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1.  Dietary Non-Esterified Oleic Acid Decreases the Jejunal Levels of Anorectic N-Acylethanolamines 
PLoS ONE  2014;9(6):e100365.
Background and Aims
Oleoylethanolamide and several other N-acylethanolamines (NAEs), e.g. linoleoylethanolamide and palmitoylethanolamide, have anorectic properties in rats, and prolonged intake of a high-fat diet decreases the levels of the anorectic NAEs in jejunum. Jejunal anorectic NAEs are thought to add to the control of food intake via activation of PPARalpha and the vagus nerve. The fat-induced decrease may explain part of the hyperphagic effect of high-fat diets. In the present study, we investigated 1) whether the reduced levels of anorectic NAEs were reversible in rats, 2) whether mice respond to dietary fat (olive oil) by reducing levels of anorectic NAEs, and 3) whether dietary non-esterified oleic acid also can decrease levels of anorectic NAEs in mice. We are searching for the fat sensor in the intestine, which mediates the decreased levels of anorectic NAEs.
Male rats and mice were fed diets high (45 energy% fat) in either triacylglycerol or free fatty acids for 7–14 days, and jejunal NAE and N-acylphosphatidylethanolamine (NAPE) levels were determined by liquid-chromatography mass spectrometry.
In rats, reduced levels of anorectic NAEs could be reversed after 3 days from changing the diet from high-fat to chow. Corresponding NAPE levels tended to show the same changes. In mice, jejunal levels of anorectic NAEs were also reduced when fed a high-fat diet. In addition, we found that non-esterified oleic acid were also able to reduce levels of anorectic NAEs in mice.
These results suggest that the down-regulation of the jejunal level of anorectic NAEs by dietary fat is not restricted to rats, and that the fatty acid component oleic acid, in dietary olive oil may be sufficient to mediate this regulation. Thus, a fatty acid sensor may mediate this effect of dietary fat.
PMCID: PMC4068999  PMID: 24959837
2.  Hypothalamic food intake regulation in a cancer-cachectic mouse model 
Appetite is frequently affected in cancer patients leading to anorexia and consequently insufficient food intake. In this study, we report on hypothalamic gene expression profile of a cancer-cachectic mouse model with increased food intake. In this model, mice bearing C26 tumour have an increased food intake subsequently to the loss of body weight. We hypothesise that in this model, appetite-regulating systems in the hypothalamus, which apparently fail in anorexia, are still able to adapt adequately to changes in energy balance. Therefore, studying changes that occur on appetite regulators in the hypothalamus might reveal targets for treatment of cancer-induced eating disorders. By applying transcriptomics, many appetite-regulating systems in the hypothalamus could be taken into account, providing an overview of changes that occur in the hypothalamus during tumour growth.
C26-colon adenocarcinoma cells were subcutaneously inoculated in 6 weeks old male CDF1 mice. Body weight and food intake were measured three times a week. On day 20, hypothalamus was dissected and used for transcriptomics using Affymetrix chips.
Food intake increased significantly in cachectic tumour-bearing mice (TB), synchronously to the loss of body weight. Hypothalamic gene expression of orexigenic neuropeptides NPY and AgRP was higher, whereas expression of anorexigenic genes CCK and POMC were lower in TB compared to controls.
In addition, serotonin and dopamine signalling pathways were found to be significantly altered in TB mice. Serotonin levels in brain showed to be lower in TB mice compared to control mice, while dopamine levels did not change. Moreover, serotonin levels inversely correlated with food intake.
Transcriptomic analysis of the hypothalamus of cachectic TB mice with an increased food intake showed changes in NPY, AgRP and serotonin signalling. Serotonin levels in the brain showed to correlate with changes in food intake. Further research has to reveal whether targeting these systems will be a good strategy to avoid the development of cancer-induced eating disorders.
Electronic supplementary material
The online version of this article (doi:10.1007/s13539-013-0121-y) contains supplementary material.
PMCID: PMC4053566  PMID: 24222472
Cancer; Hypothalamus; Appetite; Serotonin; Transcriptomics; Anorexia
3.  Tracking of a Dietary Pattern and Its Components over 10-Years in the Severely Obese 
PLoS ONE  2014;9(5):e97457.
Understanding how dietary intake changes over time is important for studies of diet and disease and may inform interventions to improve dietary intakes. We investigated how a dietary pattern (DP) tracked over 10-years in the Swedish Obese Subjects (SOS) study control group. Dietary intake was assessed at multiple time-points in 2037 severely obese individuals (BMI 41±4 kg/m2). Reduced rank regression was used to derive a dietary pattern using dietary energy density (kJ/g), saturated fat (%) and fibre density (mg/kJ) as response variables and score respondents at each follow-up. Tracking coefficients for the DP, its key foods and macronutrient response variables and corrected for time-dependent and time-independent covariates were calculated using generalised estimating equations to take into account all available data. The DP tracking coefficient was moderate for women (0.40; 95% CI: 0.38–0.42) and men (0.38; 95% CI: 0.35–0.41). Of the eleven foods key to this DP, fruit and vegetable intakes had the strongest tracking coefficient for both sexes. Fast food and candy had the lowest tracking coefficients for women and men respectively. Scores for an energy dense, high saturated fat, low fibre density DP appear moderately stable over a 10-year period in this severely obese population. Furthermore, some food groups appear more amenable to change while others, often the most healthful, appear more stable and may require intervention before adulthood.
PMCID: PMC4026226  PMID: 24841709
4.  Short Term Feeding of a High Fat Diet Exerts an Additive Effect on Hepatocellular Damage and Steatosis in Liver-Specific PTEN Knockout Mice 
PLoS ONE  2014;9(5):e96553.
Hepatospecific deletion of PTEN results in constitutive activation of Akt and increased lipogenesis. In mice, the addition of a high fat diet (HFD) downregulates lipogenesis. The aim of this study was to determine the effects of a HFD on hepatocellular damage induced by deletion of PTEN.
12 Week old male flox/flox hepatospecific PTEN mice (PTENf/f) or Alb-Cre controls were fed a HFD composed of 45% fat-derived calories (from corn oil) or a normal chow. Animals were then analyzed for hepatocellular damage, oxidative stress and expression of enzymes involved in fatty acid metabolism.
In the Alb-Cre animals, the addition of a HFD resulted in a significant increase in liver triglycerides and altered REDOX capacity as evidenced by increased GPX activity, decreased GST activity and decreased hepatic concentrations of GSSG. In addition, SCD2, ACLY and FASN were all downregulated by the addition of HFD. Furthermore, expression of PPARα and PPARα-dependent proteins Cyp4a and ACSL1 were upregulated. In the PTENf/f mice, HFD resulted in significant increased in ALT, serum triglycerides and decreased REDOX capacity. Although expression of fatty acid synthetic enzymes was elevated in the chow fed PTENf/f group, the addition of HFD resulted in SCD2, ACLY and FASN downregulation. Compared to the Alb-Cre HFD group, expression of PGC1α, PPARα and its downstream targets ACSL and Cyp4a were upregulated in PTENf/f mice.
These data suggest that during conditions of constitutive Akt activation and increased steatosis, the addition of a HFD enhances hepatocellular damage due to increased CD36 expression and altered REDOX status. In addition, this work indicates HFD-induced hepatocellular damage occurs in part, independently of Akt signaling.
PMCID: PMC4018288  PMID: 24818992
5.  Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs 
Age  2014;36(3):9648.
Aging is a progressive process that results in the accumulation of intra- and extracellular alterations that in turn contribute to a reduction in health. Age-related changes in DNA methylation have been reported before and may be responsible for aging-induced changes in gene expression, although a causal relationship has yet to be shown. Using genome-wide assays, we analyzed age-induced changes in DNA methylation and their effect on gene expression with and without transient induction with the synthetic transcription modulating agent WY14,643. To demonstrate feasibility of the approach, we isolated peripheral blood mononucleated cells (PBMCs) from five young and five old healthy male volunteers and cultured them with or without WY14,643. Infinium 450K BeadChip and Affymetrix Human Gene 1.1 ST expression array analysis revealed significant differential methylation of at least 5 % (ΔYO > 5 %) at 10,625 CpG sites between young and old subjects, but only a subset of the associated genes were also differentially expressed. Age-related differential methylation of previously reported epigenetic biomarkers of aging including ELOVL2, FHL2, PENK, and KLF14 was confirmed in our study, but these genes did not display an age-related change in gene expression in PBMCs. Bioinformatic analysis revealed that differentially methylated genes that lack an age-related expression change predominantly represent genes involved in carcinogenesis and developmental processes, and expression of most of these genes were silenced in PBMCs. No changes in DNA methylation were found in genes displaying transiently induced changes in gene expression. In conclusion, aging-induced differential methylation often targets developmental genes and occurs mostly without change in gene expression.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-014-9648-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4082572  PMID: 24789080
Molecular aging; Epigenetics; DNA methylation; Gene expression; PBMCs; Epigenetic biomarkers of aging
6.  Comparison of Liver Fat Indices for the Diagnosis of Hepatic Steatosis and Insulin Resistance 
PLoS ONE  2014;9(4):e94059.
Hepatic steatosis, defined as increased hepatocellular lipid content (HCL), associates with visceral obesity and glucose intolerance. As exact HCL quantification by 1H-magnetic resonance spectroscopy (1H-MRS) is not generally available, various clinical indices are increasingly used to predict steatosis.
The purpose of this study was to test the accuracy of NAFLD liver fat score (NAFLD-LFS), hepatic steatosis index (HSI) and fatty liver index (FLI) against 1H-MRS and their relationships with insulin sensitivity and secretion.
Design, Setting and Participants
Ninety-two non-diabetic, predominantly non-obese humans underwent clinical examination, 1H-MRS and an oral glucose tolerance test (OGTT) to calculate insulin sensitivity and β-cell function. Accuracy of indices was assessed from the area under the receiver operating characteristic curve (AROC).
Median HCL was 2.49% (0.62;4.23) and correlated with parameters of glycemia across all subjects. NAFLD-LFS, FLI and HSI yielded AROCs of 0.70, 0.72, and 0.79, respectively, and related positively to HCL, insulin resistance, fasting and post-load β-cell function normalized for insulin resistance. Upon adjustment for age, sex and HCL, regression analysis revealed that NAFLD-LFS, FLI and HSI still independently associated with both insulin sensitivity and β-cell function.
The tested indices offer modest efficacy to detect steatosis and cannot substitute for fat quantification by 1H-MRS. However, all indices might serve as surrogate parameters for liver fat content and also as rough clinical estimates of abnormal insulin sensitivity and secretion. Further validation in larger collectives such as epidemiological studies is needed.
PMCID: PMC3986069  PMID: 24732091
7.  Screw placement in percutaneous acetabular surgery: gender differences of anatomical landmarks in a cadaveric study 
International Orthopaedics  2012;37(4):673-679.
Percutaneous reduction and periarticular screw implantation techniques have been successfully introduced in acetabular surgery. The advantages of this less invasive approach are attenuated by higher risks of screw misplacement. Anatomical landmarks are strongly needed to prevent malplacement. This cadaver study was designed to identify reliable anatomical osseous landmarks in the pelvic region for screw placement in acetabular surgery. Gender differences were specifically addressed.
Twenty-seven embalmed cadaveric hemipelvic specimens (13 male, 14 female) were used. After soft-tissue removal, anterior and posterior column acetabular screw placement was conducted by one orthopaedic trauma surgeon under direct vision. Each column was addressed by antegrade and retrograde screw insertion. Radiographic verification of ideal screw placement was followed by assessment of the distance from the different entry points to adjoining anatomical osseous structures.
For anterior column screw positioning, the posterior superior iliac spine (PSIS), posterior inferior iliac spine (PIIS), iliopectineal eminence and centre of the symphysis were most reliable regarding gender differences. For posterior column screw positioning, the distance to the anterior superior iliac spine (ASIS) and the ischial tuberosity showed the lowest deviation between the different gender specimens. Highest gender differences were seen in relation to the cranial rim of the superior pubic ramus in retrograde anterior column screw positioning (p = 0.002). Most landmarks could be targeted within a 2.5-cm range in all specimens.
The findings emphasise the relevance of osseous landmarks in acetabular surgery. By adhering to easily identifiable structures, screw placement can be safely performed. Significant gender differences must be taken into consideration during preoperative planning.
PMCID: PMC3609967  PMID: 23250351
8.  Predictors of Full Enteral Feeding Achievement in Very Low Birth Weight Infants 
PLoS ONE  2014;9(3):e92235.
To elucidate the role of prenatal, neonatal and early postnatal variables in influencing the achievement of full enteral feeding (FEF) in very low birth weight (VLBW) infants and to determine whether neonatal intensive care units (NICUs) differ in this outcome.
Population-based retrospective cohort study using data on 1,864 VLBW infants drawn from the “Emilia-Romagna Perinatal Network” Registry from 2004 to 2009. The outcome of interest was time to FEF achievement. Eleven prenatal, neonatal and early postnatal variables and the study NICUs were selected as potential predictors of time to FEF. Parametric survival analysis was used to model time to FEF as a function of the predictors. Marginal effects were used to obtain adjusted estimates of median time to FEF for specific subgroups of infants.
Lower gestational age, exclusive formula feeding, higher CRIB II score, maternal hypertension, cesarean delivery, SGA and PDA predicted delayed FEF. NICUs proved to be heterogeneous in terms of FEF achievement. Newborns with PDA had a 4.2 days longer predicted median time to FEF compared to those without PDA; newborns exclusively formula-fed had a 1.4 days longer time to FEF compared to those fed human milk.
The results of our study suggest that time to FEF is influenced by clinical variables and NICU-specific practices. Knowledge of the variables associated with delayed/earlier FEF achievement could help in improving specific aspects of routine clinical management of VLBW infants and to reduce practice variability.
PMCID: PMC3960219  PMID: 24647523
9.  Usage of Plant Food Supplements across Six European Countries: Findings from the PlantLIBRA Consumer Survey 
PLoS ONE  2014;9(3):e92265.
The popularity of botanical products is on the rise in Europe, with consumers using them to complement their diets or to maintain health, and products are taken in many different forms (e.g. teas, juices, herbal medicinal products, plant food supplements (PFS)). However there is a scarcity of data on the usage of such products at European level.
To provide an overview of the characteristics and usage patterns of PFS consumers in six European countries.
Data on PFS usage were collected in a cross-sectional, retrospective survey of PFS consumers using a bespoke frequency of PFS usage questionnaire.
A total sample of 2359 adult PFS consumers from Finland, Germany, Italy, Romania, Spain and the United Kingdom.
Data analyses
Descriptive analyses were conducted, with all data stratified by gender, age, and country. Absolute frequencies, percentages and 95% confidence intervals are reported.
Overall, an estimated 18.8% of screened survey respondents used at least one PFS. Characteristics of PFS consumers included being older, well-educated, never having smoked and self-reporting health status as “good or very good”. Across countries, 491 different botanicals were identified in the PFS products used, with Ginkgo biloba (Ginkgo), Oenothera biennis (Evening primrose) and Cynara scolymus (Artichoke) being most frequently reported; the most popular dose forms were capsules and pills/tablets. Most consumers used one product and half of all users took single-botanical products. Some results varied across countries.
The PlantLIBRA consumer survey is unique in reporting on usage patterns of PFS consumers in six European countries. The survey highlights the complexity of measuring the intake of such products, particularly at pan-European level. Incorporating measures of the intake of botanicals in national dietary surveys would provide much-needed data for comprehensive risk and benefit assessments at the European level.
PMCID: PMC3958487  PMID: 24642692
10.  Acute Post-Exercise Myofibrillar Protein Synthesis Is Not Correlated with Resistance Training-Induced Muscle Hypertrophy in Young Men 
PLoS ONE  2014;9(2):e89431.
Muscle hypertrophy following resistance training (RT) involves activation of myofibrillar protein synthesis (MPS) to expand the myofibrillar protein pool. The degree of hypertrophy following RT is, however, highly variable and thus we sought to determine the relationship between the acute activation of MPS and RT-induced hypertrophy. We measured MPS and signalling protein activation after the first session of resistance exercise (RE) in untrained men (n = 23) and then examined the relation between MPS with magnetic resonance image determined hypertrophy. To measure MPS, young men (24±1 yr; body mass index  = 26.4±0.9 kg•m2) underwent a primed constant infusion of L-[ring-13C6] phenylalanine to measure MPS at rest, and acutely following their first bout of RE prior to 16 wk of RT. Rates of MPS were increased 235±38% (P<0.001) above rest 60–180 min post-exercise and 184±28% (P = 0.037) 180–360 min post exercise. Quadriceps volume increased 7.9±1.6% (−1.9–24.7%) (P<0.001) after training. There was no correlation between changes in quadriceps muscle volume and acute rates of MPS measured over 1–3 h (r = 0.02), 3–6 h (r = 0.16) or the aggregate 1–6 h post-exercise period (r = 0.10). Hypertrophy after chronic RT was correlated (r = 0.42, P = 0.05) with phosphorylation of 4E-BP1Thr37/46 at 1 hour post RE. We conclude that acute measures of MPS following an initial exposure to RE in novices are not correlated with muscle hypertrophy following chronic RT.
PMCID: PMC3933567  PMID: 24586775
11.  Hepatic Glucose Intolerance Precedes Hepatic Steatosis in the Male Aromatase Knockout (ArKO) Mouse 
PLoS ONE  2014;9(2):e87230.
Estrogens are known to play a role in modulating metabolic processes within the body. The Aromatase knockout (ArKO) mice have been shown to harbor factors of Metabolic syndrome with central adiposity, hyperinsulinemia and male-specific hepatic steatosis. To determine the effects of estrogen ablation and subsequent replacement in males on whole body glucose metabolism, three- and six-month-old male ArKO mice were subjected to whole body glucose, insulin and pyruvate tolerance tests and analyzed for ensuing metabolic changes in liver, adipose tissue, and skeletal muscle. Estrogen-deficient male ArKO mice showed increased gonadal adiposity which was significantly reduced upon 17β-estradiol (E2) treatment. Concurrently, elevated ArKO serum leptin levels were significantly reduced upon E2 treatment and lowered serum adiponectin levels were restored to wild type levels. Three-month-old male ArKO mice were hyperglycemic, and both glucose and pyruvate intolerant. These phenotypes continued through to 6 months of age, highlighting a loss of glycemic control. ArKO livers displayed changes in gluconeogenic enzyme expression, and in insulin signaling pathways upon E2 treatment. Liver triglycerides were increased in the ArKO males only after 6 months of age, which could be reversed by E2 treatment. No differences were observed in insulin-stimulated ex vivo muscle glucose uptake nor changes in ArKO adipose tissue and muscle insulin signaling pathways. Therefore, we conclude that male ArKO mice develop hepatic glucose intolerance by the age of 3 months which precedes the sex-specific development of hepatic steatosis. This can be reversed upon the administration of exogenous E2.
PMCID: PMC3919708  PMID: 24520329
12.  Nur77 Decreases Atherosclerosis Progression in apoE−/− Mice Fed a High-Fat/High-Cholesterol Diet 
PLoS ONE  2014;9(1):e87313.
It is clear that lipid disorder and inflammation are associated with cardiovascular diseases and underlying atherosclerosis. Nur77 has been shown to be involved in inflammatory response and lipid metabolism.
Here, we explored the role of Nur77 in atherosclerotic plaque progression in apoE−/− mice fed a high-fat/high cholesterol diet.
Methods and Results
The Nur77 gene, a nuclear hormone receptor, was highly induced by treatment with Cytosporone B (Csn-B, specific Nur77 agonist), recombinant plasmid over-expressing Nur77 (pcDNA-Nur77), while inhibited by treatment with siRNAs against Nur77 (si-Nur77) in THP-1 macrophage-derived foam cells, HepG2 cells and Caco-2 cells, respectively. In addition, the expression of Nur77 was highly induced by Nur77 agonist Csn-B, lentivirus encoding Nur77 (LV-Nur77), while silenced by lentivirus encoding siRNA against Nur77 (si-Nur77) in apoE−/− mice fed a high-fat/high cholesterol diet, respectively. We found that increased expression of Nur77 reduced macrophage-derived foam cells formation and hepatic lipid deposition, downregulated gene levels of inflammatory molecules, adhesion molecules and intestinal lipid absorption, and decreases atherosclerotic plaque formation.
These observations provide direct evidence that Nur77 is an important nuclear hormone receptor in regulation of atherosclerotic plaque formation and thus represents a promising target for the treatment of atherosclerosis.
PMCID: PMC3909091  PMID: 24498071
13.  Saliva from Obese Individuals Suppresses the Release of Aroma Compounds from Wine 
PLoS ONE  2014;9(1):e85611.
Recent evidence suggests that a lower extent of the retronasal aroma release correspond to a higher amount of ad libitum food intake. This has been regarded as one of the bases of behavioral choices towards food consumption in obese people. In this pilot study we investigated the hypothesis that saliva from obese individuals could be responsible for an alteration of the retro-nasal aroma release. We tested this hypothesis in vitro, by comparing the release of volatiles from a liquid food matrix (wine) after its interaction with saliva from 28 obese (O) and 28 normal-weight (N) individuals.
Methods and Findings
Amplicon sequencing of the 16S rRNA V4 region indicated that Firmicutes and Actinobacteria were more abundant in O, while Proteobacteria and Fusobacteria dominated in N. Streptococcaceae were significantly more abundant in the O subjects and constituted 34% and 19% on average of the saliva microbiota of O and N subjects, respectively. The Total Antioxidant Capacity was higher in O vs N saliva samples. A model mouth system was used to test whether the in-mouth wine aroma release differs after the interaction with O or N saliva. In O samples, a 18% to 60% significant decrease in the mean concentration of wine volatiles was detected as a result of interaction with saliva, compared with N. This suppression was linked to biochemical differences in O and N saliva composition, which include protein content.
Microbiological and biochemical differences were found in O vs N saliva samples. An impaired retronasal aroma release from white wine was detected in vitro and linked to compositional differences between saliva from obese and normal-weight subjects. Additional in vivo investigations on diverse food matrices could contribute to understanding whether a lower olfactory stimulation due to saliva composition can be a co-factor in the development/maintenance of obesity.
PMCID: PMC3899019  PMID: 24465618
14.  Longitudinal Profiling of the Tissue-Specific Expression of Genes Related with Insulin Sensitivity in Dairy Cows during Lactation Focusing on Different Fat Depots 
PLoS ONE  2014;9(1):e86211.
In dairy cows the milk associated energy output in early lactation exceeds the input via voluntary feed intake. To spare glucose for mammary lactose synthesis, peripheral insulin sensitivity (IS) is reduced and fat mobilization is stimulated. For these processes a link between IS and the endocrine functions of adipose tissue (AT) is likely; we thus aimed to characterise the mRNA expression from bovine AT derived proteins and receptors that are related to IS according to the literature in metabolically active tissues plus systemic IS throughout lactation. Conjugated linoleic acids (CLA) reduce milk fat thus decreasing the milk drain of energy and potentially dampening lipolysis, but may also affect IS. Subcutaneous (s.c.) AT and liver from pluriparous cows receiving either control fat or CLA supplement (100 g/day from 1 to 182 days in milk each) were biopsied covering week −3 to 36 relative to parturition. In an additional trial with primiparous cows treated analogously and slaughtered on days in milk 1, 42 or 105, samples from liver, udder, skeletal muscle and 3 visceral and 3 s.c. AT were obtained and assayed for mRNA abundance of adiponectin, its receptors, leptin, leptin receptor, PPARγ, PPARγ2, IL-6, and TNF-α. In pluriparous animals, the mRNA abundance of most of the target genes decreased after parturition in s.c. AT but increased in liver. In primiparous cows, AT depot specific differences were mostly related to retroperitoneal AT; adiponectin receptor 1 and TNF-α were affected predominantly. CLA effects in primiparous cows were largely limited to decreased PPARγ2 mRNA abundance in udder tissue. In pluriparous cows, insulin secretion was increased by CLA resulting in decreased systemic IS but without consistent changes in tissue target mRNA abundance. The temporal gene expression profiles from the adipokines and related receptors support their coactive function in adapting to the needs of lactation.
PMCID: PMC3897665  PMID: 24465964
15.  Long-Term Effect of Different Physical Activity Levels on Subclinical Atherosclerosis in Middle-Aged Men: A 25-Year Prospective Study 
PLoS ONE  2014;9(1):e85209.
The purpose of the study was to investigate the influence of lifetime physical activity (PA) on selected indices of atherosclerosis in longitudinal observation of middle-aged men.
The subject of the study was a cohort of 101 men (mean age 59,7±9,0 years), free of cardiovascular symptoms and treatment, participating in follow-up examinations in the years 1985/90-2011/12. Self-report PA was assessed by interviewer-administered Seven-Day PA Recall and Historical PA questionnaire. Subclinical atherosclerosis was measured by assessing the coronary artery calcification (CAC) according to Agatston's method using multi-slice computed tomography; the carotid intima-media thickness (IMT) using high-resolution B-mode ultrasound; and the reactive hyperemia index (RHI) using peripheral arterial tonometry (EndoPAT2000). The participants were initially divided into three groups according to tertiles of exercise-related energy expenditure (EE) in kcal/week at baseline, i.e. <2050 (low-to-moderate; n = 33), 2050–3840 (high; n = 34), >3840 (very high; n = 34).
The low-to-moderate, high and very high PA groups were comparable in terms of age and atherosclerosis risk factors at baseline. No linear relationship was found between PA and CAC, IMT and RHI. Men who maintained low-to-moderate (n = 26), high (n = 21) and very high (n = 15) PA level had the mean CAC of 286.1±361.9, 10.7±28.9, and 106.1±278.3 (p<0.001 for low-to moderate vs high; p<0.05 for low-to-moderate vs very high); the mean IMT of 0.751±0.19 mm, 0,641±0.26 mm, and 0.750±0.60 mm (p>0.05); and the mean RHI of 1.69±0.4, 2.00±0.4, and 2.13±0.5 (p for trend = 0.050), respectively. No cases of CAC>400, IMT ≥0.9 and RHI<1.67 were noted only among men with maintained high PA level. At final examination men with high and very high PA had more favorable cardiometabolic profile than men with lower PA.
Maintaining regular high PA level through young and middle adulthood may protect against atherosclerosis as measured by CAC, IMT and RHI.
PMCID: PMC3896363  PMID: 24465505
16.  The free radical scavenger Trolox dampens neuronal hyperexcitability, reinstates synaptic plasticity, and improves hypoxia tolerance in a mouse model of Rett syndrome 
Rett syndrome (RS) causes severe cognitive impairment, loss of speech, epilepsy, and breathing disturbances with intermittent hypoxia. Also mitochondria are affected; a subunit of respiratory complex III is dysregulated, the inner mitochondrial membrane is leaking protons, and brain ATP levels seem reduced. Our recent assessment of mitochondrial function in MeCP2 (methyl-CpG-binding protein 2)-deficient mouse (Mecp2-/y) hippocampus confirmed early metabolic alterations, an increased oxidative burden, and a more vulnerable cellular redox balance. As these changes may contribute to the manifestation of symptoms and disease progression, we now evaluated whether free radical scavengers are capable of improving neuronal and mitochondrial function in RS. Acute hippocampal slices of adult mice were incubated with the vitamin E derivative Trolox for 3–5 h. In Mecp2-/y slices this treatment dampened neuronal hyperexcitability, improved synaptic short-term plasticity, and fully restored synaptic long-term potentiation (LTP). Furthermore, Trolox specifically attenuated the increased hypoxia susceptibility of Mecp2-/y slices. Also, the anticonvulsive effects of Trolox were assessed, but the severity of 4-aminopyridine provoked seizure-like discharges was not significantly affected. Adverse side effects of Trolox on mitochondria can be excluded, but clear indications for an improvement of mitochondrial function were not found. Since several ion-channels and neurotransmitter receptors are redox modulated, the mitochondrial alterations and the associated oxidative burden may contribute to the neuronal dysfunction in RS. We confirmed in Mecp2-/y hippocampus that Trolox dampens neuronal hyperexcitability, reinstates synaptic plasticity, and improves the hypoxia tolerance. Therefore, radical scavengers are promising compounds for the treatment of neuronal dysfunction in RS and deserve further detailed evaluation.
PMCID: PMC3932407  PMID: 24605086
oxidative stress; redox signaling; reactive oxygen species (ROS); mitochondrial metabolism; free radical scavenger; neurodevelopmental disorder; synaptic dysfunction; vitamin E
17.  Inhibitor of differentiation proteins do not influence prognosis of biliary tract cancer 
AIM: To investigate the expression and clinical relevance of inhibitor of differentiation (ID) proteins in biliary tract cancer.
METHODS: ID protein expression was analyzed in 129 samples from patients with advanced biliary tract cancer (BTC) (45 extrahepatic, 50 intrahepatic, and 34 gallbladder cancers), compared to normal controls and correlated with clinical an pathological parameters.
RESULTS: ID1-3 proteins are frequently overexpressed in all BTC subtypes analyzed. No correlation between increased ID protein expression and tumor grading, tumor subtype or treatment response was detected. Survival was influenced primary tumor localization (extrahepatic vs intrahepatic and gall bladder cancer, OS 1.5 years vs 0.9 years vs 0.7 years, P = 0.002), by stage at diagnosis (OS 2.7 years in stage I vs 0.6 years in stage IV, P < 0.001), resection status and response to systemic chemotherapy. In a multivariate model, ID protein expression did not correlate with clinical prognosis. Nevertheless, there was a trend of shorter OS in patients with loss of cytoplasmic ID4 protein expression (P = 0.076).
CONCLUSION: ID protein expression is frequently deregulated in BTC but does not influence clinical prognosis. Their usefulness as prognostic biomarkers in BTC is very limited.
PMCID: PMC3882406  PMID: 24409060
Biliary tract cancer; Cholangiocarcinoma; Inhibitor of differentiation; Prognostic factors
18.  A Metabolomic Analysis of Omega-3 Fatty Acid-Mediated Attenuation of Western Diet-Induced Nonalcoholic Steatohepatitis in LDLR-/- Mice 
PLoS ONE  2013;8(12):e83756.
Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease and a risk factor for cirrhosis, hepatocellular carcinoma and liver failure. Previously, we reported that dietary docosahexaenoic acid (DHA, 22:6,n-3) was more effective than eicosapentaenoic acid (EPA, 20:5,n-3) at reversing western diet (WD) induced NASH in LDLR-/- mice.
Using livers from our previous study, we carried out a global non-targeted metabolomic approach to quantify diet-induced changes in hepatic metabolism.
Livers from WD + olive oil (WD + O)-fed mice displayed histological and gene expression features consistent with NASH. The metabolomic analysis of 320 metabolites established that the WD and n-3 polyunsaturated fatty acid (PUFA) supplementation had broad effects on all major metabolic pathways. Livers from WD + O-fed mice were enriched in saturated (SFA) and monounsaturated fatty acids (MUFA), palmitoyl-sphingomyelin, cholesterol, n-6 PUFA, n-6 PUFA-containing phosphoglycerolipids, n-6 PUFA-derived oxidized lipids (12-HETE) and depleted of C20-22 n-3 PUFA-containing phosphoglycerolipids, C20-22 n-3 PUFA-derived oxidized lipids (18-HEPE, 17,18-DiHETE) and S-lactoylglutathione, a methylglyoxal detoxification product. WD + DHA was more effective than WD + EPA at attenuating WD + O-induced changes in NASH gene expression markers, n-6 PUFA and oxidized lipids, citrate and S-lactosyl glutathione. Diet-induced changes in hepatic MUFA and sphingolipid content were associated with changes in expression of enzymes involved in MUFA and sphingolipid synthesis. Changes in hepatic oxidized fatty acids and S-lactoylglutathione, however, correlated with hepatic n-3 and n-6 C20-22 PUFA content. Hepatic C20-22 n-3 PUFA content was inversely associated with hepatic α-tocopherol and ascorbate content and positively associated with urinary F2- and F3-isoprostanes, revealing diet effects on whole body oxidative stress.
DHA regulation of hepatic SFA, MUFA, PUFA, sphingomyelin, PUFA-derived oxidized lipids and S-lactoylglutathione may explain the protective effects of DHA against WD-induced NASH in LDLR-/- mice.
PMCID: PMC3866250  PMID: 24358308
19.  Inflammation but Not Dietary Macronutrients Insufficiency Associated with the Malnutrition-Inflammation Score in Hemodialysis Population 
PLoS ONE  2013;8(12):e83233.
Malnutrition is associated with increased risk of mortality in hemodialysis patients. And insufficient dietary intake is the common cause for malnutrition. So, in order to survey the dietary intake of hemodialysis patients and study the relationship between the dietary feature and nutritional status, a cross-sectional study was performed. 75 hemodialysis patients from South China participated in the dietary intake survey and nutrition assessment. A three-day diet diary record was used to estimate the major dietary macronutrients. Nutritional status was assessed by malnutrition-inflammation score (MIS) in addition to several related anthropometric measurements. Serum albumin, transferrin, and high-sensitivity C-reactive protein (CRP) were measured. Receiver operating characteristic (ROC) curve analysis was used to quantify the assessing value of independent parameters for nutritional status. The results showed that 48% patients were malnourished according to the MIS. The malnourished patients had a lower body mass index (BMI), fat mass (FM), albumin and a higher level of CRP, compared with normal nourished patients (P < 0.05). However, no significant differences of macronutrients (calories, protein, fat, carbohydrates, etc) were found between the two nutrition groups (P > 0.05). The multivariate regression analysis showed that the major macronutrients had no significant association with MIS (P > 0.05). In conclusion, malnutrition is very common in South China hemodialysis population and these data indicated that inflammation but not dietary macronutrients insufficiency might be the candidate cause for malnutrition in hemodialysis population.
PMCID: PMC3859660  PMID: 24349471
20.  Dietary Omega-3 Polyunsaturated Fatty Acids Alter the Fatty Acid Composition of Hepatic and Plasma Bioactive Lipids in C57BL/6 Mice: A Lipidomic Approach 
PLoS ONE  2013;8(11):e82399.
Omega (n)-3 polyunsaturated fatty acids (PUFA) are converted to bioactive lipid components that are important mediators in metabolic and physiological pathways; however, which bioactive compounds are metabolically active, and their mechanisms of action are still not clear. We investigated using lipidomic techniques, the effects of diets high in n-3 PUFA on the fatty acid composition of various bioactive lipids in plasma and liver.
Methodology and Principal Findings
Female C57BL/6 mice were fed semi-purified diets (20% w/w fat) containing varying amounts of n-3 PUFA before mating, during gestation and lactation, and until weaning. Male offspring were continued on their mothers’ diets for 16 weeks. Hepatic and plasma lipids were extracted in the presence of non-naturally occurring internal standards, and tandem electrospray ionization mass spectrometry methods were used to measure the fatty acyl compositions. There was no significant difference in total concentrations of phospholipids in both groups. However, there was a significantly higher concentration of eicosapentaenoic acid containing phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and cholesteryl esters (CE) (p < 0.01) in the high n-3 PUFA group compared to the low n-3 PUFA group in both liver and plasma. Plasma and liver from the high n-3 PUFA group also had a higher concentration of free n-3 PUFA (p < 0.05). There were no significant differences in plasma concentrations of different fatty acyl species of phosphatidylethanolamine, triglycerides, sphingomyelin and ceramides.
Our findings reveal for the first time that a diet high in n-3 PUFA caused enrichment of n-3 PUFA in PC, LPC, CE and free fatty acids in the plasma and liver of C57BL/6 mice. PC, LPC, and unesterified free n-3 PUFA are important bioactive lipids, thus altering their fatty acyl composition will have important metabolic and physiological roles.
PMCID: PMC3836780  PMID: 24278481
21.  Antidiabetic Effects of Chamomile Flowers Extract in Obese Mice through Transcriptional Stimulation of Nutrient Sensors of the Peroxisome Proliferator-Activated Receptor (PPAR) Family 
PLoS ONE  2013;8(11):e80335.
Given the significant increases in the incidence of metabolic diseases, efficient strategies for preventing and treating of these common disorders are urgently needed. This includes the development of phytopharmaceutical products or functional foods to prevent or cure metabolic diseases. Plant extracts from edible biomaterial provide a potential resource of structurally diverse molecules that can synergistically interfere with complex disorders. In this study we describe the safe application of ethanolic chamomile (Matricaria recutita) flowers extract (CFE) for the treatment and prevention of type 2 diabetes and associated disorders. We show in vitro that this extract activates in particular nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) and its isotypes. In a cellular context, in human primary adipocytes CFE administration (300 µg/ml) led to specific expression of target genes of PPARγ, whereas in human hepatocytes CFE-induced we detected expression changes of genes that were regulated by PPARα. In vivo treatment of insulin-resistant high-fat diet (HFD)-fed C57BL/6 mice with CFE (200 mg/kg/d) for 6 weeks considerably reduced insulin resistance, glucose intolerance, plasma triacylglycerol, non-esterified fatty acids (NEFA) and LDL/VLDL cholesterol. Co-feeding of lean C57BL/6 mice a HFD with 200 mg/kg/d CFE for 20 weeks showed effective prevention of fatty liver formation and hepatic inflammation, indicating additionally hepatoprotective effects of the extract. Moreover, CFE treatment did not reveal side effects, which have otherwise been associated with strong synthetic PPAR-targeting molecules, such as weight gain, liver disorders, hemodilution or bone cell turnover. Taken together, modulation of PPARs and other factors by chamomile flowers extract has the potential to prevent or treat type 2 diabetes and related disorders.
PMCID: PMC3827197  PMID: 24265809
22.  Dietary Behaviour and Socioeconomic Position: The Role of Physical Activity Patterns 
PLoS ONE  2013;8(11):e78390.
The positive association between education level and health outcomes can be partly explained by dietary behaviour. We investigated the associations between education and several indices of food intake and potential influencing factors, placing special emphasis on physical-activity patterns, using a representative sample of the German adult population.
The German National Health Interview and Examination Survey 1998 (GNHIES98) involved 7,124 participants aged between 18 and 79. Complete information on the exposure (education) and outcome (nutrition) variables was available for 6,767 persons. The associations between ‘education’ and indices of ‘sugar-rich food’, ‘fat-rich food’, ‘fruit-and-vegetable’ and ‘alcohol’ intake were analysed separately for men and women using multivariate logistic regression analysis. Odds ratios (OR) of education level on nutrition outcomes were calculated and adjusted for age, region (former East/West Germany), occupation, income and other influencing factors such as physical activity indicators.
Men and women with only a primary education had a more frequent intake of sugar-rich and fat-rich foods and a less frequent intake of fruit and vegetables and alcohol than people with a tertiary education. ‘Physical work activity’ partly explained the associations between education and sugar-rich food intake. The interference with physical work activity was stronger among men than women. No significant associations between education and energy-dense food intake were observed in the retirement-age group of persons aged 65+ and among persons with low energy expenditure.
In Germany, adults with a low level of education report that they consume energy-dense foods more frequently – and fruit and vegetables and alcohol less frequently – than adults with a high education level. High levels of physical work activity among adults with a low education level may partly explain why they consume more energy-dense foods.
PMCID: PMC3819390  PMID: 24223150
23.  Maternal Western-Style High Fat Diet Induces Sex-Specific Physiological and Molecular Changes in Two-Week-Old Mouse Offspring 
PLoS ONE  2013;8(11):e78623.
Maternal diet is associated with the development of metabolism-related and other non-communicable diseases in offspring. Underlying mechanisms, functional profiles, and molecular markers are only starting to be revealed. Here, we explored the physiological and molecular impact of maternal Western-style diet on the liver of male and female offspring. C57BL/6 dams were exposed to either a low fat/low cholesterol diet (LFD) or a Western-style high fat/high cholesterol diet (WSD) for six weeks before mating, as well as during gestation and lactation. Dams and offspring were sacrificed at postnatal day 14, and body, liver, and blood parameters were assessed. The impact of maternal WSD on the pups’ liver gene expression was characterised by whole-transcriptome microarray analysis. Exclusively male offspring had significantly higher body weight upon maternal WSD. In offspring of both sexes of WSD dams, liver and blood parameters, as well as hepatic gene expression profiles were changed. In total, 686 and 604 genes were differentially expressed in liver (p≤0.01) of males and females, respectively. Only 10% of these significantly changed genes overlapped in both sexes. In males, in particular alterations of gene expression with respect to developmental functions and processes were observed, such as Wnt/beta-catenin signalling. In females, mainly genes important for lipid metabolism, including cholesterol synthesis, were changed. We conclude that maternal WSD affects physiological parameters and induces substantial changes in the molecular profile of the liver in two-week-old pups. Remarkably, the observed biological responses of the offspring reveal pronounced sex-specificity.
PMCID: PMC3818485  PMID: 24223833
24.  Liver Glycerol Permeability and Aquaporin-9 Are Dysregulated in a Murine Model of Non-Alcoholic Fatty Liver Disease 
PLoS ONE  2013;8(10):e78139.
One form of liver steatosis, namely Non-Alcoholic Fatty Liver Disease (NAFLD), is a worrisome health problem worldwide characterized by intrahepatic triacylglycerol (TG) overaccumulation. NAFLD is a common feature of metabolic syndrome being often associated with obesity, dyslipidemia and diabetes and mostly closely linked to insulin resistance. The mechanism of NAFLD pathogenesis is object of intense investigation especially regarding complex systems ultimately resulting in excessive TG deposition in hepatocytes. However, scarce is the attention about the relevance of hepatic import of glycerol, the other primary source (as glycerol-3-phosphate) of increased TG in hepatocytes. Obese leptin-deficient (ob/ob) mice, an animal model of NAFLD, were used to evaluate the functional involvement of Aquaporin-9 (AQP9), the major pathway of liver glycerol entry, in hepatosteatosis. By RT-PCR and qPCR, the level of Aqp9 mRNA in the liver of starved obese mice was comparable with the corresponding control lean littermates. By immunoblotting, the AQP9 protein at the hepatocyte sinusoidal plasma membrane of obese mice was markedly lower (33%) than lean mice, a finding fully confirmed by immunohistochemistry. By stopped-flow light scattering, the liver glycerol permeability of ob/ob mice was significantly lower (53%) than lean mice, a finding consistent with both the observed down-regulation of AQP9 protein and increased level of plasma glycerol characterizing obese mice. In summary, our results suggest implication of AQP9 in liver steatosis. The reduction of hepatocyte AQP9 and, consequently, glycerol permeability might be a defensive mechanism to counteract further fat infiltration in liver parenchyma.
PMCID: PMC3813550  PMID: 24205128
25.  Maternal Malnutrition and Offspring Sex Determine Juvenile Obesity and Metabolic Disorders in a Swine Model of Leptin Resistance 
PLoS ONE  2013;8(10):e78424.
The present study aimed to determine, in a swine model of leptin resistance, the effects of type and timing of maternal malnutrition on growth patterns, adiposity and metabolic features of the progeny when exposed to an obesogenic diet during their juvenile development and possible concomitant effects of the offspring sex. Thus, four groups were considered. A CONTROL group involved pigs born from sows fed with a diet fulfilling their daily maintenance requirements for pregnancy. The treated groups involved the progeny of females fed with the same diet but fulfilling either 160% or 50% of pregnancy requirements during the entire gestation (OVERFED and UNDERFED, respectively) or 100% of requirements until Day 35 of pregnancy and 50% of such amount from Day 36 onwards (LATE-UNDERFED). OVERFED and UNDERFED offspring were more prone to higher corpulence and fat deposition from early postnatal stages, during breast-feeding; adiposity increased significantly when exposed to obesogenic diets, especially in females. The effects of sex were even more remarkable in LATE-UNDERFED offspring, which had similar corpulence to CONTROL piglets; however, females showed a clear predisposition to obesity. Furthermore, the three groups of pigs with maternal malnutrition showed evidences of metabolic syndrome and, in the case of individuals born from OVERFED sows, even of insulin resistance and the prodrome of type-2 diabetes. These findings support the main role of early nutritional programming in the current rise of obesity and associated diseases in ethnics with leptin resistance.
PMCID: PMC3813450  PMID: 24205230

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