Reactive oxygen species‐induced changes in glucose and lipid metabolism contribute to the accumulation of cholesterol in the liver during aging

Aging is a major risk factor for many chronic diseases due to increased vulnerability to external stress and susceptibility to disease. Aging is associated with metabolic liver disease such as nonalcoholic fatty liver. In this study, we investigated changes in lipid metabolism during aging in mice and the mechanisms involved. Lipid accumulation was increased in liver tissues of aged mice, particularly cholesterol. Increased uptake of both cholesterol and glucose was observed in hepatocytes of aged mice as compared with younger mice. The mRNA expression of GLUT2, GK, SREBP2, HMGCR, and HMGCS, genes for cholesterol synthesis, was gradually increased in liver tissues during aging. Reactive oxygen species (ROS) increase with aging and are closely related to various aging‐related diseases. When we treated HepG2 cells and primary hepatocytes with the ROS inducer, H₂O₂, lipid accumulation increased significantly compared to the case for untreated HepG2 cells. H₂O₂ treatment significantly increased glucose uptake and acetyl‐CoA production, which results in glycolysis and lipid synthesis. Treatment with H₂O₂ significantly increased the expression of mRNA for genes related to cholesterol synthesis and uptake. These results suggest that ROS play an important role in altering cholesterol metabolism and consequently contribute to the accumulation of cholesterol in the liver during the aging process.     

Membrane cholesterol depletion as a trigger of Nav1.9 channel‐mediated inflammatory pain

Cholesterol is a major lipid component of the mammalian plasma membrane. While much is known about its metabolism, its transport, and its role in atherosclerotic vascular disease, less is known about its role in neuronal pathophysiology. This study reveals an unexpected function of cholesterol in controlling pain transmission. We show that inflammation lowers cholesterol content in skin tissue and sensory DRG culture. Pharmacological depletion of cellular cholesterol entails sensitization of nociceptive neurons and promotes mechanical and thermal hyperalgesia through the activation of voltage‐gated Nav1.9 channels. Inflammatory mediators enhance the production of reactive oxygen species and induce partitioning of Nav1.9 channels from cholesterol‐rich lipid rafts to cholesterol‐poor non‐raft regions of the membrane. Low‐cholesterol environment enhances voltage‐dependent activation of Nav1.9 channels leading to enhanced neuronal excitability, whereas cholesterol replenishment reversed these effects. Consistently, we show that transcutaneous delivery of cholesterol alleviates hypersensitivity in animal models of acute and chronic inflammatory pain. In conclusion, our data establish that membrane cholesterol is a modulator of pain transmission and shed a new light on the relationship between cholesterol homeostasis, inflammation, and pain.     

Rapid depletion of dissolved oxygen in 96‐well microtiter plate Staphylococcus epidermidis biofilm assays promotes biofilm development and is influenced by inoculum cell concentration

Biofilm‐related research using 96‐well microtiter plates involves static incubation of plates indiscriminate of environmental conditions, making oxygen availability an important variable which has not been considered to date. By directly measuring dissolved oxygen concentration over time we report here that dissolved oxygen is rapidly consumed in Staphylococcus epidermidis biofilm cultures grown in 96‐well plates irrespective of the oxygen concentration in the gaseous environment in which the plates are incubated. These data indicate that depletion of dissolved oxygen during growth of bacterial biofilm cultures in 96‐well plates may significantly influence biofilm production. Furthermore higher inoculum cell concentrations are associated with more rapid consumption of dissolved oxygen and higher levels of S. epidermidis biofilm production. Our data reveal that oxygen depletion during bacterial growth in 96‐well plates may significantly influence biofilm production and should be considered in the interpretation of experimental data using this biofilm model. Biotechnol. Bioeng. 2009;103: 1042–1047. © 2009 Wiley Periodicals, Inc.     

C1q/TNF‐related protein 9 inhibits the cholesterol‐induced Vascular smooth muscle cell phenotype switch and cell dysfunction by activating AMP‐dependent kinase

Vascular smooth muscle cells (VSMCs) switch to macrophage‐like cells after cholesterol loading, and this change may play an important role in the progression of atherosclerosis. C1q/TNF‐related protein 9 (CTRP9) is a recently discovered adipokine that has been shown to have beneficial effects on glucose metabolism and vascular function, particularly in regard to cardiovascular disease. The question of whether CTRP9 can protect VSMCs from cholesterol damage has not been addressed. In this study, the impact of CTRP9 on cholesterol‐damaged VSMCs was observed. Our data show that in cholesterol‐treated VSMCs, CTRP9 significantly reversed the cholesterol‐induced increases in pro‐inflammatory factor secretion, monocyte adhesion, cholesterol uptake and expression of the macrophage marker CD68. Meanwhile, CTRP9 prevented the cholesterol‐induced activation of the TLR4–MyD88–p65 pathway and upregulated the expression of proteins important for cholesterol efflux. Mechanistically, as siRNA‐induced selective gene ablation of AMPKα1 abolished these effects of CTRP9, we concluded that CTRP9 achieves these protective effects in VSMCs through the AMP‐dependent kinase (AMPK) pathway.     

Tissue lipid peroxidation and glutathione‐dependent enzyme status in patients with oral squamous cell carcinoma

We examined the extent of lipid peroxidation and the status of reduced glutathione (GSH) and the GSH‐dependent enzymes—glutathione peroxidase (GPx) and glutathione‐S‐transferase (GST)—in oral tumour tissues from 33 adult oral cancer patients and an equal number of age‐ and sex‐matched normal subjects. Diminished lipid peroxidation in the oral tumour tissue was accompanied by a significant decrease in phospholipids and an increase in the cholesterol/phospholipid (C/P) ratio. The concentration of glutathione and the activities of GPx and GST were elevated in oral tumour tissues. These findings suggest that GSH‐ and GSH‐dependent enzymes play a crucial role in tobacco‐related tumourigenesis and may be considered as markers of carcinogen exposure. Copyright © 1999 John Wiley & Sons, Ltd.     

Up‐regulation of β‐amyloidogenesis in neuron‐like human cells by both 24‐ and 27‐hydroxycholesterol: protective effect of N‐acetyl‐cysteine

An abnormal accumulation of cholesterol oxidation products in the brain of patients with Alzheimer's disease (AD) would further link an impaired cholesterol metabolism in the pathogenesis of the disease. The first evidence stemming from the content of oxysterols in autopsy samples from AD and normal brains points to an increase in both 27‐hydroxycholesterol (27‐OH) and 24‐hydroxycholesterol (24‐OH) in the frontal cortex of AD brains, with a trend that appears related to the disease severity. The challenge of differentiated SK‐N‐BE human neuroblastoma cells with patho‐physiologically relevant amounts of 27‐OH and 24‐OH showed that both oxysterols induce a net synthesis of Aβ_1‐42 by up‐regulating expression levels of amyloid precursor protein and β‐secretase, as well as the β‐secretase activity. Interestingly, cell pretreatment with N‐acetyl‐cysteine (NAC) fully prevented the enhancement of β‐amyloidogenesis induced by the two oxysterols. The reported findings link an impaired cholesterol oxidative metabolism to an excessive β‐amyloidogenesis and point to NAC as an efficient inhibitor of oxysterols‐induced Aβ toxic peptide accumulation in the brain.     

Respiration rate in human primary renal proximal and early distal tubular cells in vitro: considerations for biohybrid renal devices

Background: For biotechnological use of cells in tissue engineered applications, such as biohybrid renal devices, optimal culture conditions are required. Oxygen delivery is one of the most important cell determined system criterion for ex vivo applications. It is involved in the maintenance of highly oxygen‐dependent renal tubular epithelial cells, affecting metabolic state, differentiation, and desired transport functions. The purpose of this study was to examine respiratory patterns such as basal oxygen consumption, solute transport‐related oxygen demand, and oxygen concentration‐dependent oxygen uptake of renal tubular epithelial cells in vitro. Methods: Respiratory patterns of highly purified human primary renal proximal (hPTC) and early distal tubular cells (hTALDC) were analyzed by perfusion respirometry. Spontaneous oxygen consumptions and maximum respirations after carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) uncoupling were measured. Respiration fractions contributing to basolateral Na⁺/K⁺‐ATPase transport activities were assessed via ouabain inhibition and Na⁺‐free medium. Furthermore, we determined oxygen uptake in dependency of oxygen concentration and morphology in various culture conditions (shaken, static). Results: Respiration of solely hPTC strongly depended on oxygen concentration in a Michaelis‐Menten pattern at noncritical oxygen concentrations. Respiration of both cell types was significantly increased by CCCP, whereas average Na⁺/K⁺‐ATPase‐based oxygen uptake fractions differ significantly between the two cell types. Nevertheless, no significant differences were found in spontaneous respiration between hPTC and hTALDC. Conclusions: Our results clearly indicate that cell‐specific oxygen consumption parameters have to be considered in the design of biotechnological devices intended to support kidney function by cell‐supported renal replacement therapy. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Phospholipid peroxidation as a factor in gallstone pathogenesis

Phospholipid peroxidation markedly reduces the stability of mixed micellar systems composed of cholate, phosphatidylcholine and supersaturating levels of cholesterol. This suggests that lipid peroxidation is likely to play a significant role in the precipitation of cholesterol from gallbladder bile, thus in the pathogenesis of cholesterol gallstones. This conclusion is supported by studies of the nucleation time of cholesterol in gallbladder biles, which was significantly reduced by exposure to a stream of oxygen. This effect of phospholipid peroxidation on cholesterol solubility may occur in other biological fluids as well. In view of the increased lipid peroxidation in the elderly, it may explain the effect of age on the frequency of various diseases related to cholesterol precipitation.     

Sex‐dependent Associations of Leptin With Metabolic Syndrome–related Variables: The Stanislas Study

Serum leptin has been reported to be associated in a sex‐dependent manner with C‐reactive protein (CRP), independently of adiposity. We tested the hypothesis that leptin is associated, independently of anthropometry indexes and in a sex‐dependent way, with other inflammatory markers and variables related to metabolic syndrome (MS). In 384 healthy middle‐aged adults (192 men and 192 women) total fat mass (FM), waist circumference (WC), serum leptin and 15 MS‐related parameters (systolic and diastolic blood pressure, triglycerides, cholesterol, high density lipoprotein (HDL)‐cholesterol, apo AI and B, fasting glucose, uric acid, CRP, orosomucoid and haptoglobin levels and aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and γ‐glutamyl transferase (GGT) activities) were measured. After adjustment for age, alcohol and cigarette consumption, WC, and total FM, leptin concentration was significantly associated with serum triglycerides, total cholesterol, apo B, uric acid and haptoglobin concentrations and liver enzyme activity only in men, and with apo AI, HDL‐cholesterol (only borderline) and CRP only in women. Sex interaction terms were significant for total cholesterol, apo B, HDL cholesterol, uric acid, ALAT and GGT, and borderline significant for triglycerides, apo AI and ASAT. In this healthy population, leptin is significantly associated with various MS factors, independently of WC and total FM, depending on gender. Our study provides further evidence of sex‐related differences mediated by leptin in inflammatory mechanisms and other MS‐related metabolic pathways.     

Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism

The effect of the cholesterol content of the plasma membrane on the intracellular concentration of oxygen in Chinese hamster ovary (CHO) cells and their mutants was investigated by EPR oximetry. Total and free cholesterol content was significantly higher in 25 RA CHO cells as compared to wild-type and M 19 CHO cells, with most of the free cholesterol in normal and mutant CHO cells located in the plasma membrane. The plasma membrane cholesterol content also was altered by various biochemical means, and the effect on the oxygen gradient was studied. Comparing the three cell lines, the gradient was larger with increased content of cholesterol in the plasma cell membrane. This result also is supported by an additional increase in the oxygen gradients with the incorporation of additional cholesterol in the plasma membrane and a decrease in the oxygen gradient when the cholesterol was depleted from the plasma membrane. The results indicate that the concentration of cholesterol in the plasma membrane can be an important factor for the magnitude of the oxygen gradient observed across the cell membrane.     

Defining Health‐Related Obesity in Prepubertal Children

Objective: The purpose of this study was to develop percentage of fat and waist circumference cut‐points in prepubertal children with the intention of defining obesity associated with cardiovascular disease (CVD) risk. Research Methods and Procedures: A cross‐sectional analysis of 87 prepubertal children aged 4 to 11 years was used. Percentage of body fat was determined by DXA. Waist circumference was measured to the nearest millimeter. Receiver Operating Characteristic analyses of percentage of fat and waist circumference were used to develop cut‐points for individuals with adverse levels of CVD risk factors. Results: The risk factors selected for analyses (i.e., fasting insulin, high‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol, triglycerides, and total cholesterol/high‐density lipoprotein cholesterol) were significantly related to percentage of body fat and waist circumference. Likelihood ratios were used to identify percentage of fat and waist circumference cut‐points associated with adverse cardiovascular risk profiles. Two cut‐points, an upper cut‐point of 33% body fat and a lower cut‐point of 20% body fat, were derived. Waist circumference cut‐points indicative of adverse and normal risk‐factor profiles were 71 cm and 61 cm, respectively. Discussion: The data indicate that children with ≥33% body fat and children with a waist circumference ≥71 cm were more likely to possess an adverse CVD risk‐factor profile than a normal risk‐factor profile. The likelihood of children with <20% body fat or a waist circumference <61 cm possessing an adverse CVD risk‐factor profile as opposed to a normal risk‐factor profile was small. The cut‐points describe an adequate health‐related definition of childhood obesity.     

The influence of oxygen supply on metabolism of neural cells cultured on a gas‐permeable PTFE foil

The influence of oxygen on neural stem cell proliferation, differentiation, and apoptosis is of great interest for regenerative therapies in neurodegenerative disorders, such as Parkinson's disease. These oxygen depending mechanisms have to been considered for the optimization of neural cell culture conditions. In this study, we used a cell culture system with an oxygen‐permeable polytetrafluorethylene (PTFE) foil to investigate the effect of oxygen on metabolism and survival of neural cell lines in vitro. Human glial astrocytoma‐derived cells (GOS‐3) and rat pheochromacytoma cells (PC12) were cultured on the gas‐permeable PTFE foil as well as a conventional non oxygen‐permeable cell culture substrate at various oxygen concentrations. Analyses of metabolic activity, gene expression of apoptotic grade, and dopamine synthesis were performed. Under low oxygen partial pressure (2%, 5%) the anaerobic metabolism and apoptotic rate of cultured cells is diminished on PTFE foil when compared with the conventional culture dishes. In contrast, under higher oxygen atmosphere (21%) the number of apoptotic cells on the PTFE foil was enhanced. This culture model demonstrates a suitable model for the improvement of oxygen dependent metabolism under low oxygen conditions as well as for induction of oxidative stress by high oxygen atmosphere without supplementation of neurotoxins. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Oxysterols, cholesterol biosynthesis, and vascular endothelial cell monolayer barrier function

A spectrum of cholesterol oxidation derivatives (oxysterols) is generated in food products exposed to heat or radiation in the presence of oxygen. One of these derivatives (cholestan-3 beta,5 alpha,6 beta-triol) was shown to compromise the selective barrier function of cultured vascular endothelial cell monolayers, an action that may initiate atherosclerotic lesion formation. This study sought to investigate the relationship of cholesterol synthesis inhibition by several naturally occurring oxysterols to depression of vascular endothelial cell monolayer barrier function, determined as an increase in albumin transfer across cultured endothelial monolayers. All oxysterols tested caused a variable time- and dose-dependent elevation in trans-endothelial albumin transfer, and they were also able to inhibit cholesterol biosynthesis to varying degrees. Pure cholesterol was without effect on both counts. The correlation between the increase in albumin transfer related to oxysterol exposure and the ability of oxysterols to suppress cholesterol biosynthesis was, however, poor. Moreover, mevinolin, a water-soluble competitive inhibitor of cholesterol synthesis, reduced the rate of cholesterol synthesis to 0.9% of control but did not significantly increase albumin transfer. Cholestan-3 beta,5 alpha,6 beta-triol caused a 660% elevation in albumin transfer while cholesterol synthesis remained at 11% of control. We conclude that changes in endothelial barrier function caused by exposure to the oxysterols examined, but not pure cholesterol, are probably related to factors other than the well-known action of cholesterol biosynthesis inhibition. These findings may have implications in the development of atherosclerosis.     

FGF21 induces autophagy‐mediated cholesterol efflux to inhibit atherogenesis via RACK1 up‐regulation

Fibroblast growth factor 21 (FGF21) acts as an anti‐atherosclerotic agent. However, the specific mechanisms governing this regulatory activity are unclear. Autophagy is a highly conserved cell stress response which regulates atherosclerosis (AS) by reducing lipid droplet degradation in foam cells. We sought to assess whether FGF21 could inhibit AS by regulating cholesterol metabolism in foam cells via autophagy and to elucidate the underlying molecular mechanisms. In this study, ApoE^?/? mice were fed a high‐fat diet (HFD) with or without FGF21 and FGF21 + 3‐Methyladenine (3MA) for 12 weeks. Our results showed that FGF21 inhibited AS in HFD‐fed ApoE^?/? mice, which was reversed by 3MA treatment. Moreover, FGF21 increased plaque RACK1 and autophagy‐related protein (LC3 and beclin‐1) expression in ApoE^?/? mice, thus preventing AS. However, these proteins were inhibited by LV‐RACK1 shRNA injection. Foam cell development is a crucial determinant of AS, and cholesterol efflux from foam cells represents an important defensive measure of AS. In this study, foam cells were treated with FGF21 for 24 hours after a pre‐treatment with 3MA, ATG5 siRNA or RACK1 siRNA. Our results indicated that FGF21‐induced autophagy promoted cholesterol efflux to reduce cholesterol accumulation in foam cells by up‐regulating RACK1 expression. Interestingly, immunoprecipitation results showed that RACK1 was able to activate AMPK and interact with ATG5. Taken together, our results indicated that FGF21 induces autophagy to promote cholesterol efflux and reduce cholesterol accumulation in foam cells through RACK1‐mediated AMPK activation and ATG5 interaction. These results provided new insights into the molecular mechanisms of FGF21 in the treatment of AS.     

Pex11a deficiency causes dyslipidaemia and obesity in mice

Peroxisomes play a central role in lipid metabolism. We previously demonstrated that Pex11a deficiency impairs peroxisome abundance and fatty acid β‐oxidation and results in hepatic triglyceride accumulation. The role of Pex11a in dyslipidaemia and obesity is investigated here with Pex11a knockout mice (Pex11a^?/?). Metabolic phenotypes including tissue weight, glucose tolerance, insulin sensitivity, cholesterol levels, fatty acid profile, oxygen consumption, physical activity were assessed in wild‐type (WT) and Pex11a^?/? fed with a high‐fat diet. Molecular changes and peroxisome abundance in adipose tissue were evaluated through qRT‐PCR, Western blotting, and Immunofluorescence. Pex11a^?/? showed increased fat mass, decreased skeletal muscle, higher cholesterol levels, and more severely impaired glucose and insulin tolerance. Pex11a^?/? consumed less oxygen, indicating a decrease in fatty acid oxidation, which is consistent with the accumulation of very long‐ and long‐chain fatty acids. Adipose palmitic acid (C16:0) levels were elevated in Pex11a^?/?, which may be because of dramatically increased fatty acid synthase mRNA and protein levels. Furthermore, Pex11a deficiency increased ventricle size and macrophage infiltration, which are related to the reduced physical activity. These data demonstrate that Pex11a deficiency impairs physical activity and energy expenditure, decreases fatty acid β‐oxidation, increases de novo lipogenesis and results in dyslipidaemia and obesity.     

STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

StAR‐related lipid transfer domain‐3 (STARD3) is a sterol‐binding protein that creates endoplasmic reticulum (ER)–endosome contact sites. How this protein, at the crossroad between sterol uptake and synthesis pathways, impacts the intracellular distribution of this lipid was ill‐defined. Here, by using in situ cholesterol labeling and quantification, we demonstrated that STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane. STARD3‐mediated cholesterol routing depends both on its lipid transfer activity and its ability to create ER–endosome contacts. Corroborating this, in vitro reconstitution assays indicated that STARD3 and its ER‐anchored partner, Vesicle‐associated membrane protein‐associated protein (VAP), assemble into a machine that allows a highly efficient transport of cholesterol within membrane contacts. Thus, STARD3 is a cholesterol transporter scaffolding ER–endosome contacts and modulating cellular cholesterol repartition by delivering cholesterol to endosomes.     

Hyperglycemia accelerates ATP‐binding cassette transporter A1 degradation via an ERK‐dependent pathway in macrophages

An elevation in blood glucose concentration leads to increased risk of developing diabetes‐associated atherosclerotic cardiovascular disease due to an excessive accumulation of cholesterol in arterial macrophages. ATP‐binding cassette transporter A1 (ABCA1) is an atheroprotective protein that mediates the export of cholesterol from macrophages. The present study aims to investigate the effect of hyperglycemia on the regulation of ABCA1 expression and to explore its underlying mechanisms of regulation in macrophages. Our results show that high glucose activates the extracellular signal‐regulated kinases (ERK) signaling pathway via reactive oxygen species (ROS) production, which in turn down‐regulates ABCA1 mRNA and protein expression. This down‐regulation is mediated by accelerating ABCA1 mRNA and protein degradation in macrophages exposed to high concentrations of glucose. Our results provide evidence for the first time that hyperglycemia inhibits ABCA1 expression by ERK‐modulated ABCA1 mRNA and protein stability. Overall, these results provide a mechanism for hyperglycemia‐induced reduction in ABCA1 expression, which suggests a promising strategy for the treatment of diabetes‐associated atherosclerosis. J. Cell. Biochem. 114: 1364–1373, 2013. © 2012 Wiley Periodicals, Inc.     

Long‐term Exercise Training in Overweight Adolescents Improves Plasma Peptide YY and Resistin

The objective of this study was to investigate the effect of long‐term exercise training on concentrations of five hormones related to appetite and insulin resistance in overweight adolescents. In addition, we were interested in the relationships of these hormones with each other and with anthropometric and/or cardiovascular disease marker changes. Participants were ≥ the 85th percentile for BMI for age and sex and participated in an 8‐month supervised aerobic training program. Anthropometrics, cardiovascular fitness assessment, and fasting blood samples were taken pre‐ and post‐training. Glucose, insulin, total cholesterol (TC), high‐density lipoprotein (HDL) cholesterol, low‐density lipoprotein (LDL) cholesterol, triglycerides, leptin, active ghrelin, total peptide YY (PYY), adiponectin, and resistin concentrations were measured. The participants increased their time to exhaustion on an incremental treadmill test and decreased both percent body fat and blood triglyceride concentrations. Total PYY concentration increased and resistin concentration decreased after long‐term exercise training, which are favorable outcomes. Leptin concentrations were related to weight, percent body fat, waist circumference, and triglyceride concentrations pre‐ and post‐training. The changes in resistin concentrations were related to the changes in triglyceride concentrations. We conclude that long‐term exercise training has beneficial effects for overweight adolescents with respect to PYY and resistin, hormones related to appetite and insulin sensitivity.