35 Da and Fraction II was 14,315.86 +/- 0.34 Da. The peptide map for each fraction was determined by limited digestion of each fraction with either trypsin, Asp-N, or chymotrypsin to yield overlapping peptide fragments. Mass spectral analysis of these digests indicated the two proteins had identical amino acid fragments and that Cys69 was reduced and there were no Asn to Asp exchanges. Hence, these two forms of L-FABP were not isoforms and were not the result of differences in bound fatty acid. It is proposed that these two distinct forms of rat L-FABP were structural conformers based on two alternative folding pathways.

Roles of multiple oxidized LDL lipids in cellular injury: dominance of 7 beta-hydroperoxycholesterol.

The relative toxicities of several lipid oxidation products formed on oxidized LDL, their presence on oxidized LDL, and potential mechanisms of cell injury compared to oxidized LDL were examined. Toxicities to fibroblasts, with lipoprotein-deficient serum supplementation, were: 7 beta-hydroperoxycholesterol > 7 beta-hydroxycholesterol = 4-hydroxynonenal > 7-ketocholesterol > 5 alpha, 6 alpha-epoxycholesterol. Lysophosphatidylcholine was only significantly cytotoxic in the absence of lipoprotein-deficient serum. Without serum, relative toxicities were: 7 beta-hydroperoxycholesterol > lysophosphatidylcholine > 4-hydroxynonenal > 7 beta-hydroxycholesterol. Similar relative potencies were observed in smooth muscle and endothelial cell cultures. 7 beta-Hydroperoxycholesterol accumulated on oxidized LDL to greater amounts than other oxysterols and 4-hydroxynonenal, but less than lysophosphatidylcholine. Cell injury by 7 beta-hydroperoxycholesterol and oxidized LDL was inhibitable by antioxidants but not by exogenous cholesterol or cycloheximide. In contrast, a) toxicities by 7 beta-hydroxycholesterol, 7-ketocholesterol, 5 alpha, 6 alpha-epoxycholesterol, and 4-hydroxynonenal were not inhibited by antioxidants; b) 7 beta-hydroxycholesterol and lysophosphatidylcholine toxicities were inhibited by exogenous cholesterol; and c) 7 beta-hydroxycholesterol toxicity was inhibited by cycloheximide. Injury by lysophosphatidylcholine was reduced by vitamin E and not affected by altering the cellular exposure to selenium; reduced selenium enhanced toxicity by oxidized LDL and 7 beta-hydroperoxycholesterol. The high relative toxicity of 7 beta-hydroperoxycholesterol, the level of its accumulation on oxidized LDL, and its mechanism of action similar to oxidized LDL suggest that it is the compound predominantly responsible for oxidized LDL induced cytotoxicity.

In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals.

Mounting evidence supports current theories linking lipoprotein oxidation to atherosclerosis. We sought the cellular biochemical mechanism by which oxidized LDL inflicts cell injury. Inhibitors of candidate pathways of cell death were used to treat human fibroblast target cells exposed to oxidized LDL.. Ebselen, which degrades lipid hydroperoxides, inhibited oxidized LDL toxicity, consistent with our recent report that 7 beta-hydroperoxycholesterol (7 beta-OOH chol) is the major cytotoxin of oxidized LDL. Intracellular chelation of metal ions inhibited, while preloading cells with iron enhanced, toxicity, Inhibition of oxidized LDL and 7 beta-OOH chol toxicity by 2-keto-4-thiolmethyl butyric acid, a putative alkoxyl radical scavenger and by vitamin E, probucol and diphenylphenylenediamine, putative scavengers of peroxyl radicals was consistent with the involvement of these radicals in the lethal sequence. Cell death was thus postulated to occur due to lipid peroxidation via a sequence involving lipid hydroperoxide-induced, iron-mediated formation of alkoxyl, lipid, and peroxyl radicals. Pathways involving other reactive oxygen species, new protein synthesis, or altered cholesterol metabolism were considered less likely, since putative inhibitors failed to lessen toxicity. Understanding the mechanism of cell injury by oxidized LDL and its toxic moiety, 7 beta-OOH chol, may indicate specific interventions in the cell injury believed to accompany vascular lesion development.

Cholesterol interaction with recombinant human sterol carrier protein-2.

The interaction of human recombinant sterol carrier protein-2 (SCP-2) with sterols was examined. Two independent ligand binding methods, Lipidex 1000 binding of [3H]cholesterol and a fluorescent dehydroergosterol binding assay, were used to determine the affinity of SCP-2 for sterols. Binding analysis indicated SCP-2 bound [3H]cholesterol and dehydroergosterol with a Kd of 0.3 and 1.7 microM, respectively, and suggested the presence of a single binding site. Phase fluorometry and circular dichroism were used to characterize the SCP-2 sterol binding site. Alterations in dehydroergosterol lifetime, SCP-2 tryptophan lifetime, and SCP-2 tryptophan quenching by acrylamide upon cholesterol binding demonstrated a shielding of the SCP-2 tryptophan from the aqueous solvent by bound sterol. Differential polarized phase fluorometry revealed decreased SCP-2 tryptophan rotational correlation time upon cholesterol binding. Circular dichroism of SCP-2 indicated that cholesterol elicited a small decrease in SCP-2 alpha helical content. The data suggest that SCP-2 binds sterols with affinity consistent with a lipid transfer protein that may act either as an aqueous carrier or at a membrane surface to enhance sterol desorption.

Structure and polarity of mouse brain synaptic plasma membrane: effects of ethanol in vitro and in vivo.

Structural and dielectric alteration by ethanol in vitro and chronic ethanol consumption were examined in synaptic plasma membranes (SPM) using diphenylhexatriene and charged diphenylhexatriene derivatives. These fluorophores, in combination with multifrequency phase and modulation fluorometry, allowed the examination of the surface and interior core of SPM. Limiting anisotropy and rotational relaxation time demonstrated that the synaptosomal plasma membrane surface domain was more rigid than the interior core domain. Ethanol in vitro fluidized the interior core and surface domains in SPM of the control, but not chronic ethanol-treated mice. Although the latter membranes were more rigid than control membranes, the intrinsic rigidity of the interior core of the synaptosomal plasma membrane did not strictly correlate with effects of ethanol in vitro. SPM of irradiated membranes were more rigid, but ethanol fluidized those membranes. Diphenylhexatriene lifetime and photoreactivity were sensitive to the range of dielectric constants in the SPM interior core. Ethanol in vitro increased both the surface and interior core range of dielectric constants of SPM from control but not chronic ethanol-treated animals. Thus, ethanol in vitro altered not only the fluidity but also the range of dielectric constants in both the surface and interior core domains in SPM of control but not chronic ethanol-treated mice.

Sterol carrier protein-2 stimulates intermembrane sterol transfer by direct membrane interaction.

It is unclear how the cytosolic sterol carrier protein-2 (SCP-2) binds sterols and enhances sterol transfer between membranes. Therefore, human recombinant SCP-2 was used in conjunction with phase fluorometry, dialysis, and chemical labeling techniques to show if a direct membrane effect accounted for this activity. SCP-2 directly interacted with L-cell fibroblast plasma membrane vesicles as determined by increased fluorescence anisotropy of coumarin-labeled protein (CPM-SCP-2). Furthermore, a new fluorescence lifetime component due to plasma membrane-bound CPM-SCP-2 was observed. Dialysis studies with 3H- cholesterol loaded plasma membranes indicated that SCP-2, added to the donor compartment, stimulated sterol transfer whether or not the dialysis membrane was permeable to SCP-2. Nevertheless, ligand-binding experiments indicated that chemically blocking the SCP-2 sterol binding site inhibited the ability of SCP-2 to enhance sterol transfer between plasma membrane vesicles. SCP-2 did not stimulate plasma membrane fusion. Addition of SCP-2 to plasma membranes increased the anisotropy plasma membrane proteins covalently reacted with CPM, but not that of lipids labeled with the fatty acid analogue octadecyl rhodamine B. In conclusion, the data are consistent with SCP-2 stimulating intermembrane sterol transfer by direct interaction with sterol in the membrane and enhancing its desorption from the membrane.

Expression of liver fatty acid binding protein in L-cells: plasma membrane response to ethanol.

Expression of liver fatty acid binding protein (L-FABP) in transfected L-cell fibroblasts modifies plasma membrane structure and function [Incerpi et al., 1992, Arch. Biochem. Biophys. 298, 35-42]. The effect of L-FABP expression on ethanol induced fluidization of plasma membranes was examined. Ethanol in vitro selectively fluidized the exofacial leaflet of the plasma membranes from L-cells expressing low amounts of L-FABP. In contrast, the plasma membranes from L-cells expressing high amounts of L-FABP were resistant to the actions of ethanol. Furthermore, diphenylhexatriene lifetime distributional analysis demonstrated that the plasma membrane exofacial leaflet had a lower range of apparent dielectric constants than the cytofacial leaflet for both low- and high-expression cells. Both the center of lifetime and the lifetime distributional width of diphenylhexatriene in the bulk plasma membrane versus the cytofacial leaflet were consistent with significantly lower apparent dielectric constant in the exofacial leaflet of high-expression versus low-expression cells. Ethanol in vitro preferentially increased the exofacial leaflet apparent dielectric properties of the plasma membranes from low-expression but not high-expression cells. In conclusion, ethanol appears to dehydrate the lipid headgroups of plasma membranes from high-expression cells, thereby conferring resistance to ethanol fluidization. In contrast, ethanol may not dehydrate the plasma membrane lipid head groups of low-expression cells, resulting in ethanol fluidizing the outer leaflet of the plasma membrane.

Synaptic plasma membrane structure and polarity of long-sleep and short-sleep mice.

Membrane dielectric as a primary basis for effects of ethanol was examined in synaptic plasma membranes (SPM) of genetically selected ethanol-sensitive long-sleep (LS) and ethanol-resistant short-sleep (SS) mice. Multifrequency phase and modulation of fluorometry of diphenylhexatriene (DPH) was used to resolve structural and dielectric differences in the membrane interior core. Fluorescence spectral peak ratios, fluorescence lifetime analysis, and initial rates of photoreaction of DPH in SPM provided sensitive measures of SPM interior core dielectric properties. The membrane microenvironment sensed by DPH was more polar in SPM from SS mice than in SPM from LS mice. Physiological concentrations of ethanol in vitro (25-75 mM) increased the SPM interior core dielectric and potentiated photoreaction of DPH with other membrane components of SPM from LS, but not SS, mice. These effects of ethanol in vitro were maximal by 75 mM ethanol and/or exacerbated at higher ethanol. In addition, ethanol in vitro increased the fraction of DPH associated with photoreaction products with lipids from SPM of ethanol-sensitive LS mice. The data were consistent with ethanol in vitro increasing the polar molecules (ethanol and/or water) of SPM from LS but not SS mice. It is suggested that ethanol alters the polarity and increases reactivity of the interior core lipid-protein interface.

Expression of rat L-FABP in mouse fibroblasts: role in fat absorption.

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose levels is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigated in vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accommodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtained in vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.

Analysis of cholesterol and desmosterol in cultured cells without organic solvent extraction.

Cultured cell sterols such as cholesterol and desmosterol are usually extracted into organic solvents before they are quantified with cholesterol esterase and oxidase. A method to quantify these cultured cell sterols using cholesterol enzymes without prior organic solvent extraction is described. In this method, a suspension or monolayer of cultured L-M, U-937, or PC-12 cells is digested with 0.1% sodium dodecyl sulfate (SDS), and the digest treated with microbial cholesterol enzymes. The quantity of oxidized sterols produced by the reaction can be measured easily with high-pressure liquid chromatography, when a mixture of sterols is present, or by the production of hydrogen peroxide when only one sterol is present. This method is easier and safer to use than solvent extraction and can greatly expedite the quantitation of cultured cell sterols. Preliminary data show that other lipids such as choline phospholipids, triglycerides, and fatty acids can also be directly quantified in SDS cell digest by using specific enzymes to transform these lipids into hydrogen peroxides.

HPLC analysis of desmosterol, 7-dehydrocholesterol, and cholesterol.

A simple and sensitive method to analyze mixtures of desmosterol, 7-dehydrocholesterol and cholesterol is described. The method involves the oxidative conversion of the sterols with cholesterol oxidase, followed by high performance liquid chromatographic (HPLC) analysis. A C18 reversed phase column (3 microns, 75 X 4.6 mm) and a mixture of methanol and acetonitrile (1:1, v/v) at a rate of 1 ml/min are used to separate the sterols. The eluted sterols are quantified by measuring UV absorption at 240 nm. As little as 10 pmoles of sterol can be quantified under these conditions.