Cellular cholesterol accumulation modulates high fat high sucrose (HFHS) diet-induced ER stress and hepatic inflammasome activation in the development of non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH), is the form of non-alcoholic fatty liver disease posing risk to progress into serious long term complications. Human and pre-clinical models implicate cellular cholesterol dysregulation playing important role in its development. Mouse model studies suggest synergism between dietary cholesterol and fat in contributing to NASH but the mechanisms remain poorly understood. Our laboratory previously reported the primary importance of hepatic endoplasmic reticulum cholesterol (ER-Chol) in regulating hepatic ER stress by comparing the responses of wild type, Ldlr −/− xLcat +/+ and Ldlr −/− xLcat −/− mice, to a 2% high cholesterol diet (HCD). Here we further investigated the roles of ER-Chol and ER stress in HFHS diet-induced NASH using the same strains. With HFHS diet feeding, both WT and Ldlr −/− xLcat +/+ accumulate ER-Chol in association with ER stress and inflammasome activation but the Ldlr −/− xLcat −/− mice are protected. By contrast, all three strains accumulate cholesterol crystal, in correlation with ER-Chol, albeit less so in Ldlr −/− xLcat −/− mice. By comparison, HCD feeding per se (i) is sufficient to promote steatosis and activate inflammasomes, and (ii) results in dramatic accumulation of cholesterol crystal which is linked to inflammasome activation in Ldlr −/− xLcat −/− mice, independent of ER-Chol. Our data suggest that both dietary fat and cholesterol each independently promote steatosis, cholesterol crystal accumulation and inflammasome activation through distinct but complementary pathways. In vitro studies using palmitate-induced hepatic steatosis in HepG2 cells confirm the key roles by cellular cholesterol in the induction of steatosis and inflammasome activations. These novel findings provide opportunities for exploring a cellular cholesterol-focused strategy for treatment of NASH.     

Cathepsin G activity lowers plasma LDL and reduces atherosclerosis

Cathepsin G (CatG), a serine protease present in mast cells and neutrophils, can produce angiotensin-II (Ang-II) and degrade elastin. Here we demonstrate increased CatG expression in smooth muscle cells (SMCs), endothelial cells (ECs), macrophages, and T cells from human atherosclerotic lesions. In low-density lipoprotein (LDL) receptor-deficient (Ldlr^–/–) mice, the absence of CatG reduces arterial wall elastin degradation and attenuates early atherosclerosis when mice consume a Western diet for 3 months. When mice consume this diet for 6 months, however, CatG deficiency exacerbates atherosclerosis in aortic arch without affecting lesion inflammatory cell content or extracellular matrix accumulation, but raises plasma total cholesterol and LDL levels without affecting high-density lipoprotein (HDL) or triglyceride levels. Patients with atherosclerosis also have significantly reduced plasma CatG levels that correlate inversely with total cholesterol (r = –0.535, P < 0.0001) and LDL cholesterol (r = –0.559, P < 0.0001), but not with HDL cholesterol (P = 0.901) or triglycerides (P = 0.186). Such inverse correlations with total cholesterol (r = –0.504, P < 0.0001) and LDL cholesterol (r = –0.502, P < 0.0001) remain significant after adjusting for lipid lowering treatments among this patient population. Human CatG degrades purified human LDL, but not HDL. This study suggests that CatG promotes early atherogenesis through its elastinolytic activity, but suppresses late progression of atherosclerosis by degrading LDL without affecting HDL or triglycerides.     

Lipodystrophic gene Agpat2 deficiency aggravates hyperlipidemia and atherosclerosis in Ldlr−/− mice

AimsDysfunction of adipose tissue increases the risk of cardiovascular disease. It was well established that obesity aggravates atherosclerosis, but the effect of adipose tissue loss on atherosclerosis has been less studied. AGPAT2 is the first causative gene of congenital generalized lipodystrophy (CGL), but the role of AGPAT2 on atherosclerosis has not been reported. Hypertriglyceridemia is one of the clinical manifestations of CGL patients, but it is usually absent in CGL mouse model on a normal diet. This study will investigate the effect of Agpat2 on hyperlipidemia and atherosclerosis.Methods and resultsIn this study, Agpat2 knockout (Agpat2^−/−) mice were generated using CRISPR/Cas system, which showed severe loss of adipose tissue and fatty liver, consistent with previous reports. Agpat2^−/− mice were then crossed with hypercholesterolemic and atherosclerotic prone LDL receptor knockout (Ldlr^−/−) mice to obtain double knockout mouse model (Agpat2^−/−Ldlr^−/−). Plasma lipid profile, insulin resistance, fatty liver, and atherosclerotic lesions were observed after 12 weeks of the atherogenic high-fat diet (HFD) feeding. We found that compared with Ldlr^−/− mice, Agpat2^−/−Ldlr^−/− mice showed significantly higher plasma total cholesterol and triglycerides after HFD feeding. Agpat2^−/−Ldlr^−/− mice also developed hyperglycemia and hyperinsulinemia, with increased pancreatic islet area. The liver weight of Agpat2^−/−Ldlr^−/− mice was about 4 times higher than that of Ldlr^−/− mice. The liver lipid deposition was severe and Sirius red staining showed liver fibrosis. In addition, in Agpat2^−/−Ldlr^−/− mice, the area of atherosclerotic lesions in aortic arch and aortic root was significantly increased.ConclusionsOur results show that Agpat2 deficiency led to more severe hyperlipidemia, liver fibrosis and aggravation of atherosclerosis in Ldlr^−/− mice. This study provided additional insights into the role of adipose tissue in hyperlipidemia and atherosclerosis.     

Endothelial permeability in vitro and in vivo: Protective actions of ANP and omapatrilat in experimental atherosclerosis

Increased arterial endothelial cell permeability (ECP) is considered an initial step in atherosclerosis. Atrial natriuretic peptide (ANP) which is rapidly degraded by neprilysin (NEP) may reduce injury-induced endothelial cell leakiness. Omapatrilat represents a first in class of pharmacological agents which inhibits both NEP and angiotensin converting enzyme (ACE). We hypothesized that ANP prevents thrombin-induced increases of ECP in human aortic ECs (HAECs) and that omapatrilat would reduce aortic leakiness and atherogenesis and enhance ANP mediated vasorelaxation of isolated aortas. Thrombin induced ECP determined by I¹²⁵ albumin flux was assessed in HAECs with and without ANP pretreatment. Next we examined the effects of chronic oral administration of omapatrilat (12 mg/kg/day, n = 13) or placebo (n = 13) for 8 weeks on aortic leakiness, atherogenesis and ANP-mediated vasorelaxation in isolated aortas in a rabbit model of atherosclerosis produced by high cholesterol diet. In HAECs, thrombin-induced increases in ECP were prevented by ANP. Omapatrilat reduced the area of increased aortic leakiness determined by Evans-blue dye and area of atheroma formation assessed by Oil-Red staining compared to placebo. In isolated arterial rings, omapatrilat enhanced vasorelaxation to ANP compared to placebo with and without the endothelium. ANP prevents thrombin-induced increases in ECP in HAECs. Chronic oral administration of omapatrilat reduces aortic leakiness and atheroma formation with enhanced endothelial independent vasorelaxation to ANP. These studies support the therapeutic potential of dual inhibition of NEP and ACE in the prevention of increased arterial ECP and atherogenesis which may be linked to the ANP/cGMP system.     

Hypolipidemic effect of Goami-3 rice (Oryza sativa L. cv. Goami-3) on C57BL/6J mice is mediated by the regulation of peroxisome proliferator-activated receptor-α and -γ

We investigated the hypolipidemic effects of Goami-3 rice (GR; Oryza sativa L. cv. Goami-3), a newly developed strain with high levels of amylose and fibers. Diet-induced obese mice were fed three types of isocaloric diets for 8 weeks: a high-fat diet, a high-fat diet with GR or control rice (CR; O. sativa L. cv. Ilpumbyeo). Mice fed GR exhibited a significant reduction in body fat (–23%), total cholesterol (–20%) and triglyceride concentrations (–30%) compared to mice fed CR. The mice fed GR showed induction of peroxisome proliferator-activated receptor (PPAR)-α and inhibition of γ expressions in the liver and adipose tissue. The reduced adiposity of mice fed GC was supported by changes in the expression of genes related to lipid accumulation and hydrolysis in adipose tissues and the plasma concentrations of insulin, adiponectin and leptin. Principal components analysis with gas chromatography–time-of-flight mass spectrometry-based metabolomic data revealed that the average level of specific plasma metabolites in the GR group was statistically different from that in the other groups after 4 weeks. These metabolites included propionic acid, valine, leucine and proline. Based on partial least-squares analysis, the plasma concentrations of valine were inversely correlated with the high-density lipoprotein (HDL) to non-HDL and HDL to total cholesterol ratios. In conclusion, GR feeding for 8 weeks significantly improved dyslipidemia and adiposity in diet-induced obese mice by regulating gene expression of PPARs and its target genes. Key plasma metabolites (including valine) were significantly altered by the hypolipidemic effects of GR.     

Inhibition of intestinal cholesterol absorption decreases atherosclerosis but not adipose tissue inflammation

Adipose tissue inflammation is associated with insulin resistance and increased cardiovascular disease risk in obesity. We previously showed that addition of cholesterol to a diet rich in saturated fat and refined carbohydrate significantly worsens dyslipidemia, insulin resistance, adipose tissue macrophage accumulation, systemic inflammation, and atherosclerosis in LDL receptor-deficient (Ldlr^−/−) mice. To test whether inhibition of intestinal cholesterol absorption would improve metabolic abnormalities and adipose tissue inflammation in obesity, we administered ezetimibe, a dietary and endogenous cholesterol absorption inhibitor, to Ldlr^−/− mice fed chow or high-fat, high-sucrose (HFHS) diets without or with 0.15% cholesterol (HFHS+C). Ezetimibe blunted weight gain and markedly reduced plasma lipids in the HFHS+C group. Ezetimibe had no effect on glucose homeostasis or visceral adipose tissue macrophage gene expression in the HFHS+C fed mice, although circulating inflammatory markers serum amyloid A (SSA) and serum amyloid P (SSP) levels decreased. Nevertheless, ezetimibe treatment led to a striking (>85%) reduction in atherosclerotic lesion area with reduced lesion lipid and macrophage content in the HFHS+C group. Thus, in the presence of dietary cholesterol, ezetimibe did not improve adipose tissue inflammation in obese Ldlr^−/− mice, but it led to a major reduction in atherosclerotic lesions associated with improved plasma lipids and lipoproteins.     

Sap flow and water use in African baobab (Adansonia digitata L.) seedlings in response to drought stress

The African baobab (Adansonia digitata L.) is an important multi-purpose fruit tree with high potential for domestication in drier Africa. Although adult individuals are well-known to be drought resistant, only little has been reported on how young baobab trees can survive drought. Therefore, the aim of this study was to examine short-term soil drought effects on water relations of baobab seedlings. Baobab seedlings used a limited amount of stored water to buffer daily water deficits (~ 8.5 g d^− 1), which contributed up to only ~ 17.5% of daily water use and ~ 6% of total plant water. Under drought, a strong reduction in stomatal conductance (~ 85%) resulted in a midday leaf water potential of − 1 MPa and zero stem sap flow followed by significant leaf loss. Plant anatomy evidenced the presence of water storage tissues and the vulnerability to xylem embolism. The taproot was the most important plant part for water storage (68% of total plant water), suggesting root-succulence rather than stem-succulence. When drought intensified, limitation of leaf transpiration and/or root water uptake led to drought-enforced dormancy. Despite the large amounts of water stored in the taproot (~ 90%) and the stem (~ 75%), only a limited amount of stored water appeared to be used to sustain upper leaves and plant metabolism during the dormant period, and to facilitate recovery following water supply. Drought avoidance, conservative water use and the presence of internal stored water allow baobab seedlings to survive drought.     

Dietary coenzyme Q10 does not protect against cigarette smoke-augmented atherosclerosis in apoE-deficient mice

Dietary coenzyme Q10 reduces spontaneous atherosclerosis in the apoE-deficient mouse model of experimental atherosclerosis. We have shown previously that exposure to sidestream cigarette smoke (SSCS) enhances atherosclerotic lesion formation in apoE-deficient mice. The aim of the present study was to determine if CoQ10 protected against SSCS-mediated atherosclerosis. Female apoE-deficient mice were fed a saturated fat-enriched diet (SFD) alone, or supplemented with 1% wt/wt coenzyme Q10 (SFD-Q10). Mice in each diet group were exposed to SSCS for 4 hrs/day, 5 days/week in a whole-body exposure chamber maintained at 35 ± 4 mg smoke particulates/m³. Mice kept in filtered ambient air served as controls. Mice were euthanized after either 6 or 15 weeks of SSCS exposure and following measurements were performed: i) lung 7-ethoxyresorufin-O-deethylase (EROD) activity; ii) plasma cholesterol and CoQ10 concentrations; iii) aortic intimal area covered by atherosclerotic lesions; and, iv) pathological characterization of lesions. Lung EROD activity increased in SSCS mice of both diet groups, confirming SSCS exposure. Plasma concentrations of CoQ10 in SFD-Q10-fed mice were increased markedly in comparison to SFD-fed mice. Plasma cholesterol concentrations and distributions of cholesterol in lipoprotein fractions were unaffected by SSCS exposure. Dietary supplementation with CoQ10 significantly reduced atherosclerotic lesions in control mice. As reported previously, exposure to SSCS increased the size of lesions in apoE-/- mice at both time points. However, dietary supplementation with CoQ10 had no effect on atherosclerotic lesions augmented by SSCS exposure. The results suggest a role of oxidative processes in smoke-augmented atherosclerosis that are different than those mitigated by CoQ10.     

Non-phytin phosphorus requirements of commercial broilers and White Leghorn layers

Experiments were conducted to determine the optimum requirements of non-phytin phosphorus (NPP) in commercial broilers and White Leghorn layers. Five levels of NPP (2.5, 3.0, 3.5, 4.0 and 4.5 g kg⁻¹ diet) were tested to assess the NPP requirement of commercial broilers (3–30 days of age) fed maize–soya diets containing 10 g Ca kg⁻¹. Each level of NPP was fed to quadruplicate groups of ten chicks each. Inclusion of graded levels of NPP significantly (P < 0.01) influenced body weight gain, feed intake, tibia ash content, phosphorus content in serum, tibia ash and phosphorus retention. The predicted NPP requirements for body weight gain, P content in serum and tibia ash were 4.4, 4.48 and 4.1 g kg⁻¹ diet, respectively. The NPP requirement for tibia ash was the highest (7.4 g kg⁻¹ diet). Similarly, four levels of NPP (2.0, 2.5, 3.0 and 3.5 g kg⁻¹ diet) were tested with maize–soya diets containing 35 g Ca kg⁻¹ for White Leghorn layers (266–350 days of age). Each diet was tested on four groups of 12 hens in each. Egg production was not influenced by the variation in dietary NPP levels. The predicted NPP requirements for better egg weight and shell thickness were 2.6 and 2.4 g kg⁻¹ diet, respectively, while for the serum inorganic P level the value was 3.42 g kg⁻¹ diet. Therefore, it can be concluded that commercial broilers need about 4.4 g NPP kg⁻¹ diet for better performance, whereas, White Leghorn layers need not more than 2.0 g NPP kg⁻¹ diet for better egg production. However, layers require 2.6 g NPP kg⁻¹ diet to produce eggs with better egg size and shell quality.     

Growth and loss of distal tissue in blades of Lessonia nigrescens and Lessonia trabeculata (Laminariales)

Meristematic growth and loss of distal tissue from blades of two ecologically important species in the south-east Pacific, Lessonia nigrescens and Lessonia trabeculata, was evaluated during 1 year. Comparative growth was determined by a hole-punch method, loss of distal tissue from the blades was determined by subtracting final blade length (with loss) from expected blade lengths (without loss); growth and tissue loss were transformed to fresh biomass units for calculation of inter-algae differences. The results showed that blade elongation rate increased at the beginning of spring, and declined towards the end of summer, with mean values between 0.40 and 0.08 cm day⁻¹ for L. nigrescens, and 0.65–0.17 cm day⁻¹ for L. trabeculata. Loss of distal tissue varied seasonally when examined as length units for both species; with mean values between 0.24 and 0.10 cm day⁻¹ for L. nigrescens, and 0.51–0.25 cm day⁻¹ for L. trabeculata. Variations in fresh biomass units were only observed in Lessonia trabeculata, increasing in spring, with mean values to 0.13 g (fresh weight) day⁻¹. Annual growth and loss of distal tissue were higher in L. trabeculata (0.41 and 0.39 cm day⁻¹, respectively) than in L nigrescens (0.19 and 0.15 cm day⁻¹). When growth and tissue loss were considered as fresh biomass, monthly gains significantly outweighed loss of distal tissue in both species, but parallel results based on length data followed a different trend. L. trabeculata released about 50% of its growth biomass as particulate organic matter, while the comparative value for L. nigrescens was about 20%.     

Sulforaphane improves dysregulated metabolic profile and inhibits leptin-induced VSMC proliferation: Implications toward suppression of neointima formation after arterial injury in western diet-fed obese mice

Sulforaphane (SFN), a dietary phase-2 enzyme inducer that mitigates cellular oxidative stress through nuclear factor erythroid 2-related factor 2 (Nrf2) activation, is known to exhibit beneficial effects in the vessel wall. For instance, it inhibits vascular smooth muscle cell (VSMC) proliferation, a major event in atherosclerosis and restenosis after angioplasty. In particular, SFN attenuates the mitogenic and pro-inflammatory actions of platelet-derived growth factor (PDGF) and tumor necrosis factor-α (TNFα), respectively, in VSMCs. Nevertheless, the vasoprotective role of SFN has not been examined in the setting of obesity characterized by hyperleptinemia and insulin resistance. Using the mouse model of western diet-induced obesity, the present study demonstrates for the first time that subcutaneous delivery of SFN (0.5 mg/Kg/day) for ~ 3 weeks significantly attenuates neointima formation in the injured femoral artery [↓ (decrease) neointima/media ratio by ~ 60%; n = 5–8]. This was associated with significant improvements in metabolic parameters, including ↓ weight gain by ~ 52%, ↓ plasma leptin by ~ 42%, ↓ plasma insulin by ~ 63%, insulin resistance [↓ homeostasis model assessment of insulin resistance (HOMA-IR) index by ~ 73%], glucose tolerance (↓ AUC_GTT by ~ 24%), and plasma lipid profile (e.g., ↓ triglycerides). Under in vitro conditions, SFN significantly decreased leptin-induced VSMC proliferation by ~ 23% (n = 5) with associated diminutions in leptin-induced cyclin D1 expression and the phosphorylation of p70S6kinase and ribosomal S6 protein (n = 3–4). The present findings reveal that, in addition to improving systemic metabolic parameters, SFN inhibits leptin-induced VSMC proliferative signaling that may contribute in part to the suppression of injury-induced neointima formation in diet-induced obesity.     

Lecithin/cholesterol acyltransferase modulates diet-induced hepatic deposition of triglycerides in mice

Lecithin/cholesterol acyltransferase (LCAT) is responsible for the esterification of the free cholesterol of plasma lipoproteins. Here, we investigated the involvement of LCAT in mechanisms associated with diet-induced hepatic triglyceride accumulation in mice. LCAT-deficient (LCAT^?/?) and control C57BL/6 mice were placed on a Western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% cholesterol, 4.5 kcal/g) for 24 weeks, then histopathological and biochemical analyses were performed. We report that, in our experimental setup, male LCAT^?/? mice are characterized by increased diet-induced hepatic triglyceride deposition and impaired hepatic histology and architecture. Mechanistic analyses indicated that LCAT deficiency was associated with enhanced intestinal absorption of dietary triglycerides (3.6±0.5 mg/dl per minute for LCAT^?/? vs. 2.0±0.7 mg/dl per minute for C57BL/6 mice; P<.05), accelerated clearance of postprandial triglycerides and a reduced rate of hepatic very low density lipoprotein triglyceride secretion (9.8±1.1 mg/dl per minute for LCAT^?/? vs. 12.5±1.3 mg/dl per minute for C57BL/6 mice, P<.05). No statistical difference in the average daily food consumption between mouse strains was observed. Adenovirus-mediated gene transfer of LCAT in LCAT^?/? mice that were fed a Western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content (121.2±5.9 mg/g for control infected mice vs. 95.1±5.8 mg/g for mice infected with Ad-LCAT, P<.05) and a great improvement of hepatic histology and architecture. Our data extend the current knowledge on the functions of LCAT, indicating that LCAT activity is an important modulator of processes associated with diet-induced hepatic lipid deposition.     

Haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice

AimsTo investigate whether haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice.Methods and ResultsExperiments were performed using irradiated LDL receptor-deficient (LDLR^−/−) mice with marrow from either TLR4-deficient (TLR4^−/−) or age-matched wild-type (WT) mice. After 12 weeks of being fed a high-cholesterol diet, TLR4^−/− → LDLR^−/− mice developed fewer atherosclerotic lesions in the aorta compared to WT → LDLR^−/− mice. This effect was associated with an increase in multilocular lipid droplets and mitochondria in perivascular adipose tissue (PVAT). Immunofluorescence analysis confirmed that there was an increase in capillary density and M2 macrophage infiltration, accompanied by a decrease in tumour necrosis factor (TNF)-α expression in the localized PVAT of TLR4^−/− → LDLR^−/− mice. In vitro studies indicated that bone marrow-derived macrophages (BMDMs) from WT mice demonstrated an M1-like phenotype and expression of inflammatory cytokines induced by palmitate. These effects were attenuated in BMDMs isolated from TLR4^−/− mice. Furthermore, brown adipocytes incubated with conditioned medium (CM) derived from palmitate-treated BMDMs, exhibited larger and more unilocular lipid droplets, and reduced expression of brown adipocyte-specific markers and perilipin-1 compared to those observed in brown adipocytes exposed to CM from palmitate-treated BMDMs of TLR4^−/− mice. This decreased potency was primarily due to TNF-α, as demonstrated by the capacity of the TNF-α neutralizing antibody to reverse these effects.ConclusionsThese results suggest that haematopoietic-specific deletion of TLR4 promotes PVAT homeostasis, which is involved in reducing macrophage-induced TNF-α secretion and increasing mitochondrial biogenesis in brown adipocytes.     

Extent estimates and land cover relationships for functional indicators in non-wadeable rivers

Functional indicators are being increasingly used to assess waterway health but their responses to pressure in non-wadeable rivers have not been widely documented or applied in modern survey designs that provide unbiased estimates of extent. This study tests the response of river metabolism and loss in cotton strip tensile strength across a land use pressure gradient in non-wadeable rivers of northern New Zealand, and reports extent estimates for river metabolism and decomposition rates. Following adjustment for probability of selection, ecosystem respiration (ER) and gross primary production (GPP) for the target population of order 5–7 non-wadeable rivers averaged −7.3 and 4.8 g O₂ m⁻² d⁻¹, respectively, with average P/R < 1 indicating dominance by heterotrophic processes. Ecosystem respiration was <−3.3 g O₂ m⁻² d⁻¹ for 75% of non-wadeable river length with around 20% of length between −10 and −20 g O₂ m⁻² d⁻¹. Cumulative distribution functions of cotton strength loss estimates indicated a more-or-less linear relationship with river km reflecting an even spread of decay rates (range in k 0.0007–0.2875 d⁻¹) across non-wadeable rivers regionally. A non-linear relationship with land cover was detected for GPP which was typically <5 g O₂ m⁻² d⁻¹ where natural vegetation cover was below 20% and greater than 80% of upstream catchment area. For cotton strength loss, the relationship with land cover was wedge-shaped such that sites with >60% natural cover had low decay rates (<0.02 d⁻¹) with variability below this increasing as natural cover declined. Using published criteria for assessing waterway health based on ER and GPP, 232–298 km (20–29%) of non-wadeable river length was considered to have severely impaired ecosystem functioning, and 436–530 km (42–50%) had no evidence of impact on river metabolism.     

Position—Specific contribution of interface tryptophans on membrane protein energetics

Interface tryptophans are key residues that facilitate the folding and stability of membrane proteins. Escherichia coli OmpX possesses two unique interface tryptophans, namely Trp76, which is present at the interface and is solvent-exposed, and Trp140, which is relatively more lipid solvated than Trp76 in symmetric lipid membranes. Here, we address the requirement for tryptophan and the consequences of aromatic amino acid substitutions on the folding and stability of OmpX. Using spectroscopic measurements of OmpX-Trp/Tyr/Phe mutants, we show that the specific mutation W76 → Y allows barrel assembly > 1.5-fold faster than native OmpX, and increases stability by ~ 0.4 kcal mol^− 1. In contrast, mutating W140 → F/Y lowers OmpX thermodynamic stability by ~ 0.4 kcal mol^− 1, without affecting the folding kinetics. We conclude that the stabilizing effect of tryptophan at the membrane interface can be position—and local environment—specific. We propose that the thermodynamic contributions for interface residues be interpreted with caution.     

Ecosystem functions as indicators for heathland responses to nitrogen fertilisation

Anthropogenic deposition of reactive nitrogen (N) has increased during the 20th century, and is considered an important driver of shifts in ecosystem functions and biodiversity loss. The objective of the present study was to identify those ecosystem functions that best evidence a target ecosystem’s sensitivity to N deposition, taking coastal heathlands as an example. We conducted a three-year field experiment in heathlands of the island Fehmarn (Baltic Sea, North Germany), which currently are subject to a background deposition of 9 kg N ha⁻¹ yr⁻¹. We experimentally applied six levels of N fertilisation (application of 0, 2.5, 5, 10, 20, and 50 kg N ha⁻¹ yr⁻¹), and quantified the growth responses of different plant species of different life forms (dwarf shrubs, graminoids, bryophytes, lichens) as well as shifts in the C:N ratios of plant tissue and humus horizons. For an applicability of the experimental findings (in terms of heathland management and critical load assessment) fertilisation effects on response variables were visualised by calculating the treatment ‘effect sizes’. The current year’s shoot increment of the dominant dwarf shrub Calluna vulgaris proved to be the most sensitive indicator to N fertilisation. Shoot increment significantly responded to additions of ≥ 5 kg N ha⁻¹ yr⁻¹ already in the first year, whereas flower formation of Calluna vulgaris increased only in the high-N treatments. Similarly, tissue C:N ratios of vascular plants (Calluna vulgaris and the graminoids Carex arenaria and Festuca ovina agg.) only decreased in the highest N treatments (50 and 20 kg N ha⁻¹ yr⁻¹, respectively). In contrast, tissue C:N ratios of cryptogams responded more quickly and sensitively than vascular plants. For example, Cladonia spp. tissue C:N ratios responded to N additions ≥ 5 kg N ha⁻¹ yr⁻¹ in the second study year. After three years we observed an increase in cover of graminoids and a corresponding decrease of cryptogams at N fertilisation rates of ≥ 10 kg N ha⁻¹ yr⁻¹. Soil C:N ratios proved to be an inappropriate indicator for N fertilisation at least within our three-year study period. Although current critical N loads for heathlands (10−20 kg N ha⁻¹ yr⁻¹) were confirmed in our experiment, the immediate and highly sensitive response of the current year’s shoots of Calluna vulgaris suggests that at least some ecosystem functions (e.g. dwarf shrub growth) also might respond to low (i.e. < 10 kg N ha⁻¹ yr⁻¹) but chronic inputs of N.     

Comparison of Zostera marina and maerl community metabolism

The photosynthetic and repiratory metabolism of Zostera marina and maerl communities was compared, in the same area of the Bay of Brest in March–April, using benthic chambers. P–E curves for both oxygen and carbon were established for bottom irradiances between 0 and 525 μmol m⁻² s⁻¹. An exponential function was fitted to calculate daily production. Community metabolic quotients did not differ for maerl and seagrass beds. Community photosynthetic quotients were significantly higher (1.19) whereas community respiratory quotients were lower (0.70) than 1. Maerl and seagrass bed P–E curves mainly differed by the minimum saturating irradiance (E_k). Net community production was estimated to 26.8 mmol C m⁻² d⁻¹ for Z. marina meadows and 8.6 mmol C m⁻² d⁻¹ for maerl beds. The two communities can, therefore, be considered as autotrophic during the March–April period. Community respiration did not differ between Z. marina meadows and maerl beds, with an average value of 53.8 mmol C m⁻² d⁻¹ during a day. In similar environmental conditions, the production of maerl beds corresponds to approximately one third that of seagrass meadows. The maerl communities, therefore, form productive ecosystems, relevant to temperate coastal ecosystems functioning.     

Monomerization and aggregation of β-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation

β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH 7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~ 55 Å, ~ 29 Å and ~ 37 Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57 kcal mol^− 1 and 8.93 kcal mol^− 1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~ 77 Å and ~ 117 Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69 kcal mol^− 1 and 9.09 kcal mol^− 1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.     

The low density lipoprotein receptor modulates the effects of hypogonadism on diet-induced obesity and related metabolic perturbations

Here, we investigated how LDL receptor deficiency (Ldlr^−/−) modulates the effects of testosterone on obesity and related metabolic dysfunctions. Though sham-operated Ldlr^−/− mice fed Western-type diet for 12 weeks became obese and showed disturbed plasma glucose metabolism and plasma cholesterol and TG profiles, castrated mice were resistant to diet-induced obesity and had improved glucose metabolism and reduced plasma TG levels, despite a further deterioration in their plasma cholesterol profile. The effect of hypogonadism on diet-induced weight gain of Ldlr^−/− mice was independent of ApoE and Lrp1. Indirect calorimetry analysis indicated that hypogonadism in Ldlr^−/− mice was associated with increased metabolic rate. Indeed, mitochondrial cytochrome c and uncoupling protein 1 expression were elevated, primarily in white adipose tissue, confirming increased mitochondrial metabolic activity due to thermogenesis. Testosterone replacement in castrated Ldlr^−/− mice for a period of 8 weeks promoted diet-induced obesity, indicating a direct role of testosterone in the observed phenotype. Treatment of sham-operated Ldlr^−/− mice with the aromatase inhibitor exemestane for 8 weeks showed that the obesity of castrated Ldlr^−/− mice is independent of estrogens. Overall, our data reveal a novel role of Ldlr as functional modulator of metabolic alterations associated with hypogonadism.