Cow Milk Consumption Increases Iodine Status in Women of Childbearing Age in a Randomized Controlled Trial.

Background: Recent evidence has highlighted the prevalence of mild-to-moderate iodine deficiency in women of childbearing age and pregnant women, with important public health ramifications due to the role of iodine, which is required for thyroid hormone production, in neurodevelopment. Cow milk contributes the greatest amount to iodine intakes in several countries. Objective: The objective of this study was to investigate the effect of increased cow milk consumption on iodine status, thyroid hormone concentrations, and selenium status. Methods: A 12-wk randomized controlled trial was conducted in 78 low-moderate milk-consuming (<250 mL/d) healthy women (aged 18-45 y). The intervention group was asked to consume 3 L semiskimmed milk/wk, whereas the control group continued their usual milk consumption (baseline median: 140 mL/d; IQR: 40-240 mL/d). At baseline and weeks 6 and 12, participants provided a spot urine sample [urinary iodine concentration (UIC), creatinine] and a fasting blood sample (thyroid hormone concentrations, serum total selenium, selenoprotein P). Results: At baseline, the median (IQR) UIC of all participants was 78.5 µg/L (39.1-126.1 µg/L). Changes in the median UIC from baseline to week 6 (35.4 compared with 0.6 µg/L; P = 0.014) and week 12 (51.6 compared with -3.8 µg/L; P = 0.045) were significantly greater in the intervention group compared with the control group. However, despite being higher within the intervention group at weeks 6 and 12, the change in the iodine:creatinine ratio from baseline was not significantly different between groups at either week 6 (P = 0.637) or week 12 (P = 0.178). There were no significant differences in thyroid hormone concentrations or selenium status between groups at any time point. Conclusions: The present study shows that the consumption of additional cow milk can significantly increase UIC in women of childbearing age. These results suggest that cow milk is a potentially important dietary source of iodine in this population group. This trial was registered at www.clinicaltrials.gov as NCT02767167.

Dietary macronutrients and the aging liver sinusoidal endothelial cell.

Fenestrations are pores within the liver sinusoidal endothelial cells (LSECs) that line the sinusoids of the highly vascularized liver. Fenestrations facilitate the transfer of substrates between blood and hepatocytes. With pseudocapillarization of the hepatic sinusoid in old age, there is a loss of fenestrations. LSECs are uniquely exposed to gut-derived dietary and microbial substrates delivered by the portal circulation to the liver. Here we studied the effect of 25 diets varying in content of macronutrients and energy on LSEC fenestrations using the Geometric Framework method in a large cohort of mice aged 15 mo. Macronutrient distribution rather than total food or energy intake was associated with changes in fenestrations. Porosity and frequency were inversely associated with dietary fat intake, while fenestration diameter was inversely associated with protein or carbohydrate intake. Fenestrations were also linked to diet-induced changes in gut microbiome, with increased fenestrations associated with higher abundance of Firmicutes and reduced abundance of Bacteroidetes Diet-induced changes in levels of several fatty acids (C16:0, C19:0, and C20:4) were also significantly inversely associated with fenestrations, suggesting a link between dietary fat and modulation of lipid rafts in the LSECs. Diet influences fenestrations and these data reflect both the key role of the LSECs in clearing gut-derived molecules from the vascular circulation and the impact these molecules have on LSEC morphology.

Vitamin C restores healthy aging in a mouse model for Werner syndrome.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS, including a prooxidant status and a shorter mean life span compared to wild-type animals. Here, we show that Wrn mutant mice also develop premature liver sinusoidal endothelial defenestration along with inflammation and metabolic syndrome. Vitamin C supplementation rescued the shorter mean life span of Wrn mutant mice and reversed several age-related abnormalities in adipose tissues and liver endothelial defenestration, genomic integrity, and inflammatory status. At the molecular level, phosphorylation of age-related stress markers like Akt kinase-specific substrates and the transcription factor NF-kappaB, as well as protein kinase Cdelta and Hif-1alpha transcription factor levels, which are increased in the liver of Wrn mutants, were normalized by vitamin C. Vitamin C also increased the transcriptional regulator of lipid metabolism PPARalpha. Finally, microarray and gene set enrichment analyses on liver tissues revealed that vitamin C decreased genes normally up-regulated in human WS fibroblasts and cancers, and it increased genes involved in tissue injury response and adipocyte dedifferentiation in obese mice. Vitamin C did not have such effect on wild-type mice. These results indicate that vitamin C supplementation could be beneficial for patients with WS.

Contractility of permeabilized rat vastus intermedius muscle fibres following high-fat, high-sucrose diet consumption.

Obesity is a worldwide health concern associated with impaired physical function. It is not clear if contractile protein dysfunction contributes to the impairment of muscle function observed with obesity. The purpose of this study was to examine if diet-induced obesity affects contractile function of chemically permeabilized vastus intermedius fibres of male Sprague-Dawley rats expressing fast myosin heavy chain (MHC) IIa or slow MHC I. Rats consumed either a high-fat, high sucrose (HFHS) diet or a standard (CHOW) diet beginning as either weanlings (7-week duration: WEAN7 cohort, or 14-week duration: WEAN14 cohort) or young adults (12-week duration: ADULT12 cohort, 24-week duration: ADULT24 cohort). HFHS-fed rats had higher (P < 0.05) whole-body adiposity (derived from dual-energy X-ray absorptiometry) than CHOW-fed rats in all cohorts. Relative to CHOW diet groups, the HFHS diet was associated with impaired force production in (a) MHC I fibres in the ADULT24 cohort; and (b) MHC IIa fibres in the ADULT12 and ADULT24 cohorts combined. However, the HFHS diet did not significantly affect the Ca2+-sensitivity of force production, unloaded shortening velocity, or ratio of active force to active stiffness in any cohort. We conclude that diet-induced obesity can impair force output of permeabilized muscle fibres of adult rats. Novelty: We assessed contractile function of permeabilized skeletal muscle fibres in a rat model of diet-induced obesity. The high-fat, high-sucrose diet was associated with impaired force output of fibres expressing MHC I or MHC IIa in some cohorts of rats. Other measures of contractile function were not significantly affected by diet.

Rapid ecosystem recovery from diffuse pollution after the Great Irish Famine.

Remarkably little is known about the effectiveness or rates of recovery of aquatic ecosystems from reductions in human-associated pressures at landscape scales. The retention of anthropogenic contaminants within ecosystems can retard rates of recovery considerably, while the trajectories of recovery processes vary with the extent of disturbance and the resilience of biotic assemblages. The Great Irish Famine of 1845-1850 comprised one of the most significant human disasters of the 19th century, causing the death of approximately one million people and the emigration of a further two million from the country between 1845 and 1855. We found, through analysis of detailed historical census data combined with paleolimnological investigation of sedimentary nutrient concentrations, stable isotope ratios, and diatom assemblages, that the trophic level of Lough Carra, a largely shallow calcareous lake in the west of Ireland with no urban areas or point sources of any significance in its catchment, reduced considerably during and immediately after the Great Famine, shifting to new equilibria within just 2-10 years. Our results demonstrate that the reduction of human pressures from diffuse sources at landscape scales can result in the rapid and monotonic recovery of aquatic ecosystems. Moreover, the recovery of ecosystems from diffuse pollution need not necessarily take longer than recovery from pollution from point sources.

Old age is associated with ultrastructural changes in isolated rat liver sinusoidal endothelial cells.

Old age is associated with ultrastructural changes in the hepatic sinusoid called pseudocapillarization, which include defenestration and thickening of the sinusoidal endothelium. We investigated whether such changes also occur in isolated and cultured liver sinusoidal endothelial cells. Liver sinusoidal endothelial cells were isolated from young (6-10 months, n = 4) and old (24-26 months, n = 4) F344 rats and fenestrations evaluated using scanning electron microscopy. Fenestration diameter was reduced in old age from 194 +/- 1 nm to 185 +/- 1 nm (P < 0.001) and there was an age-related increase in the number fused fenestrations and large gaps. Age-related changes in the diameter of fenestrations in the liver sinusoidal endothelial cell are maintained following isolation and culture. This suggests that this age-related change may be intrinsic to the liver endothelial cell and/or irreversible.

Liver sinusoidal endothelial cells and acute non-oxidative hepatic injury induced by Pseudomonas aeruginosa pyocyanin.

The liver sinusoidal endothelial cell (LSEC) is damaged by many toxins, including oxidants and bacterial toxins. Any effect on LSECs of the Pseudomonas aeruginosa virulence factor, pyocyanin, may be relevant for systemic pseudomonal infections and liver transplantation. In this study, the effects of pyocyanin on in vivo rat livers and isolated LSECs were assessed using electron microscopy, immunohistochemistry and biochemistry. In particular, the effect on fenestrations, a crucial morphological aspect of LSECs was assessed. Pyocyanin treatment induced a dose-dependent reduction in fenestrations in isolated LSECs. In the intact liver, intraportal injection of pyocyanin (11.9 microM in blood) was associated with a reduction in endothelial porosity from 3.4 +/- 0.2% (n = 5) to 1.3 +/- 0.1% (n = 7) within 30 min. There were decreases in both diameter and frequency of fenestrations in the intact endothelium. There was also a decrease in endothelial thickness from 175.8 +/- 5.8 to 156.5 +/- 4.0 nm, an endothelial pathology finding previously unreported. Hepatocyte ultrastructure, liver function tests and immunohistochemical markers of oxidative stress (3-nitrotyrosine and malondialdehyde) were not affected. Pyocyanin induces significant ultrastructural changes in the LSEC in the absence of immunohistochemical evidence of oxidative stress or hepatocyte injury pointing to a novel mechanism for pyocyanin pathogenesis.

Antipsychotic drug administration differentially affects [3H]muscimol and [3H]flunitrazepam GABA(A) receptor binding sites.

Post-mortem studies of the human brain indicate that certain GABA(A) receptor subtypes may be differentially altered in schizophrenia. Increased binding to the total population of GABA(A) receptors using [3H]muscimol is observed in the post-mortem schizophrenic brain, yet a proportion of these receptors which bind benzodiazepines and are labelled with [3H]flunitrazepam, show decreased or unaltered expression. Data from animal studies suggest that antipsychotic drugs alter GABA(A) receptor expression in a subtype selective manner, but in the opposite direction to that observed in schizophrenia. To broaden our understanding of the effects of antipsychotic drugs on GABA(A) receptors, we examined the saturation binding maximum (B(max)) and binding affinity (K(D)) of [3H]muscimol and [3H]flunitrazepam in the prefrontal cortex (PFC), hippocampus and thalamus of male SD rats that received a sucrose solution containing either haloperidol (1.5 mg/kg), olanzapine (6.5 mg/kg) or no drug daily for up to 28 days using quantitative receptor autoradiography. [3H]Muscimol binding density was increased most prominently in the PFC after 7 days, with larger and more prolonged effects being induced by the atypical antipsychotic drug olanzapine in subcortical regions. While no changes were observed in [3H]muscimol binding in any region after 28 days of drug administration, [3H]flunitrazepam binding density (B(max)) was increased for both antipsychotic treatments in the PFC only. These findings confirm that the subset of GABA(A) receptors sensitive to benzodiazepines are regulated differently from other GABA(A) receptor subtypes following antipsychotic drug administration, in a time- and region-dependent manner.

The Effect of Processing and Seasonality on the Iodine and Selenium Concentration of Cow's Milk Produced in Northern Ireland (NI): Implications for Population Dietary Intake.

Cow's milk is the most important dietary source of iodine in the UK and Ireland, and also contributes to dietary selenium intakes. The aim of this study was to investigate the effect of season, milk fat class (whole; semi-skimmed; skimmed) and pasteurisation on iodine and selenium concentrations in Northern Ireland (NI) milk, and to estimate the contribution of this milk to consumer iodine and selenium intakes. Milk samples (unpasteurised, whole, semi-skimmed and skimmed) were collected weekly from two large NI creameries between May 2013 and April 2014 and were analysed by inductively coupled plasma-mass spectrometry (ICP-MS). Using milk consumption data from the National Diet and Nutrition Survey (NDNS) Rolling Programme, the contribution of milk (at iodine and selenium concentrations measured in the present study) to UK dietary intakes was estimated. The mean ± standard deviation (SD) iodine concentration of milk was 475.9 ± 63.5 µg/kg and the mean selenium concentration of milk was 17.8 ± 2.7 µg/kg. Season had an important determining effect on the iodine, but not the selenium, content of cow's milk, where iodine concentrations were highest in milk produced in spring compared to autumn months (534.3 ± 53.7 vs. 433.6 ± 57.8 µg/kg, respectively; p = 0.001). The measured iodine and selenium concentrations of NI milk were higher than those listed in current UK Food Composition Databases (Food Standards Agency (FSA) (2002); FSA (2015)). The dietary modelling analysis confirmed that milk makes an important contribution to iodine and selenium intakes. This contribution may be higher than previously estimated if iodine and selenium (+25.0 and +1.1 µg/day respectively) concentrations measured in the present study were replicable across the UK at the current level of milk consumption. Iodine intakes were theoretically shown to vary by season concurrent with the seasonal variation in NI milk iodine concentrations. Routine monitoring of milk iodine concentrations is required and efforts should be made to understand reasons for fluctuations in milk iodine concentrations, in order to realise the nutritional impact to consumers.

The effect of old age on liver oxygenation and the hepatic expression of VEGF and VEGFR2.

In old age, the liver contains less ATP and hypoxia-responsive genes are upregulated. Age-related changes in hepatic perfusion and the liver sinusoidal endothelial cell (LSEC) could contribute to this altered hepatic oxygen-dependent metabolism by causing intrahepatocytic hypoxia. Furthermore, age-related changes in the LSEC ('pseudocapillarization') have been partially induced by ATP depletion. To investigate whether there is intracellular hypoxia in the old rat liver, pimonidazole immunohistochemistry in intact livers and ATP levels in isolated LSECs were studied from young and old rats. There were no age-related changes. To determine whether defenestration of the LSEC could impair oxygen diffusion, pimonidazole immunohistochemistry was performed in rats treated with poloxamer 407. Despite defenestration, there was no change in pimonidazole staining. Immunohistochemistry was then performed to determine whether there are age-related changes in VEGF and VEGFR2. VEGF staining was not associated with age. However, there was an increase in perisinusoidal VEGFR2 expression with increasing age. In conclusion, liver hypoxia does not occur in old age and LSEC pseudocapillarization does not constitute an oxygen-diffusion barrier. There are no age-related changes in VEGF expression but an increase in perisinusoidal VEGFR2 expression, which has implications for the effects of aging on the hepatic sinusoid.

The effects of antipsychotic drugs on GABAA receptor binding depend on period of drug treatment and binding site examined.

Changes in GABA(A) receptors are observed in schizophrenia, with benzodiazepine-sensitive GABA(A) receptor subtypes being affected differently to other subtypes. However, long-term antipsychotic drug use in schizophrenia may underlie these changes. To test this, we examined the effects of administering a typical (haloperidol) and an atypical (olanzapine) antipsychotic drug on the GABA(A) receptor agonist (orthosteric) and benzodiazepine (allosteric) binding sites in rat prefrontal cortex. As antipsychotic drugs have delayed maximal therapeutic effects we also examined different drug treatment periods. Male SD rats received a sucrose solution containing either haloperidol (1.5 mg/kg), olanzapine (6.5 mg/kg) or no drug daily for either 7, 14 or 28 days. Sections of rat brain were then labelled with [(3)H]muscimol, which labels the total population of GABA(A) receptors, or the benzodiazepine site ligand [(3)H]flunitrazepam in separate saturation binding experiments using quantitative receptor autoradiography. [(3)H]Muscimol binding was enhanced in the prefrontal cortex after 7 days but no differences were observed after longer periods of drug administration. In contrast there was a delayed increase in density of benzodiazepine-sensitive GABA(A) receptors in the PFC, suggesting that antipsychotic drugs have different effects on different GABA(A) receptor subtypes. These changes in the properties of GABA(A) receptor binding following antipsychotic drug administration are not consistent with those observed in schizophrenia and suggest a 'reshuffling' in GABA(A) receptor subtypes over time.

A standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy.

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures--both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

Liver aging and pseudocapillarization in a Werner syndrome mouse model.

Werner syndrome is a progeric syndrome characterized by premature atherosclerosis, diabetes, cancer, and death in humans. The knockout mouse model created by deletion of the RecQ helicase domain of the mouse Wrn homologue gene (Wrn(∆hel/∆hel)) is of great interest because it develops atherosclerosis and hypertriglyceridemia, conditions associated with aging liver and sinusoidal changes. Here, we show that Wrn(∆hel/∆hel) mice exhibit increased extracellular matrix, defenestration, decreased fenestration diameter, and changes in markers of liver sinusoidal endothelial cell inflammation, consistent with age-related pseudocapilliarization. In addition, hepatocytes are larger, have increased lipofuscin deposition, more frequent nuclear morphological anomalies, decreased mitochondria number, and increased mitochondrial diameter compared to wild-type mice. The Wrn(∆hel/∆hel) mice also have altered mitochondrial function and altered nuclei. Microarray data revealed that the Wrn(∆hel/∆hel) genotype does not affect the expression of many genes within the isolated hepatocytes or liver sinusoidal endothelial cells. This study reveals that Wrn(∆hel/∆hel) mice have accelerated typical age-related liver changes including pseudocapillarization. This confirms that pseudocapillarization of the liver sinusoid is a consistent feature of various aging models. Moreover, it implies that DNA repair may be implicated in normal aging changes in the liver.

The effect of old age on apolipoprotein E and its receptors in rat liver.

Apolipoprotein E (apoE) is associated with aging and some age-related diseases. The majority of apoE is produced by hepatocytes for the receptor-mediated uptake of lipoproteins. Here, the effects of age on the hepatic expression and distribution of apoE and its receptors were determined using immunofluorescence, Western blots, and quantitative PCR in rat liver tissue and isolated hepatocytes. The expression of apoE mRNA and protein was not influenced significantly by aging. Immunofluorescence studies in isolated hepatocytes showed that apoE was more likely to be co-localized with early endosomes, golgi, and microtubules in isolated old hepatocytes. The mRNA expression of the receptor involved in sequestration of apoE, heparan sulfate proteoglycan was reduced in old age, without any significant effect on the expression of either the low-density lipoprotein receptor or low density-lipoprotein receptor-related protein. Old age is associated with changes in hepatic apoE intracellular trafficking and heparan sulfate proteoglycan expression that might contribute to age-related disease.

The effect of aging on the response of isolated hepatocytes to hydrogen peroxide and tert-butyl hydroperoxide.

Aging is associated with increased susceptibility to oxidative stress. To study this in the liver and to elucidate underlying mechanisms, hepatocytes from young (4-6 months) and old (24-26 months) rats were exposed to two oxidants, hydrogen peroxide and tert-butyl hydroperoxide. ATP content and mitochondrial activity were lower in old hepatocytes and decreased further with oxidative stress. Expression of Cu/Zn superoxide dismutase, Mn superoxide dismutase and catalase was not substantially influenced by oxidative stress in young and old hepatocytes, whereas glutathione peroxidase 1 expression was markedly increased only in young hepatocytes. Oxidative stress in young hepatocytes led to increased expression of apoE and movement of apoE to the early endosomes. In old hepatocytes, oxidative stress did not increase apoE expression and apoE was co-localized with early endosomes under control conditions. The results show that old age is associated with impaired hepatocyte responses of mitochondria, ATP, glutathione peroxidase 1 and apoE to oxidative stress.

The effect of feeding and fasting on fenestrations in the liver sinusoidal endothelial cell.

AIMS: Fenestrations are pores in the liver sinusoidal endothelial cell that facilitate the transfer of substrates between blood and hepatocytes. The aim of this study was to determine the effect of nutritional state on the morphology of fenestrations. METHODS: Scanning electron microscopy was used to investigate fenestrations in livers from fasted and fed rats. RESULTS: Fasting for 48 hours in rats was associated with an increase in the diameter of fenestrations from 90.7 +/- 11.7 nm in the fed state to 99.0 +/- 12.11 nm (p < 0.005). There was a concomitant reduction in the frequency of fenestrations from 8.45 +/- 2.43 to 7.39 +/- 2.28 fenestrations per microm(2) (p =0.05). CONCLUSIONS: Fasting was associated with increased diameter of fenestrations. The results provide evidence that fenestrations are dynamic structures that respond in vivo to physiological stimuli such as nutritional status.