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Extrahepatic tissues which oxidise ketone bodies also have the capacity to accumulate them under particular conditions. We hypothesised that acetyl-coenzyme A (acetyl-CoA) accumulation and altered redox status during low-flow ischaemia would support ketone body production in the heart. Combining a Langendorff heart model of low-flow ischaemia/reperfusion with liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), we show that β-hydroxybutyrate (β-OHB) accumulated in the ischaemic heart to 23.9 nmol/gww and was secreted into the coronary effluent. Sodium oxamate, a lactate dehydrogenase (LDH) inhibitor, increased ischaemic β-OHB levels 5.3-fold and slowed contractile recovery. Inhibition of β-hydroxy-β-methylglutaryl (HMG)-CoA synthase (HMGCS2) with hymeglusin lowered ischaemic β-OHB accumulation by 40%, despite increased flux through succinyl-CoA-3-oxaloacid CoA transferase (SCOT), resulting in greater contractile recovery. Hymeglusin also protected cardiac mitochondrial respiratory capacity during ischaemia/reperfusion. In conclusion, net ketone generation occurs in the heart under conditions of low-flow ischaemia. The process is driven by flux through both HMGCS2 and SCOT, and impacts on cardiac functional recovery from ischaemia/reperfusion.

Data supporting "β-Hydroxybutyrate Accumulates in the Rat Heart during Low-Flow Ischaemia with Implications for Functional Recovery". See README file for description.

Background Evidence-based pharmacological treatments for posttraumatic stress disorder (PTSD) are few and of limited efficacy. Previous work suggests that angiotensin type 1 receptor inhibition facilitates fear inhibition and extinction, important for recovery from PTSD. This study tests the efficacy of the angiotensin type 1 receptor antagonist losartan, an antihypertensive drug, repurposed for the treatment of PTSD. Methods A randomized controlled trial was conducted for 10 weeks in 149 men and women meeting DSM-5 PTSD criteria. Losartan (vs. placebo) was flexibly titrated from 25 to 100 mg/day by week 6 and held at highest tolerated dose until week 10. Primary outcome was the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5) change score at 10 weeks from baseline. A key secondary outcome was change in CAPS-5 associated with a single nucleotide polymorphism of the ACE gene. Additional secondary outcomes included changes in the PTSD Checklist for DSM-5 and the Patient Health Questionnaire-9, and proportion of responders with a Clinical Global Impressions–Improvement scale of “much improved” or “very much improved.” Results Both groups had robust improvement in PTSD symptoms, but there was no significant difference on the primary end point, CAPS-5 measured as week 10 change from baseline, between losartan and placebo (mean change difference, 0.9, 95% confidence interval, −3.2 to 5.0). There was no significant difference in the proportion of Clinical Global Impressions–Improvement scale responders for losartan (58.6%) versus placebo (57.9%), no significant differences in changes in PTSD Checklist for DSM-5 or Patient Health Questionnaire-9, and no association between ACE genotype and CAPS-5 improvement on losartan. Conclusions At these doses and durations, there was no significant benefit of losartan compared with placebo for the treatment of PTSD. We discuss implications for failure to determine the benefit of a repurposed drug with strong a priori expectations of success based on preclinical and epidemiological data.

Funder: Danish Council for Strategic Research; Grant(s): “LIFE-DNP: hyperpolarized magnetic resonance for in vivo quantification of lipid, sugar and amino acid metabolism in lifestyle related diseases”, The diabetic heart is energetically and metabolically abnormal, with increased fatty acid oxidation and decreased glucose oxidation. One factor contributing to the metabolic dysfunction in diabetes may be abnormal handling of acetyl and acyl groups by the mitochondria. L-carnitine is responsible for their transfer across the mitochondrial membrane, therefore, supplementation with L-carnitine may provide a route to improve the metabolic state of the diabetic heart. The primary aim of this study was to use hyperpolarized magnetic resonance imaging (MRI) to investigate the effects of L-carnitine supplementation on the in vivo metabolism of [1-13C]pyruvate in diabetes. Male Wistar rats were injected with either vehicle or streptozotocin (55 mg/kg) to induce type-1 diabetes. Three weeks of daily i.p. treatment with either saline or L-carnitine (3 g/kg/day) was subsequently undertaken. In vivo cardiac function and metabolism were assessed with CINE and hyperpolarized MRI, respectively. L-carnitine supplementation prevented the progression of hyperglycemia, which was observed in untreated streptozotocin injected animals and led to reductions in plasma triglyceride and ß-hydroxybutyrate concentrations. Hyperpolarized MRI revealed that L-carnitine treatment elevated pyruvate dehydrogenase flux by 3-fold in the diabetic animals, potentially through increased buffering of excess acetyl-CoA units in the mitochondria. Improved functional recovery following ischemia was also observed in the L-carnitine treated diabetic animals.

Objective Current pharmacologic treatments for posttraumatic stress disorder (PTSD) have shown limited efficacy, prompting a call to investigate new classes of medications. The current study investigated the efficacy of glutamate modulation with riluzole augmentation for combat-related PTSD symptoms resistant to treatment with selective serotonin reuptake inhibitors (SSRIs) or serotonin-norepinephrine reuptake inhibitors (SNRIs). Methods A randomized, double-blind, placebo-controlled, parallel trial was conducted at Walter Reed National Military Medical Center and Syracuse VA Medical Center between December 2013 and November 2017. Veterans and active duty service members with combat-related PTSD (per the Clinician Administered PTSD Scale [CAPS]) who were not responsive to SSRI or SNRI pharmacotherapy were randomized to 8-week augmentation with a starting dose of 100 mg/d of riluzole (n = 36) or placebo (n = 38) and assessed weekly for PTSD symptoms, anxiety, depression, disability, and side effects. Results Intent-to-treat analyses (N = 74) of the primary outcome (CAPS for DSM-IV) showed no significant between-group difference in change in overall PTSD symptoms (F = 0.64, P = .422), with a small effect size (d = 0.25). There was clinically significant within-group improvement in overall PTSD symptoms in both groups, with a greater mean (SD) decrease in CAPS score in the riluzole group (-21.1 [18.9]) than in the placebo group (-16.7 [17.2]). Exploratory analyses of PTSD symptom clusters showed significantly greater improvement on hyperarousal symptoms in the riluzole group as measured by the PTSD Checklist-Specific-Subscale D (d = 0.48) and near-significant findings on the CAPS Subscale D. Riluzole augmentation was not superior to placebo on change in depression, anxiety, or disability severity. Conclusions Although preliminary, the exploratory findings of this study offer some evidence that riluzole augmentation of an SSRI or SNRI may selectively improve PTSD hyperarousal symptoms without changes in overall PTSD symptoms, depression, anxiety, or disability. Additional investigation of the mechanism of the efficacy of riluzole for hyperarousal symptoms is warranted. Trial registration ClinicalTrials.gov identifier: NCT02155829.

Introduction: In a cohort of 1038 patients with pulmonary arterial hypertension (PAH) we identified 11 new mutations in ATP13A3. ATP13A3 is a poorly characterized ATPase, but recent studies suggest a role in polyamine (PA) transport. We sought to determine the impact of ATP13A3 deficiency on PA homeostasis in human pulmonary arterial endothelial cells (hPAECs). Further, we assessed if the introduction of a homolog of human disease-causing truncating mutation (P452Lfs) increased the susceptibility to PAH in a mouse model. Methods: ATP13A3 knockdown (KD) in hPAECs was achieved by siRNA transfection. P452Lfs mutant mice were generated via CRISPR-Cas9 (MRC Harwell). Echocardiography, invasive haemodynamic measurements, organ harvest were performed at 6 months of age. Polyamine content of the lungs and hPAECs was assessed by mass spectrometry. Results: ATP13A3 KD in hPAECs reduced intracellular PA content. mRNA expression of ornithine decarboxylase, rate-limiting enzyme of PA biosynthesis, was not altered, while its protein level was increased. ATP13A3 KD predisposed hPAECs to apoptosis, inhibited proliferation. P452Lfs mice developed PAH compared to wild type controls: exhibited shortened pulmonary artery acceleration time (12.1±0.8 vs 16.6±0.8ms), elevated right ventricular systolic pressure (30.2±0.5 vs 25.6±0.6mmHg) with no change in systemic pressures. Moreover, P452Lfs mice demonstrated right ventricular anterior wall thickening 0.65±0.05 vs 0.41±0.06mm and a decreased PA content in their lungs. Conclusions: Our initial findings provide further evidence that loss of function mutations in ATP13A3 are directly involved in causation of PAH and the mechanism involves alterations in cellular polyamine levels.

Introduction: Recently, our group identified 11 heterozygous mutations in ATP13A3 in a cohort of 1038 patients with pulmonary arterial hypertension (PAH). Of these, 6 variants were predicted to cause protein truncation. ATP13A3 is a poorly characterized P5-type ATPase, however studies in C. elegans suggest a role in polyamine transport and metabolism. Polyamines are known to be important in the regulation of basic cellular functions: cell proliferation, differentiation and apoptosis. In a genetically modified mouse, harbouring a human disease-associated truncating mutation (P452Lfs) of ATP13A3 we aimed to assess the development of PAH. Methods: P452Lfs knock-in mice were generated via CRISPR-Cas9. Wild type and mutant animals of both genders were assessed. Non-invasive echocardiography was carried out at 3 and 6 months of age. Invasive haemodynamic measurement followed by blood and organ harvesting for histological and molecular studies was performed at 6 months. Polyamine content of the lungs was assessed by mass spectrometry. Results: By 6 months male Atp13a3 mutant mice developed PAH compared to their wild type counterparts: they exhibited shortened pulmonary artery acceleration time (14.6±0.6 vs. 17.1±1.1 ms), elevated right ventricular systolic pressure (30.2±0.5 vs 25.6±0.6 mmHg) with no change in systemic pressures. In addition they are showing right ventricular dilatation as assessed by ECHO: right ventricular end-diastolic diameter of 1.86±0.17 vs 0.96±0.17 mm. In addition, male mutant mice demonstrated decreased polyamine content in their lungs. Conclusions: Our initial findings provide further evidence that loss of function mutations in ATP13A3 are directly involved in causation of PAH.

Background In the diagnosis of venous stenosis associated with idiopathic intracranial hypertension, previous studies have identified significant differences when venous pressures are measured under conscious sedation versus general anesthesia. No previous reports have investigated the effect that respiratory parameters may have on cerebral venous sinus pressure and the associated trans-stenosis pressure gradient. Case Description Two patients with idiopathic intracranial hypertension were retrospectively identified from a prospective database wherein venous manometry was performed as part of a venous stenting procedure with waveform recording during changes in end-tidal carbon dioxide level (EtCO2). Upon microcatheterization of the superior sagittal sinus after induction of general anesthesia, both patients were noted to have an EtCO2 of 29 mm Hg. After EtCO2 was corrected to 40 mm Hg, repeat venous manometry was conducted, which demonstrated increased SSS venous pressures from 12.6 to 21.1 mm Hg and 18.4 to 30.3 mm Hg in patients 1 and 2, respectively. In addition, the waveform amplitude increased in both patients after EtCO2 correction. Conclusions This series demonstrates that EtCO2 changes have an immediate and pronounced effect on venous sinus pressure measurements with waveform changes that may correlate to increased intracranial pressure. These findings underscore the need to perform measurements of venous sinus pressure gradients under normal awake conditions.

IntroductionThe relationship between degree of angiographic venous sinus stenosis and the trans-stenosis pressure gradient magnitude in idiopathic intracranial hypertension (IIH) is poorly understood. The present study aimed to assess the utility of angiography, venography, and non-invasive imaging (MRV or CTV) for the diagnosis and characterization of clinically significant VSS.MethodsRetrospective analysis of a prospectively collected database was performed to identify patients with medically refractory IIH who were evaluated by angiography and venous manometry for the presence of VSS with associated clinically significant pressure gradient. Angiographic stenosis was measured by two independent raters using novel methodology.ResultsThirty-seven patients met inclusion criteria for the study. In total, 70% of patients had clinically significant pressure gradients and were selected for stenting. The optimal percentage stenosis for detection of a significant pressure gradient was 34% stenosis on venous phase arteriography (sensitivity 0.81 and specificity 0.91) and 31% stenosis on venography (0.92 and 0.73). For every 10% increase in stenosis, an approximate increase in pressure gradient of 3.5 mmHg is seen. MRV/CTV had a calculated sensitivity of 0.42, and a negative predictive value of 22%.ConclusionThe degree of stenosis predictive of a clinically significant pressure gradient (30–35%) in the venous sinuses is considerably lower than the arterial stenosis at which pathologic hemodynamic alterations occur. While highly predictive of a venous pressure gradient when a stenosis is identified, non-invasive imaging does not appear to be a suitable diagnostic evaluation for the purpose of ruling out clinically significant cerebral VSS.

IntroductionVenous outflow obstruction is recognized as a contributing factor in a subset of patients with idiopathic intracranial hypertension (IIH). Little is known about venous sinus waveform morphology or how it changes after stenting.MethodsFifteen patients with IIH underwent waveform recording during catheter venography and manometry. Ten patients (Group A) with venous sinus stenosis and pressure gradient ≥7 mm Hg underwent waveform recording during awake venography and during stenting under general anesthesia. Five control IIH patients (Group B) without a gradient underwent awake recording only.ResultsGroup A patients underwent successful stenting with reduction of their gradient from 15.1±6.19 mm Hg to 1.2±0.60 mm Hg. This resulted in an amplitude reduction from 8.3 mm Hg to 2.8 mm Hg (P=0.02). Qualitative evaluation of the waveform yielded a number of novel findings. In Group A before stenting, the observed waveform progressed from an intracranial pressure (ICP)-dominated to central venous pressure (CVP)-dominated waveform. Stenting abolished the high amplitude waveform and smoothed the transition from the intracranial to central venous measurement points. Group B displayed primarily CVP-influenced waveforms distal and proximal to the transverse-sigmoid junction along with respiratory variability of the waveform, absent in 8/10 Group A patients. General anesthesia appeared to blunt the waveform in 5/10 Group A patients.ConclusionThe cerebral venous waveform appears to be influenced by both the ICP and CVP waveforms. As measurement moves proximally, the waveform progressively changes to mirror the CVP waveform. Venous sinus stenosis results in a high amplitude waveform which improves with treatment of the stenosis.

Ketosis, the metabolic response to energy crisis, is a mechanism to sustain life by altering oxidative fuel selection. Often overlooked for its metabolic potential, ketosis is poorly understood outside of starvation or diabetic crisis. Thus, we studied the biochemical advantages of ketosis in humans using a ketone ester-based form of nutrition without the unwanted milieu of endogenous ketone body production by caloric or carbohydrate restriction. In five separate studies of 39 high-performance athletes, we show how this unique metabolic state improves physical endurance by altering fuel competition for oxidative respiration. Ketosis decreased muscle glycolysis and plasma lactate concentrations, while providing an alternative substrate for oxidative phosphorylation. Ketosis increased intramuscular triacylglycerol oxidation during exercise, even in the presence of normal muscle glycogen, co-ingested carbohydrate and elevated insulin. These findings may hold clues to greater human potential and a better understanding of fuel metabolism in health and disease.

The objective was to study the effects of a commercial pyrethroid-based pour-on product, permethrin, on reproductive performance in superovulated beef heifers by assessing steroid biosynthesis and embryo quality. Nonpregnant, yearling beef heifers (n = 10; 418 ± 33 kg; 5.5 ± 0.2 body conditioning scores) were assigned by body weight and breed to either (1) saline control or (2) permethrin pour-on administered at label dose (PYR). Superovulation was achieved on all heifers using a timed, 17-day, CIDR-based protocol with GnRH and PGF2α and decreasing total dosage of 240-mg FSH administered twice daily for 4 days. Heifers were artificially inseminated twice (at onset of estrus and 12 hours later) by same technician with frozen semen from single bull collection. To determine short- and long-term effects of permethrin on embryo quality and steroid biosynthesis, superovulation was initiated twice with collection of embryos occurring at 17 and 51 days after treatment. Embryos were recovered 6.5 days after first artificial insemination via nonsurgical flush and were evaluated by International Embryo Transfer Society standards. Blood was collected at standing estrus and day of embryo recovery. Estradiol (E2) and progesterone (P4) concentrations were analyzed via RIA. MIXED and GLIMMIX procedures of SAS were used to analyze continuous and categorical data, respectively. Heifer per flush was the experimental unit. Total embryos recovered did not differ because of treatment (P = 0.30), but did decrease in flush 2 compared with flush 1 (P = 0.02). Quality grade, total transferable quality embryos, and overall flush success did not differ because of treatment (P ≥ 0.16). However, transferable quality embryos were decreased in flush 2 compared with flush 1 (P = 0.05). Total unfertilized oocytes were greater in saline control (P = 0.04). The PYR heifers tended to have less total P4 (P = 0.15) and P4 per CL (P = 0.06) at recovery. E2 per ovulated follicle and E2 per total ovarian structure was greater in flush 2 (P ≤ 0.03) but did not differ because of treatment (P ≥ 0.23). In summary, these data indicate that permethrin administration at label dose in superovulated beef heifers has a tendency to reduce P4, but embryo quality is not affected.

The peroxisome proliferator-activated receptors (PPARs) are ligand activated nuclear receptors that regulate cellular homoeostasis and metabolism. PPARs control the expression of genes involved in fatty-acid and lipid metabolism. Despite evidence showing beneficial effects of their activation in the treatment of metabolic diseases, particularly dyslipidaemias and type 2 diabetes, PPAR agonists have also been associated with a variety of side effects and adverse pathological changes. Agonists have been developed that simultaneously activate the three PPAR receptors (PPARα, γ and δ) in the hope that the beneficial effects can be harnessed while avoiding some of the negative side effects. In this study, the hepatic effects of a discontinued PPAR-pan agonist (a triple agonist of PPAR-α, -γ, and -δ), was investigated after dietary treatment of male Sprague–Dawley (SD) rats. The agonist induced liver enlargement in conjunction with metabolomic and lipidomic remodelling. Increased concentrations of several metabolites related to processes of oxidation, such as oxo-methionine, methyl-cytosine and adenosyl-methionine indicated increased stress and immune status. These changes are reflected in lipidomic changes, and increased energy demands as determined by free fatty acid (decreased 18:3 n−3, 20:5 n−3 and increased ratios of n−6/n−3 fatty acids) triacylglycerol, phospholipid (decreased and increased bulk changes respectively) and eicosanoid content (increases in PGB2 and 15-deoxy PGJ2). We conclude that the investigated PPAR agonist, GW625019, induces liver enlargement, accompanied by lipidomic remodelling, oxidative stress and increases in several pro-inflammatory eicosanoids. This suggests that such pathways should be monitored in the drug development process and also outline how PPAR agonists induce liver proliferation., Highlights • The investigated PPAR-pan agonist increased oxidative damage in the rat liver. • Metabolomics can be used to follow the dose response nature of PPAR-pan agonists. • Metabolomics is a versatile tool in following oxidative stress.

Objective Insulin signalling via phosphoinositide 3-kinase (PI3K) requires PIK3R1-encoded regulatory subunits. C-terminal PIK3R1 mutations cause SHORT syndrome, as well as lipodystrophy and insulin resistance (IR), surprisingly without fatty liver or metabolic dyslipidaemia. We sought to investigate this discordance. Methods The human pathogenic Pik3r1 Y657∗ mutation was knocked into mice by homologous recombination. Growth, body composition, bioenergetic and metabolic profiles were investigated on chow and high-fat diet (HFD). We examined adipose and liver histology, and assessed liver responses to fasting and refeeding transcriptomically. Results Like humans with SHORT syndrome, Pik3r1WT/Y657∗ mice were small with severe IR, and adipose expansion on HFD was markedly reduced. Also as in humans, plasma lipid concentrations were low, and insulin-stimulated hepatic lipogenesis was not increased despite hyperinsulinemia. At odds with lipodystrophy, however, no adipocyte hypertrophy nor adipose inflammation was found. Liver lipogenic gene expression was not significantly altered, and unbiased transcriptomics showed only minor changes, including evidence of reduced endoplasmic reticulum stress in the fed state and diminished Rictor-dependent transcription on fasting. Increased energy expenditure, which was not explained by hyperglycaemia nor intestinal malabsorption, provided an alternative explanation for the uncoupling of IR from dyslipidaemia. Conclusions Pik3r1 dysfunction in mice phenocopies the IR and reduced adiposity without lipotoxicity of human SHORT syndrome. Decreased adiposity may not reflect bona fide lipodystrophy, but rather, increased energy expenditure, and we suggest that further study of brown adipose tissue in both humans and mice is warranted., Highlights • SHORT syndrome features insulin resistance and reduced adiposity without dyslipidaemia and fatty liver. • A mouse model with a pathogenic human PI 3-Kinase mutation recapitulates this uncoupling. • Surprisingly, no adipose injury nor increased liver de novo lipogenesis is seen. • Energy expenditure is increased, causing resistance to diet-induced obesity. • This increases evidence for some beneficial metabolic effects of PI 3-Kinase inhibition.

BACKGROUND Dietary changes have led to the growing prevalence of type 2 diabetes and nonalcoholic fatty liver disease. A hallmark of both disorders is hepatic lipid accumulation, derived in part from increased de novo lipogenesis. Despite the popularity of high-protein diets for weight loss, the effect of dietary protein on de novo lipogenesis is poorly studied. We aimed to characterize the effect of dietary protein on de novo lipid synthesis. METHODS We use a 3-way crossover interventional study in healthy males to determine the effect of high-protein feeding on de novo lipogenesis, combined with in vitro models to determine the lipogenic effects of specific amino acids. The primary outcome was a change in de novo lipogenesis–associated triglycerides in response to protein feeding. RESULTS We demonstrate that high-protein feeding, rich in glutamate, increases de novo lipogenesis–associated triglycerides in plasma (1.5-fold compared with control; P < 0.0001) and liver-derived very low-density lipoprotein particles (1.8-fold; P < 0.0001) in samples from human subjects (n = 9 per group). In hepatocytes, we show that glutamate-derived carbon is incorporated into triglycerides via palmitate. In addition, supplementation with glutamate, glutamine, and leucine, but not lysine, increased triglyceride synthesis and decreased glucose uptake. Glutamate, glutamine, and leucine increased activation of protein kinase B, suggesting that induction of de novo lipogenesis occurs via the insulin signaling cascade. CONCLUSION These findings provide mechanistic insight into how select amino acids induce de novo lipogenesis and insulin resistance, suggesting that high-protein feeding to tackle diabetes and obesity requires greater consideration. FUNDING The research was supported by UK Medical Research Council grants MR/P011705/1, MC_UP_A090_1006 and MR/P01836X/1. JLG is supported by the Imperial Biomedical Research Centre, National Institute for Health Research (NIHR)., A subset of amino acids may induce de novo lipogenesis in humans, suggesting that use of high-protein diets to tackle diabetes requires greater consideration.

Introduction/purpose Idiopathic intracranial hypertension (IIH), otherwise known as pseudotumor cerebri or benign intracranial hypertension, is a syndrome defined by a severe headache in the setting of increased intracranial pressure (ICP) in the absence of an intracranial mass. The pathophysiology of IIH remains poorly understood. However, venous outflow obstruction detected by cerebral venous manometry at the transverse sinus has been recognized as a contributing factor in a subset of patients with IIH, and stent placement in this subset of patients is associated with good clinical outcomes. Surgeons often determine candidacy for stent placement based on the magnitude of the venous pressure gradient across the site of stenosis. Furthermore, there is little known about venous sinus pressure waveform morphology in patients with this condition or how it changes after stenting. The purpose of this study was to prospectively analyze patients’ venous sinus waveforms, their component peaks, and the influence of stenosis or anesthesia on waveform morphology with a goal of ultimately providing insight into the waveform and pathological morphologic changes seen with venous sinus stenosis. Materials and methods Fifteen patients with IIH were enrolled prospectively and underwent waveform recording during catheter venography and manometry. Ten patients (Group A) with venous sinus stenosis and pressure gradient >8 mmHg underwent waveform recording during awake venography and then pre- and post-stent manometry while under general anesthesia. Five control IIH patients (Group B) without a trans-stenosis gradient underwent awake recording only. Results All Group A patients underwent successful stenting with reduction of their gradient from 15.1 (+6.19) mmHg to 1.2 (+0.60) mmHg. This resulted in statistically significant reduction of the transverse sinus amplitude from 8.3 mmHg to 2.8 mmHg (p Conclusion In conclusion, the cerebral venous waveform morphology appears to be influenced by both the intracranial pressure (ICP) waveform and the central venous pressure (CVP) waveform. Additionally, venous sinus stenosis with significant venous outflow obstruction results in morphologic changes to the waveforms. After performing stenting in patients with stenosis, waveforms return to match the control group. Future studies should expand on the qualitative patterns described in this study and place a significant amount of consideration in determining the clinical and hemodynamic ramifications of these findings. Disclosures J. West: None. R. Garner: None. G. Greeneway: None. J. Traunero: None. C. Aschenbrenner: None. J. Singh: None. S. Wolfe: None. K. Fargen: None.

BACKGROUND: Inflammatory bowel disease is a group of pathologies characterised by chronic inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn's disease and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and metabolomics could represent a less invasive approach to identify biomarkers of disease presence and progression. OBJECTIVES: The lipid and the polar metabolite profile of plasma samples of patients affected by inflammatory bowel disease have been compared with healthy individuals with the aim to find their metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase extraction to further characterise differences between pathological samples. METHODS: A total of 200 plasma samples were analysed using drift tube ion mobility coupled with time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, while liquid chromatography coupled with triple quadrupole mass spectrometry was used for the polar metabolite profile analysis. RESULTS: Variations in the lipid profile between inflammatory bowel disease and healthy individuals were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly changed among pathological samples suggesting changes in phospholipase A2 and arachidonic acid metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory bowel disease. CONCLUSIONS: Given good statistical results and predictive power of the model produced in our study, metabolomics can be considered as a valid tool to investigate inflammatory bowel disease., This study was funded by Agilent Technologies, Regione Autonoma della Sardegna (L.R.7/2007, Grant Number F71J12001180002), and the Medical Research Council UK (Grant Number MR/P011705/1).

Background— Myocardial infarction (MI) is one of the leading causes of heart failure. An increasing body of evidence links alterations in cardiac metabolism and mitochondrial function with the progression of heart disease. The aim of this work was to, therefore, follow the in vivo mitochondrial metabolic alterations caused by MI, thereby allowing a greater understanding of the interplay between metabolic and functional abnormalities. Methods and Results— Using hyperpolarized carbon-13 ( 13 C)-magnetic resonance spectroscopy, in vivo alterations in mitochondrial metabolism were assessed for 22 weeks after surgically induced MI with reperfusion in female Wister rats. One week after MI, there were no detectable alterations in in vivo cardiac mitochondrial metabolism over the range of ejection fractions observed (from 28% to 84%). At 6 weeks after MI, in vivo mitochondrial Krebs cycle activity was impaired, with decreased 13 C-label flux into citrate, glutamate, and acetylcarnitine, which correlated with the degree of cardiac dysfunction. These changes were independent of alterations in pyruvate dehydrogenase flux. By 22 weeks, alterations were also seen in pyruvate dehydrogenase flux, which decreased at lower ejection fractions. These results were confirmed using in vitro analysis of enzyme activities and metabolomic profiles of key intermediates. Conclusions— The in vivo decrease in Krebs cycle activity in the 6-week post-MI heart may represent an early maladaptive phase in the metabolic alterations after MI in which reductions in Krebs cycle activity precede a reduction in pyruvate dehydrogenase flux. Changes in mitochondrial metabolism in heart disease are progressive and proportional to the degree of cardiac impairment.

Cerebral hypoxia has been proposed as a mechanism by which prenatal ethanol exposure causes fetal alcohol spectrum disorder (FASD) in children, but no study had tested this hypothesis using a chronic exposure model that mimicks a common human exposure pattern. Pregnant sheep were exposed to ethanol, 0.75 or 1.75 g kg(-1) (to create blood ethanol concentrations of 85 and 185 mg dl(-1), respectively), or saline 3 days per week in succession (a 'binge drinking' model) from gestational day (GD) 109 until GD 132. Fetuses were instrumented on GD 119-120 and studied on GD 132. The 1.75 g kg(-1) dose resulted in a significant increase in fetal biventricular output (measured by radiolabelled microsphere technique) and heart rate, and a reduction of mean arterial pressure and total peripheral resistance at 1 h, the end of ethanol infusion. The arterial partial pressure of CO(2) was increased, arterial pH was decreased and arterial partial pressure of O(2) did not change. Fetal whole-brain blood flow increased by 37% compared with the control group at 1 h, resulting in increased cerebral oxygen delivery. The elevation in brain blood flow was region specific, occurring preferentially in the ethanol-sensitive cerebellum, increasing by 44% compared with the control group at 1 h. There were no changes in the lower dose group. Assessment of regional differences in the teratogenic effects of ethanol by stereological cell-counting technique showed a reduced number of cerebellar Purkinje cells in response to the 1.75 g kg(-1) dose compared with the control brains. However, no such differences in neuronal numbers were observed in the hippocampus or the olfactory bulb. We conclude that repeated exposure to moderate doses of ethanol during the third trimester alters fetal cerebral vascular function and increases blood flow in brain regions that are vulnerable to ethanol in the presence of acidaemia and hypercapnia, and in the absence of hypoxia.

The ectopic deposition of fat is thought to lead to lipotoxicity and has been associated with mitochondrial dysfunction and diabetic cardiomyopathy. We have measured mitochondrial respiratory capacities in the hearts of ob/ob and wild-type mice on either a regular chow (RCD) or high-fat (HFD) diet across four age groups to investigate the impact of diet and age on mitochondrial function alongside a comprehensive strategy for metabolic profiling of the tissue. Myocardial mitochondrial dysfunction was only evident in ob/ob mice on RCD at 14 months, but it was detectable at 3 months on the HFD. Liquid chromatography-mass spectrometry (LC-MS) was used to study the profiles of acylcarnitines and the accumulation of triglycerides, but neither class of lipid was associated with mitochondrial dysfunction. However, a targeted LC-MS/MS analysis of markers of oxidative stress demonstrated increases in GSSG/GSH and 8-oxoguanine, in addition to the accumulation of diacylglycerols, which are lipid species linked to lipotoxicity. Our results demonstrate that myocardial mitochondria in ob/ob mice on RCD maintained a similar respiratory capacity to that of wild type until a late stage in aging. However, on a HFD, unlike wild-type mice, ob/ob mice failed to increase mitochondrial respiration, which may be associated with a complex I defect following increased oxidative damage.

Background: In spite of the fact that drinking and smoking often occur together, little is known about the pharmacokinetic interaction between alcohol and nicotine. Previous research in neonatal rats demonstrated that nicotine reduces blood alcohol concentrations (BACs) if alcohol and nicotine are administered simultaneously. However, it is unclear whether such a phenomenon can be observed in adult subjects, given the fact that there is an ontogenetic difference in alcohol metabolism. Methods: A range of nicotine doses (0, 0.25, 0.5, 1, 2, 4, and 6 mg/kg) were administered individually with an alcohol dose (4 g/kg) via intragastric (IG) intubation to adult female rats, and the resultant BACs were measured at various time points following drug administration. Furthermore, the hypothesis that nicotine's role in reducing BACs is mediated through factors related to gastric function was examined by comparing the resultant BACs after an IG intubation or intraperitoneal (IP) injection of alcohol. Results: The results from this study showed significant nicotine dose–related decreases in BACs with 0.5, 1, 2, 4, and 6 mg/kg doses of nicotine at the various time points assessed. This effect, however, occurred only when alcohol was administered via IG intubation, but not after an IP injection of alcohol. Conclusions: These results suggest that the nicotine-induced decrease in BAC may be related to gastric function. One possible explanation was related to nicotine's action in delaying gastric emptying. The longer the alcohol was retained in the stomach, the more likely that the alcohol would be metabolized by gastric alcohol dehydrogenase before its absorption into the bloodstream by the small intestine (the major site of alcohol absorption).

In rats, damage to neuronal populations in some brain regions occurs in response to neonatal alcohol exposure coinciding with the period of rapid brain growth. These alcohol-induced defects in brain development may persist into adulthood and thus have long-term implications for the functional characteristics of damaged neuronal populations. The present study examined the effects of neonatal alcohol exposure on endogenous rhythmicity of the circadian clock located in the rat suprachiasmatic nucleus (SCN). Specifically, experiments were conducted to determine whether neonatal alcohol exposure alters the circadian rhythm of brain-derived neurotrophic factor (BDNF) content in the rat SCN because this neurotrophin is an important rhythmic output from the SCN clock. Male rat pups were exposed to alcohol (4.5 g/kg/day) or isocaloric milk formula on postnatal days 4-9 using artificial rearing methods. At 5-6 months of age, SCN and hippocampal tissue was harvested and subsequently examined for content of BDNF protein. Time-dependent fluctuations in BDNF protein levels were assessed by enzyme-linked immunosorbent assay (ELISA). In alcohol-treated rats, SCN levels of BDNF were significantly decreased and were characterized by a loss of circadian rhythmicity relative to those observed in control animals. In comparison, hippocampal levels of BDNF displayed no evidence of circadian regulation in all three treatment groups, but were slightly lower in alcohol-treated animals than in control groups. Importantly, these observations suggest that alcohol exposure during the period of rapid brain development may cause permanent changes in the SCN circadian clock.

Children with fetal alcohol syndrome (FAS) display altered performance in tasks of learning and memory, behaviours thought to be associated with the hippocampus. Altered hippocampal structure has been reported in some FAS children; therefore, a rat model system was used to determine whether the size and numbers of pyramidal cells in regions CA1 and CA3 of the hippocampal formation and granule cells in the dentate gyrus were altered by alcohol exposure during different periods of development. Rat pups were exposed to alcohol in utero during the second trimester-equivalent (E10-20), the first two trimesters-equivalent (E1-20), during the time of hippocampal pyramidal cell neurogenesis (E16-20), part of the third trimester-equivalent (P4-9), and all three trimesters-equivalent (E1-20+P4-9). Control animals (nutritional and untreated) were reared for all treatment conditions. All pups were perfused on P10. CA1 volume, pyramidal cell density, and number were reduced in pups treated with alcohol during the third trimester-equivalent, whether unique or as exposure during all three trimesters-equivalent. CA3 volume was reduced in alcohol-treated animals across all gestational ages; however, pyramidal cell density and number in this region were only reduced in animals treated with alcohol during the third trimester-equivalent. Volume of the dentate gyrus did not appear to be affected by alcohol treatment. Granule cell density and number in this region were reduced in animals treated with alcohol during the third trimester-equivalent. The third trimester-equivalent in the rat appears to be a developmental period during which the hippocampus is particularly susceptible to the effects of alcohol consumption. The resulting damage to the hippocampus may contribute to the behavioural deficits related to learning and memory noted in children with FAS.

It is important to select an appropriate model system for studies examining the mechanisms of ethanol-induced injury. The most common model systems use either mice or rats with ethanol administered by means of intragastric gavage or intraperitoneal injection, yet few studies have compared directly the blood ethanol concentration (BEC) profiles that result from each of these model systems. In the current study, Sprague-Dawley rats and C57BL/6J mice were given ethanol by means of intragastric gavage or intraperitoneal injection at 40 days of age. Blood samples were collected at consistent time intervals to determine BECs. Blood ethanol concentrations in mice were sharper, with a more rapid rise to a sharp peak BEC, followed by a relatively rapid decline. In contrast, rat BEC profiles showed an initial rapid rise, followed by a more gradual rise to peak concentrations, and, then, a relatively gradual decline. This difference was particularly evident in rats receiving ethanol intragastrically. The differences found in BEC profiles between rats and mice and between ethanol administration paradigms may yield differences in the extent or mechanism of damage induced by ethanol, an important consideration when selecting an appropriate model for the investigation of ethanol-induced tissue damage.

Background: Abnormal thyroid hormone system function in the mother or fetus during pregnancy can result in brain defects, some of which resemble those found in children with fetal alcohol syndrome. It has been hypothesized that ethanol may act to mediate alcohol-related birth defects in part by altering thyroid hormone system function. We investigated whether a binge pattern of maternal ethanol consumption over the last trimester equivalent of gestation in sheep results in an alteration in fetal or maternal thyroid function. Methods: Pregnant ewes received saline or ethanol beginning on day 109 of gestation (term, 145 days) for three consecutive days per week followed by 4 days without exposure. The fetuses were surgically instrumented on day 113, and experiments were performed on days 118 or 132. Fetal and maternal blood samples were collected, and plasma tri-iodothyronine (T3), thyroxine (T4), and free T4 concentrations were measured. Results: Fetal T3 and T4 on day 118, fetal T3 on day 132, and maternal T3 on day 132 were lower in response to ethanol. Fetal and maternal free T4 and maternal T4 did not change in response to ethanol. Fetal thymus and adrenal weights were reduced in response to ethanol. Conclusions: We conclude that, in sheep, maternal ethanol exposure during the third trimester equivalent resulting in blood ethanol concentrations that are commonly achieved by ethanol abusers decreases circulating thyroid hormone concentrations in the mother and fetus and fetal thyroid and thymus mass.

Background Evidence from several recent metabolomic studies suggests that increased concentrations of triacylglycerols with shorter (14–16 carbon atoms), saturated fatty acids are associated with insulin resistance and the risk of type 2 diabetes. Although causality cannot be inferred from association studies, patients in whom the primary cause of insulin resistance can be genetically defined offer unique opportunities to address this challenge. Methods We compared metabolite profiles in patients with congenital lipodystrophy or loss-of-function insulin resistance (INSR gene) mutations with healthy controls. Results The absence of significant differences in triacylglycerol species in the INSR group suggest that changes previously observed in epidemiological studies are not purely a consequence of insulin resistance. The presence of triacylglycerols with lower carbon numbers and high saturation in patients with lipodystrophy suggests that these metabolite changes may be associated with primary adipose tissue dysfunction. The observed pattern of triacylglycerol species is indicative of increased de novo lipogenesis in the liver. To test this we investigated the distribution of these triacylglycerols in lipoprotein fractions using size exclusion chromatography prior to mass spectrometry. This associated these triacylglycerols with very low-density lipoprotein particles, and hence release of triacylglycerols into the blood from the liver. To test further the hepatic origin of these triacylglycerols we induced de novo lipogenesis in the mouse, comparing ob/ob and wild-type mice on a chow or high fat diet, confirming that de novo lipogenesis induced an increase in relatively shorter, more saturated fatty acids. Conclusions Overall, these studies highlight hepatic de novo lipogenesis in the pathogenesis of metabolic dyslipidaemia in states where energy intake exceeds the capacity of adipose tissue. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0179-6) contains supplementary material, which is available to authorized users.

Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l(-1) NaCl (as control) or 0.7 mmol l(-1) NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics.

Background : Although the mechanisms that underlie fetal alcohol-induced neuronal loss have not been determined, hypoxia/hypoxemia has been considered a leading candidate. This study was designed to test the hypothesis that neuronal loss could occur in the developing brain in the absence of fetal hypoxemia. Methods: Three groups of pregnant sheep were used: a control group, a binge-drinking group, and a pair-fed group. The alcohol and pair-fed animals were anesthetized on day 113 of pregnancy, and the mothers and fetuses were instrumented with arterial and venous catheters. All animals were killed on day 133. Stereological cell counting techniques were used to estimate the total number of Purkinje cells in the fetal cerebellum. Results: Peak maternal and fetal blood alcohol concentrations did not produce fetal hypoxemia. Nevertheless, there was a 25% loss of Purkinje cells of the cerebellum in the alcohol-exposed fetuses compared with that in the pair-fed controls. The loss of neurons was not accompanied by microencephaly or a concomitant decrease in either cerebellar weight or volume of the fetal cerebellum. Conclusions: Neuronal loss can be observed after alcohol exposure during the third trimester equivalent in fetal sheep in the absence of alcohol-induced hypoxemia. Furthermore, cell loss in the absence of deficits in gross brain weight or regional brain volume indicates that the lack of gross brain volume deficits from magnetic resonance imaging techniques is not a reliable indication that the brain is unaffected by the alcohol exposure.

Background : We tested the hypothesis that in utero ethanol exposure results in changes in fetal and maternal adrenocorticotropin (ACTH) and cortisol during the third trimester equivalent, by using a chronically instrumented fetal sheep model. Methods: Pregnant ewes received saline or ethanol intravenously 3 consecutive days per week from day 109 to day 132 of gestation. Fetal and maternal blood samples were collected on days 118 and 132. Results: Maternal and fetal ACTH and cortisol values increased on days 118 and 132 of gestation in response to ethanol infusions that created blood ethanol concentrations (BECs) that are easily achievable by human drinkers. Peak ACTH and cortisol values were detected 30 to 60 min after peak BECs were achieved. Conclusions: Chronic ethanol exposure during the third trimester equivalent in sheep resulted in repeated activation of the hypothalamus-pituitary-adrenal axis in both the mother and fetus. Temporally, the patterns of maternal and fetal responses to ethanol infusion were similar. We conclude that ovine maternal ethanol exposure during the third trimester equivalent increases fetal ACTH and cortisol concentrations, hormonal responses that may play a role in mediating alcohol-related birth defects.

Background: Prenatal alcohol exposure disrupts motor performance in affected offspring. The ventrolateral nucleus (VLN) of the thalamus functions to relay information between the cerebellum and motor cortex. Reductions in the size of the thalamus have been found in children with fetal alcohol syndrome, and therefore a rat model system was used to determine whether VLN size and neuronal number were altered by alcohol exposure during development. Methods: Rat pups were exposed to alcohol in utero during the first 10 days of gestation (first trimester equivalent), the second 10 days of gestation (second trimester equivalent), or the first two trimesters equivalent combined. Some pups were exposed to alcohol in utero during the time of VLN neurogenesis. In addition, offspring from some of the dams treated during the first two trimesters equivalent were reared artificially from postnatal day (P) 4 through P9 (part of the third trimester equivalent) and received binge-like alcohol during this time, resulting in offspring exposed to alcohol during all three trimesters equivalent. Other offspring from untreated dams were reared in the same manner but received alcohol only during the third trimester equivalent. Control animals (nutritional and untreated) were reared for all treatment conditions. All pups were perfused on P10. Results: A unique effect of alcohol treatment was not found for the VLN volume or the number of neural cells within the VLN. However, the period of VLN neurogenesis was found to be sensitive to both alcohol and nutritional control treatments, resulting in significant decreases in the VLN volume and neural cell number. Conclusions: Motor deficits seen in offspring exposed prenatally to alcohol do not seem to result from direct effects on the structure of the VLN of the thalamus.

Background: Alcohol-induced zinc deficiency is one of the mechanisms proposed as a cause of developmental brain damage associated with fetal alcohol syndrome. It is known that alcohol exposure during the brain growth spurt period leads to cerebellar Purkinje cell loss. Therefore, this study examined whether zinc supplementation was capable of preventing alcohol-induced Purkinje cell loss in the cerebellar vermis in a neonatal rat model system. Methods: Sprague-Dawley rat pups were given alcohol (EtOH; 4.5 g/kg/day), zinc (Zn; 0.54 mg/ml diet; [10 times the regular diet Zn concentration]), or both from postnatal days (PD) 4 through 9 using the artificial-rearing paradigm. A gastrostomy control (GC) and a suckle control group (SC) also were included. All pups were killed on PD 10. Following perfusion, the cerebellar vermis was dissected and processed for stereological cell counting. The total number of Purkinje cells and the volume of the cerebellar vermis were determined. Results: Alcohol produced a significant loss of Purkinje cells compared with that in the GC group (no EtOH and no Zn supplement). The zinc supplementation had no effect in attenuating alcohol-induced Purkinje cell loss in the cerebellar vermis. In fact, the serum zinc concentration data indicated higher zinc concentrations following either EtOH or Zn treatment. Interestingly, the GC group showed a significantly lower zinc concentration compared with the SC group, even though no significant difference in Purkinje cell numbers was observed between these two control groups. Conclusion: These findings indicate that alcohol exposure during the third trimester equivalent did not result in zinc deficiency in this neonatal rat model system, nor did zinc supplementation rescue the alcohol-induced Purkinje cell loss in the cerebellar vermis. These findings showed clearly that the serum zinc concentration was not correlated with Purkinje cell loss, suggesting that alcohol-induced loss of cerebellar Purkinje cells in this neonatal rat model system is independent of the availability of serum zinc.

Background: The mechanisms by which maternal ethanol abuse during pregnancy causes neurodevelopmental injury in the fetus are not well understood. The purpose of this study was to use a chronically instrumented fetal sheep model system to determine if a binge pattern of ethanol exposure administered throughout the third trimester reduced fetal arterial partial pressure of oxygen (P a O 2 ); a positive finding would support the hypothesis that fetal hypoxemia may play a role in mediating ethanol-related birth defects. Methods: Pregnant ewes received saline or 0.75, 1.25, 1.5, or 1.75 g/kg of ethanol intravenously over 1 hr beginning on day 109 of gestation (term = 145 days) for 3 consecutive days per week followed by 4 days without exposure. The fetuses were surgically instrumented on day 113, and experiments were performed on days 118 or 132, the 6th and the 12th ethanol exposure, respectively. Results: Ethanol infusions resulted in peak blood ethanol concentrations of 80.8 ± 6.5, 182.5 ± 13.5, 224.4 ± 13.9, and 260.6 ± 20.0 mg/dl ± SEM (maternal) and 70.0 ± 5.9, 149.7 ± 9.0, 216.9 ± 14.0, and 233.3 ± 19.8 mg/dl ± SEM (fetal) in response to the 0.75, 1.25, 1.5, and 1.75 g/kg doses, respectively. Maternal and fetal heart rate and maternal blood pressure increased whereas fetal blood pressure decreased in a dose-dependent manner in response to ethanol infusions. Maternal and fetal arterial pH decreased and arterial partial pressures of carbon dioxide increased in response to ethanol infusions. Maternal P a O 2 decreased whereas fetal P a O 2 did not change in response to ethanol infusions. Conclusions: A binge ethanol exposure paradigm, three consecutive days per week throughout the third trimester at ethanol doses that created blood ethanol concentrations commonly achieved by human ethanol abusers, resulted in changes in maternal and fetal heart rate, changes in blood pressure, hypercapnea, acidemia, and maternal, but not fetal, hypoxemia. We conclude that in an ovine model system, ethanol doses that create blood ethanol concentrations as high as 260 mg/dl do not result in fetal hypoxemia. Remaining issues to address with this model system are whether neurodevelopmental injuries that are associated with maternal ethanol abuse are mediated by a reduction in fetal cerebral blood flow, fetal hypercapnea, or acidemia.

Background: The mechanisms that mediate fetal brain injury which results from maternal alcohol consumption are not well understood. Although fetal hypoxia is a popularly proposed mechanism, it has been difficult to assess brain oxygenation in vivo. We measured intracellular high energy phosphate concentrations and estimated intracellular pH (pH i ) in brains of unanesthetized neonatal rat pups by using in vivo 31 P-NMR spectroscopy. We reasoned that decreases in brain oxygenation sufficient to result in brain injury would also reduce high energy phosphates and pH i . Methods: On postnatal day 4, before alcohol administration, pups were placed into a 20 mm diameter NMR probe, their heads were positioned carefully in the center of the 31 P detection coil, and spectra were collected over 20 min. Animals were then fed diet with or without 4.5 g/kg of ethanol in two (in succession) of 12 daily feedings via artificial rearing methods. A second spectrum was collected at 90 min after the beginning of the second alcohol feeding, at the time that coincided with the peak blood alcohol concentration (BAC). Identical feedings were performed daily until day 9, when pre- and postfeeding spectra were again obtained. Positive control groups were fed control diet and were studied in atmospheres of 5% oxygen, 95% nitrogen or 0% oxygen, 100% nitrogen. Results: Phosphocreatine (PCr), β-adenosine triphosphate (ATP), and pH i decreased and inorganic phosphate (P i ) increased in day 4 animals subjected to 0% oxygen (20 min) compared with pretreatment and all other treatment groups. Day 9 animals did not tolerate these conditions. There were no significant changes in response to 5% oxygen on day 4, but P, increased and β-ATP decreased compared with pretreatment values and compared with alcohol and control groups on day 9. There were no changes in PCr, β-ATP, or pH, in response to alcohol treatment at either age. PCr was significantly increased in the alcohol and 5% oxygen groups and apparently increased in the control group on day 9 compared with day 4, most likely due to increases in cranial muscle mass within the NMR coil. Conclusions: We conclude that acute alcohol exposure that results in peak BACs of 315 mg/dl does not alter brain high energy phosphate concentrations or pH i in neonatal rat pups, although these BACs are known to result in significant brain injury. These findings do not support hypoxia as a mechanism of alcohol-mediated brain injury during the third trimester equivalent in the rat pup model.

Introduction: Animal studies modeling fetal alcohol syndrome have demonstrated that developmental exposure to alcohol is associated with decreased brain weight and significant neuronal loss in multiple regions of the developing brain. Our previous data suggest that the Fas/Apo [apoptosis]-1 receptor is transiently expressed in the developing cerebral cortex during the peak period of naturally occurring apoptotic cell death and maximum sensitivity to alcohol. Therefore, we hypothesized that ethanol increases the expression of suicide receptors such as Fas/Apo-1 in the developing fetal cerebral cortex and leads to an upregulation or extension of the normal period of apoptosis and consequent disorganization of the neural circuitry. Methods: Ethanol was administered in one of four doses (120, 320, 630, and 950 mg/dl) to organotypic explant cultures of the developing cerebral cortex established from postnatal day 2 rats and maintained for 6 days in vitro. The number of cells expressing Fas/Apo-1 receptor mRNA was counted. Apoptosis was measured by the use of two independent assays; a cell death enzyme-linked immunosorbent assay for DNA fragmentation and flow cytometric analysis of Annexin-V binding to phosphatidylserine externalized to the outer leaflet of the plasma membrane. Necrosis was also estimated by two independent measures, the amount of lactate dehydrogenase released into culture medium and flow cytometric analysis of cells that were positive for both Annexin-V and propidium iodide. Results: A significantly larger number of developing cortical cells expressed Fas/Apo-1 mRNA at the lower doses (120 and 320 mg/dl) than at the higher doses (630 and 950 mg/dl). Furthermore, ethanol induced apoptosis in a dose-related manner, with peak apoptosis observed at a dose of 630 mg/dl in the case of DNA fragmentation and at 630 and 950 mg/dl in the case of phosphatidylserine translocation to the outer leaflet of the plasma membrane. Ethanol did not induce necrosis at any of the administered doses of ethanol. Conclusions: Our data suggest that ethanol induces a susceptibility to apoptotic signals at low doses by upregulating the expression of mRNAs for cytotoxic receptors such as Fas/Apo-1 in the developing cerebral cortex. However, ethanol itself specifically induces apoptosis in the developing cerebral cortex only at higher doses.

Although alcohol has been shown to affect brain development adversely, the underlying mechanism of alcohol's actions are poorly understood. The present study addressed the hypothesis that alcohol affects growth factor availability during critical periods of neural growth by measuring the mRNA expression of brain-derived neurotrophic factor (BDNF), a potent developmental growth factor. Multiple offspring of timed-pregnant rat dams given alcohol (6.0 g/kg per day) or control treatments during gestation were sacrificed at either embryonic (E) day 21 or E33 (usually postnatal day 10) when their olfactory bulbs were processed for molecular analyses or neuron counting. BDNF mRNA levels were measured by reverse-transcription–polymerase chain reaction, and DNA methylation of the BDNF gene was quantified by Southern blot analyses following digestion with methylation-sensitive enzymes. Estimates of total granule cell number were obtained by counting those cells using unbiased stereological techniques. There was a significant decrease in BDNF mRNA levels in the alcohol-exposed offspring of both ages compared with controls. In addition, the number of olfactory bulb granule cells significantly decreased in the E33 but not the E21 rat pups exposed to alcohol compared with their appropriate aged controls. Finally, BDNF DNA of alcohol-exposed animals was less susceptible to digestion with the methylation-sensitive enzyme HpaII compared with controls, suggesting that the DNA of the alcohol exposed pups was hypermethylated. Our results indicate that exposure to alcohol during early brain development in the rat, a period equivalent to the first two trimesters in humans, can have a detrimental effect on normal development of the olfactory bulb by reducing the number of BDNF-synthesizing neurons. Although the exact mechanism for the alcohol-induced neuronal loss is unknown, the inappropriate transcription of the BDNF gene is one mechanism that may account for the complexity of effects observed in offspring exposed to heavy alcohol exposurein utero. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 414–423, 1999

Children of women who abuse alcohol during pregnancy may be affected by varying degrees of neurological abnormality, even if they are not diagnosed with Fetal Alcohol Syndrome. The extent of the behavioral deficits of the affected offspring may be a function of several factors, such as the differential vulnerability of the various regions of the brain-to-alcohol insult. In this study, groups of timed-pregnant rats were exposed to different doses of alcohol (EtOH 2.25, EtOH 4.5, EtOH 6.5 g/kg/day) or control conditions (maltose dextrin solution or no treatment) from embryonic day 1 (E1: sperm positive) to E20. On E33 (usually postnatal day 10), all pups were perfused. Their brains were removed, dissected into forebrain, cerebellum, and brainstem, and weighed. Blood alcohol concentrations (BACs) were measured on 4 different days of gestation, but the peak BACs across gestation for the three alcohol-treated groups averaged 142, 294, and 413 mg/dl for the EtOH 2.25, EtOH 4.5, and EtOH 6.5 g/kg groups, respectively. Analysis of the body weight data indicated that pups in the EtOH 6.5 g/kg group had a greater somatic growth deficit than pups from all other groups. Although the whole brain, forebrain, cerebellum, and brainstem weights of pups in the EtOH 6.5 g/kg group were significantly smaller than those in the control groups, within-treatment group analyses indicated that the cerebella of pups in the EtOH 6.5 g/kg group were more severely affected than were their forebrains or brainstems. The analyses of the brain region to body weight ratios revealed again that the cerebellum-to-body-weight ratio of pups in the EtOH 6.5 g/kg group was more severely affected than the forebrain or brainstem to body weight ratios. Collectively, these data lend support to the view that gross regions of the brain are differentially vulnerable to alcohol insult during the first two trimesters equivalent, and suggest that the cerebellum is vulnerable to injury from exposure to high BACs during a developmental period other than the third trimester equivalent.

Animal models have clearly established that ethanol exposure can deplete neurons in the developing nervous system. However, the mechanism by which ethanol reduces cell number is unclear. In our study, cultures of pheochromocytoma cells, a neuronal-like cell line, were maintained in media, which supported cell proliferation. Although cell numbers continued to increase in the presence of ethanol, this increase was partially inhibited by ethanol exposure. This inhibitory effect was concentration and duration dependent. Cell proliferation was still partially inhibited after removal of ethanol, but this inhibition was temporary and disappeared after a 24-hr recovery period in ethanol-free conditions. Further study indicated that ethanol partially inhibited the increase in cell numbers by two mechanisms: (1) studies with vital stains indicated that ethanol induced cell death; (2) experiments using synchronized pheochromocytoma cell cultures showed that ethanol can induce cell cycle delay, thereby lengthening the doubling time of the cells. Analysis by flow cytometry indicated that with ethanol exposure, the cells accumulated in the G1 phase of the cell cycle. Our results suggest that in the developing nervous system, ethanol may limit the numbers of proliferating, neuronal precursor cells by two simultaneous mechanisms, cell death and cell cycle delay.

Annually approximately 1:200,000 young children and adolescents are affected by juvenile dermatomyositis (JDM). Genetic factors are thought to contribute to the etiology. Since the discovery of the human leukocyte antigen class II associated DM molecule much has been learned about its role in the normal processing of HLA-class II molecules with a limited number of polymorphisms being found. Blood samples were collected from 30 patients who were seen in the clinic and 40 healthy volunteers. Exon 3 of the HLA-DM A and B genes were amplified and specific polymorphisms were identified given allele designations. The DMA∗0103 allele was found in 43% of patient alleles versus 8% in the control group, this difference reached significance at a p value of 0.0004. The DMB∗0102 allele was found in 20% of patients compared with 3% of the controls with a calculated p value of 0.037. Relative risk (RR) ratios with CI were as follows: DMA∗0103 vs control RR = 5.7 and DMB∗0102 vs control RR = 8. In conclusion, we feel that the polymorphisms represented in the DMA∗0103 and the DMB∗0102 alleles are increased in frequency in our JDM patients.

Previous research from our laboratory has shown that [ethanol (EtOH)] exposure during the brain growth spurt is detrimental to olfactory bulb development. This study extends those findings by examining the effects of EtOH, nicotine (NIC), and the combination of these drugs (EtOH/NIC) on olfactory bulb mitral cell numbers, as well as on various major neurotransmitter levels in neonatal rats. An artificial rearing paradigm was used in the present studies. These artificially reared pups were given 4 g/kg/day of EtOH and/or 6 mg/kg/day of NIC on postnatal day (PD) 4 to PD 9, except in the case of the acute neurochemistry study, in which the pups received treatment on PD 9 only. An artificially reared gastrostomy control group (GC) and a suckle control group were included. The mean total numbers of mitral cells in the EtOH and NIC groups were significantly reduced from that of the GC, as well as the volume of the left main olfactory bulb. There was no difference among any of the groups in mitral cell density. As for neurochemistry data, there was no difference in neurotransmitter levels among any of the groups in the repeat exposure regimen. There were, however, changes after the acute exposure (exposure on PD 9 only). Both serotonin and GABA levels were significantly increased only after NIC exposure. However, norepinephrine levels were significantly decreased after acute exposure in all three drug treatment groups, compared with that of the control group. Except for the GC control group, dopamine levels were not detected consistently after acute exposure to EtOH, NIC, or EtOH/NIC. Collectively, these findings demonstrate that exposure to EtOH or NIC individually during the brain growth spurt results in developmental deficits in the olfactory bulb, suggesting that both EtOH and NIC are neuroteratogens. Furthermore, this study demonstrated the capability of NIC to antagonize (protect) EtOH-induced mitral cell loss in the developing olfactory bulb.

The purpose of this study was to investigate the spontaneous activity of mature rat cerebellar neurons that had been exposed to ethanol (EtOH) during postnatal days 4 to 10, which corresponds to the third trimester in humans. Newborn Sprague-Dawley rats were implanted with gastric feeding tubes and were artificially reared from postnatal days 4 to 10 with two different diets. The experimental group received 4.5 g/kg/day of EtOH delivered in a milk solution. Controls received similar feeding with an isocaloric supplement replacing the EtOH. Electrophysiological evaluations were performed after an EtOH-free rearing period. Although lobules IX and X of the cerebellar vermis appeared morphologically smaller in the animals neonatally exposed to EtOH, compared with controls, extracellular recordings from both Purkinje cells and Golgi interneurons in adult rats showed no differences in spontaneous activity or firing pattern between the control and EtOH-exposed animals. Similarly, excitations and inhibitions of Purkinje neuron activity evoked by parallel pathway stimulation appeared unaffected by the developmental EtOH exposure. However, we did observe a significant decrease in the proportion of Purkinje neurons generating complex spike bursts in the group exposed to EtOH neonatally. These data suggest that, although fewer Purkinje neurons may survive the brain growth spurt if exposed to EtOH during this critical period of development, those that do survive appear to function normally. The observed abnormality in complex spike production may result from EtOH effects on developing neurons in the inferior olive that give rise to the climbing fibers that cause this bursting pattern in Purkinje neurons.

Neuronal cell loss is one of the most debilitating effects of fetal ethanol exposure. Cultures of cerebellar granule cells are a useful model to investigate ethanol neurotoxicity, because ethanol depletes cell numbers in these cultures, which also occurs in vivo. The primary goal of the present study was to identify and characterize agents that can ameliorate the ethanol-induced cell death that occurs in this culture system. Growth factors, such as nerve growth factor (NGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) can prevent neuronal degeneration after toxic insult in various experimental paradigms. These growth factors were investigated in the current study to determine whether or not they can mitigate ethanol-induced death of cerebellar granule cells in culture. Results indicate that NGF and bFGF significantly reduced the ethanol-induced cell loss. Both the NGF- and bFGF-mediated neuroprotection required protein and RNA synthesis, because actinomycin D (RNA synthesis inhibitor) and cycloheximide (protein synthesis inhibitor) blocked their neuroprotective effects. In addition to its neuroprotective effect, bFGF also had a neurotrophic effect and could enhance cell survival in the absence of ethanol exposure. NGF did not have a neurotrophic effect. Neither EGF nor IGF-I was neuroprotective, although the latter did have a substantial neurotrophic effect. In conclusion, bFGF and NGF have long been recognized for their role in enhancing neuronal cell survival and differentiation. This study suggests that these growth factors can also provide neuroprotection against ethanol-induced cell death.

The cerebellum is especially vulnerable to ethanol's neurotoxic effects during development, and ethanol exposure during the brain growth spurt will deplete cerebellar neurons. The mechanisms underlying this neuronal cell loss remain elusive. Nerve growth factor (NGF) is a neurotrophin that promotes cell survival in various brain areas, and there is evidence that NGF may play a role in the developing cerebellum. This study examined whether ethanol exposure of the neonatal rat cerebellum altered the levels of either NGF or the expression of p75 and trkA, which are two components of the NGF receptor. Ethanol exposure had no effect on NGF levels in the neonatal cerebellum, as determined by an NGF-specific ELISA. Immunohistochemical labeling techniques indicated that both the p75 and trkA NGF receptors were expressed on Purkinje cell dendrites in the developing cerebellum, with posterior lobules expressing higher levels of p75 and trkA NGF receptor, compared with anterior lobules. Ethanol exposure of neonatal rats reduced the expression of both p75 and trkA NGF receptors on the Purkinje cell dendrites. These results suggest that ethanol could interfere with neurotrophic support of Purkinje cells by reducing the levels of available NGF receptor.

The concurrent use of alcohol and cocaine has recently attracted attention in the medical research field due to the prevalence of this drug abuse pattern and the exclusive formation of a pharmacologically active substance, cocaethylene (CE). This is the first study to examine the neuroteratogenic effects of cocaethylene exposure during the brain growth spurt (part of the third trimester equivalent) on brain growth restrictions and neurochemical profiles. For the brain growth restrictions study, three groups of artificially reared rat pups were given daily injections of 0, 10 or 20 mg/kg cocaethylene (s.c.) from postnatal days (PDs) 4 through 9. One group of normally reared pups (suckle control) also was used. These pups were perfused on PD 10 and the brains were removed and weighed (forebrain, cerebellum and brainstem). For the neurochemistry study, five groups of artificially reared pups were used and were treated identically to those in the brain growth restrictions study, with the exceptions that animals assigned to acute cocaethylene treatment groups did not receive cocaethylene from PDs 4 through 8 and all animals in this study were sacrificed on PD 9 by decapitation. One suckle control group was included to control the possible artificial rearing effects on the neurochemical measures. Blood and fresh brain tissues (cortex, subcortical structures, cerebellum and brainstem) were collected for blood cocaethylene concentration and neurochemical analyses using GC/MS and HPLC techniques, respectively. The statistical analyses indicated that daily administration of 10 or 20 mg/kg cocaethylene, but not 0 mg/kg cocaethylene, significantly restricted the brain growth (brain weights) in all three brain regions assessed. Furthermore, cocaethylene administration from PDs 4 through 9 produced region-specific alterations in various neurotransmitter concentrations. The changes in neurotransmitter levels were not a function of the responses to the last cocaethylene injection on PD 9, since the outcomes between six days of cocaethylene treatment (PDs 4 to 9) and one day acute treatment (PD 9) were notably different. Furthermore, the artificial rearing procedure appeared to produce significant alterations in various neurotransmitter levels when compared with normally reared (suckle) controls. Collectively, these results suggest that cocaethylene is neuroteratogenic to the developing brain during the third trimester equivalent and the unique formation of cocaethylene resulting from the concurrent use of alcohol and cocaine may represent an increased risk to the developing brain beyond the intrinsic neuroteratogenic effects of cocaine and alcohol individually.

Prenatal alcohol exposure has been shown to damage the developing central nervous system (CNS) in a variety of ways, including neuroanatomical anomalies, neurochemical imbalance, and neuropharmacological dysfunction. The present study investigated one of the functional aspects of the dopaminergic system in neonatal rats exposed prenatally to a bingelike alcohol paradigm by measuring dopamine concentrations following a single postnatal cocaine challenge. Pregnant Sprague-Dawley rats were given daily intragastric intubations of 5.1 g/kg alcohol solution from embryonic day (E) 1 to 20. Pair-fed and ad lib-fed animals served as controls. On E33 (usually postnatal day 10), offspring from all groups were given injections (IP) of either 0, 20, or 40 mg/kg cocaine. Animals were sacrificed and the substantia nigra/ventral tegmental area (SN/VTA) and nucleus accumbens (NAc) were dissected for the determination of dopamine concentrations using HPLC. Basal dopamine levels (0 mg/kg cocaine group) did not alter as a function of prenatal alcohol treatment in either region. However, acute cocaine injection increased the dopamine content in NAc, but not in SN/VTA, in ad lib-fed animals, and this elevation in dopamine level was significantly attenuated by prenatal alcohol treatment in both female and male animals, and by prenatal pair-fed treatment in male animals. Taken together, these results indicate that there appears to be a regional difference in acute cocaine-induced dopamine elevation, and prenatal binge-like alcohol exposure significantly alters the functional responsiveness of dopaminergic system in NAc. Furthermore, these data suggest that male offspring may be more sensitive to stress-associated or nutritional influences during gestation.

Fetal exposure to alcohol is the major known cause of mental retardation in the Western world. For more than half of the 20th century, the placenta was widely believed to be an effective barrier against environmental agents. The discovery that offspring of pregnant women who were exposed to German measles or administered thalidomide were often malformed raised awareness that teratogens could be any environmental agent, including viruses and drugs, that caused abnormal development. Alcohol was not identified as a teratogen until the 1970s. Fetal exposure to alcohol can cause fetal alcohol syndrome (FAS), which is characterized by specific physical traits and central nervous system dysfunctions. The development of animal model systems has facilitated our study of the effects of fetal alcohol exposure and the elucidation of the mechanisms involved in alcohol-induced abnormal development. Despite our current understanding of the effects of fetal alcohol exposure, the occurrence of FAS and associated fetal alcohol spectrum disorders is still widespread and the associated health-care costs are staggering. This symposium provides an up-to-date analysis of fetal exposure to alcohol and FAS. It is directed not only to investigators working in the field but to a diverse group of scientists working in the biological and biomedical fields to stimulate cross-disciplinary awareness, interest, and collaboration.

Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia in utero. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several “fetal” genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.

Alcohol was infused intravenously into chronically cannulated adult female sheep as a 40% solution (w/v) at doses of 0.5, 1.0, 1.5, or 2.0 g/kg over 1 hr. Saline infusions, equal in volume to the highest dose, served as a control. Dose-dependent peak blood alcohol concentrations (BACs) were attained 60 min after the beginning of alcohol infusion for all doses (86.5 +/- 3.7, 213.2 +/- 11.0, 373.2 +/- 14.3, and 494.1 +/- 34.5 mg/dl +/- SE, respectively). Plasma cortisol concentrations increased in response to the 0.5 g/kg infusions (BACs less than 100 mg/dl), whereas both ACTH and cortisol concentrations increased in the 1.0 and 2.0 g/kg dose groups. Mean arterial pressure, heart rate, and Paco2 increased, whereas Pao2 decreased in response to the 1.5 and 2.0 g/kg infusions. Arterial pH declined in the highest dose group. Respiratory rate was lower in all groups receiving alcohol compared with that of the control group. Hematocrit did not change. We conclude that BACs in adult female sheep below 100 mg/dl (levels easily achieved by social drinkers) result in activation of the hypothalamus-pituitary-adrenal axis. At high BACs (> 350 mg/dl), pituitary adrenal responses are accompanied by increases in heart rate, blood pressure, and Paco2 and decreases in Pao2 and arterial pH. These findings support the hypothesis that alcohol acts directly on the brain to mediate pituitary adrenal responses and that the additional responses to high BACs (the blood gas and hemodynamic responses), might be mediated by direct actions of alcohol on the brain, by cerebral ischemia, or by alcohol-mediated suppression of ventilatory drive and hypoxemia.