The metabolic response of human trophoblasts derived from term placentas to metformin.

AIMS/HYPOTHESIS: Metformin is increasingly used therapeutically during pregnancy worldwide, particularly in the treatment of gestational diabetes, which affects a substantial proportion of pregnant women globally. However, the impact on placental metabolism remains unclear. In view of the association between metformin use in pregnancy and decreased birthweight, it is essential to understand how metformin modulates the bioenergetic and anabolic functions of the placenta. METHODS: A cohort of 55 placentas delivered by elective Caesarean section at term was collected from consenting participants. Trophoblasts were isolated from the placental samples and treated in vitro with clinically relevant doses of metformin (0.01 mmol/l or 0.1 mmol/l) or vehicle. Respiratory function was assayed using high-resolution respirometry to measure oxygen concentration and calculated [Formula: see text]. Glycolytic rate and glycolytic stress assays were performed using Agilent Seahorse XF assays. Fatty acid uptake and oxidation measurements were conducted using radioisotope-labelled assays. Lipidomic analysis was conducted using LC-MS. Gene expression and protein analysis were performed using RT-PCR and western blotting, respectively. RESULTS: Complex I-supported oxidative phosphorylation was lower in metformin-treated trophoblasts (0.01 mmol/l metformin, 61.7% of control, p<0.05; 0.1 mmol/l metformin, 43.1% of control, p<0.001). The proton efflux rate arising from glycolysis under physiological conditions was increased following metformin treatment, up to 23±5% above control conditions following treatment with 0.1 mmol/l metformin (p<0.01). There was a significant increase in triglyceride concentrations in trophoblasts treated with 0.1 mmol/l metformin (p<0.05), particularly those of esters of long-chain polyunsaturated fatty acids. Fatty acid oxidation was reduced by ~50% in trophoblasts treated with 0.1 mmol/l metformin compared with controls (p<0.001), with no difference in uptake between treatment groups. CONCLUSIONS/INTERPRETATION: In primary trophoblasts derived from term placentas metformin treatment caused a reduction in oxidative phosphorylation through partial inactivation of complex I and potentially by other mechanisms. Metformin-treated trophoblasts accumulate lipids, particularly long- and very-long-chain polyunsaturated fatty acids. Our findings raise clinically important questions about the balance of risk of metformin use during pregnancy, particularly in situations where the benefits are not clear-cut and alternative therapies are available.

Preferences Elicited and Respected for Seriously Ill Veterans through Enhanced Decision-Making (PERSIVED): a protocol for an implementation study in the Veterans Health Administration.

BACKGROUND: Empirical evidence supports the use of structured goals of care conversations and documentation of life-sustaining treatment (LST) preferences in durable, accessible, and actionable orders to improve the care for people living with serious illness. As the largest integrated healthcare system in the USA, the Veterans Health Administration (VA) provides an excellent environment to test implementation strategies that promote this evidence-based practice. The Preferences Elicited and Respected for Seriously Ill Veterans through Enhanced Decision-Making (PERSIVED) program seeks to improve care outcomes for seriously ill Veterans by supporting efforts to conduct goals of care conversations, systematically document LST preferences, and ensure timely and accurate communication about preferences across VA and non-VA settings. METHODS: PERSIVED encompasses two separate but related implementation projects that support the same evidence-based practice. Project 1 will enroll 12 VA Home Based Primary Care (HBPC) programs and Project 2 will enroll six VA Community Nursing Home (CNH) programs. Both projects begin with a pre-implementation phase during which data from diverse stakeholders are gathered to identify barriers and facilitators to adoption of the LST evidence-based practice. This baseline assessment is used to tailor quality improvement activities using audit with feedback and implementation facilitation during the implementation phase. Site champions serve as the lynchpin between the PERSIVED project team and site personnel. PERSIVED teams support site champions through monthly coaching sessions. At the end of implementation, baseline site process maps are updated to reflect new steps and procedures to ensure timely conversations and documentation of treatment preferences. During the sustainability phase, intense engagement with champions ends, at which point champions work independently to maintain and improve processes and outcomes. Ongoing process evaluation, guided by the RE-AIM framework, is used to monitor Reach, Adoption, Implementation, and Maintenance outcomes. Effectiveness will be assessed using several endorsed clinical metrics for seriously ill populations. DISCUSSION: The PERSIVED program aims to prevent potentially burdensome LSTs by consistently eliciting and documenting values, goals, and treatment preferences of seriously ill Veterans. Working with clinical operational partners, we will apply our findings to HBPC and CNH programs throughout the national VA healthcare system during a future scale-out period.

Fluorescence-estimated oil concentration (Foil) in the Deepwater Horizon subsea oil plume.

Tracking the subsea oil plume during the 2010 Deepwater Horizon Oil Spill (DWH) was conducted using in situ fluorescence via vertical profilers (n = 1157) and discrete sample chemical analyses (n = 7665). During monitoring efforts, discrete samples provided a coarse picture of the oil plume footprint, but the majority of the samples were below standard analytical detection limits for petroleum hydrocarbons. In situ fluorescence data improved the spatial and temporal resolution of the subsea oil plume characterization. Here we synthesized millions of continuous fluorescence data points from hundreds of contemporaneously discrete samples collected to demonstrate how fluorescence could serve as a proxy for Benzene-Toluene-Ethylbenzene-Xylene (BTEX) concentration. Data mined from Gulf Science Data repository were well correlated, and geographically and temporally aligned to provide direct comparisons. Described here are the methods used to calibrate the fluorescence data and to spatially approximate the three-dimensional geographic extent of the oil plume.

Inorganic nitrate, hypoxia, and the regulation of cardiac mitochondrial respiration-probing the role of PPARα.

Dietary inorganic nitrate prevents aspects of cardiac mitochondrial dysfunction induced by hypoxia, although the mechanism is not completely understood. In both heart and skeletal muscle, nitrate increases fatty acid oxidation capacity, and in the latter case, this involves up-regulation of peroxisome proliferator-activated receptor (PPAR)α expression. Here, we investigated whether dietary nitrate modifies mitochondrial function in the hypoxic heart in a PPARα-dependent manner. Wild-type (WT) mice and mice without PPARα (Ppara-/-) were given water containing 0.7 mM NaCl (control) or 0.7 mM NaNO3 for 35 d. After 7 d, mice were exposed to normoxia or hypoxia (10% O2) for the remainder of the study. Mitochondrial respiratory function and metabolism were assessed in saponin-permeabilized cardiac muscle fibers. Environmental hypoxia suppressed mass-specific mitochondrial respiration and additionally lowered the proportion of respiration supported by fatty acid oxidation by 18% (P < 0.001). This switch away from fatty acid oxidation was reversed by nitrate treatment in hypoxic WT but not Ppara-/- mice, indicating a PPARα-dependent effect. Hypoxia increased hexokinase activity by 33% in all mice, whereas lactate dehydrogenase activity increased by 71% in hypoxic WT but not Ppara-/- mice. Our findings indicate that PPARα plays a key role in mediating cardiac metabolic remodeling in response to both hypoxia and dietary nitrate supplementation.-Horscroft, J. A., O'Brien, K. A., Clark, A. D., Lindsay, R. T., Steel, A. S., Procter, N. E. K., Devaux, J., Frenneaux, M., Harridge, S. D. R., Murray, A. J. Inorganic nitrate, hypoxia, and the regulation of cardiac mitochondrial respiration-probing the role of PPARα.

PPARα-independent effects of nitrate supplementation on skeletal muscle metabolism in hypoxia.

Hypoxia is a feature of many disease states where convective oxygen delivery is impaired, and is known to suppress oxidative metabolism. Acclimation to hypoxia thus requires metabolic remodelling, however hypoxia tolerance may be aided by dietary nitrate supplementation. Nitrate improves tissue oxygenation and has been shown to modulate skeletal muscle tissue metabolism via transcriptional changes, including through the activation of peroxisome proliferator-activated receptor alpha (PPARα), a master regulator of fat metabolism. Here we investigated whether nitrate supplementation protects skeletal muscle mitochondrial function in hypoxia and whether PPARα is required for this effect. Wild-type and PPARα knockout (PPARα-/-) mice were supplemented with sodium nitrate via the drinking water or sodium chloride as control, and exposed to environmental hypoxia (10% O2) or normoxia for 4 weeks. Hypoxia suppressed mitochondrial respiratory function in mouse soleus, an effect partially alleviated through nitrate supplementation, but occurring independently of PPARα. Specifically, hypoxia resulted in 26% lower mass specific fatty acid-supported LEAK respiration and 23% lower pyruvate-supported oxidative phosphorylation capacity. Hypoxia also resulted in 24% lower citrate synthase activity in mouse soleus, possibly indicating a loss of mitochondrial content. These changes were not seen, however, in hypoxic mice when supplemented with dietary nitrate, indicating a nitrate dependent preservation of mitochondrial function. Moreover, this was observed in both wild-type and PPARα-/- mice. Our results support the notion that nitrate supplementation can aid hypoxia tolerance and indicate that nitrate can exert effects independently of PPARα.

Inorganic Nitrate Mimics Exercise-Stimulated Muscular Fiber-Type Switching and Myokine and γ-Aminobutyric Acid Release.

Exercise is an effective intervention for the prevention and treatment of type 2 diabetes. Skeletal muscle combines multiple signals that contribute to the beneficial effects of exercise on cardiometabolic health. Inorganic nitrate increases exercise efficiency, tolerance, and performance. The transcriptional regulator peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) coordinates the exercise-stimulated skeletal muscle fiber-type switch from glycolytic fast-twitch (type IIb) to oxidative slow-twitch (type I) and intermediate (type IIa) fibers, an effect reversed in insulin resistance and diabetes. We found that nitrate induces PGC1α expression and a switch toward type I and IIa fibers in rat muscle and myotubes in vitro. Nitrate induces the release of exercise/PGC1α-dependent myokine FNDC5/irisin and ß-aminoisobutyric acid from myotubes and muscle in rats and humans. Both exercise and nitrate stimulated PGC1α-mediated γ-aminobutyric acid (GABA) secretion from muscle. Circulating GABA concentrations were increased in exercising mice and nitrate-treated rats and humans; thus, GABA may function as an exercise/PGC1α-mediated myokine-like small molecule. Moreover, nitrate increased circulating growth hormone levels in humans and rodents. Nitrate induces physiological responses that mimic exercise training and may underlie the beneficial effects of this metabolite on exercise and cardiometabolic health.

Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism.

BACKGROUND: Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of ß-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms. RESULTS: Herein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARß/δ- and PPARα-dependent mechanism. Enhanced PPARß/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα(-/-) mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation. CONCLUSIONS: Nitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.

Suppression of erythropoiesis by dietary nitrate.

In mammals, hypoxia-triggered erythropoietin release increases red blood cell mass to meet tissue oxygen demands. Using male Wistar rats, we unmask a previously unrecognized regulatory pathway of erythropoiesis involving suppressor control by the NO metabolite and ubiquitous dietary component nitrate. We find that circulating hemoglobin levels are modulated by nitrate at concentrations achievable by dietary intervention under normoxic and hypoxic conditions; a moderate dose of nitrate administered via the drinking water (7 mg NaNO3/kg body weight/d) lowered hemoglobin concentration and hematocrit after 6 d compared with nonsupplemented/NaCl-supplemented controls. The underlying mechanism is suppression of hepatic erythropoietin expression associated with the downregulation of tissue hypoxia markers, suggesting increased pO2. At higher nitrate doses, however, a partial reversal of this effect occurred; this was accompanied by increased renal erythropoietin expression and stabilization of hypoxia-inducible factors, likely brought about by the relative anemia. Thus, hepatic and renal hypoxia-sensing pathways act in concert to modulate hemoglobin in response to nitrate, converging at an optimal minimal hemoglobin concentration appropriate to the environmental/physiologic situation. Suppression of hepatic erythropoietin expression by nitrate may thus act to decrease blood viscosity while matching oxygen supply to demand, whereas renal oxygen sensing could act as a brake, averting a potentially detrimental fall in hematocrit.

Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart.

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.

Oral Coenzyme Q10 supplementation does not prevent cardiac alterations during a high altitude trek to everest base cAMP.

Exposure to high altitude is associated with sustained, but reversible, changes in cardiac mass, diastolic function, and high-energy phosphate metabolism. Whilst the underlying mechanisms remain elusive, tissue hypoxia increases generation of reactive oxygen species (ROS), which can stabilize hypoxia-inducible factor (HIF) transcription factors, bringing about transcriptional changes that suppress oxidative phosphorylation and activate autophagy. We therefore investigated whether oral supplementation with an antioxidant, Coenzyme Q10, prevented the cardiac perturbations associated with altitude exposure. Twenty-three volunteers (10 male, 13 female, 46±3 years) were recruited from the 2009 Caudwell Xtreme Everest Research Treks and studied before, and within 48 h of return from, a 17-day trek to Everest Base Camp, with subjects receiving either no intervention (controls) or 300 mg Coenzyme Q10 per day throughout altitude exposure. Cardiac magnetic resonance imaging and echocardiography were used to assess cardiac morphology and function. Following altitude exposure, body mass fell by 3 kg in all subjects (p<0.001), associated with a loss of body fat and a fall in BMI. Post-trek, left ventricular mass had decreased by 11% in controls (p<0.05) and by 16% in Coenzyme Q10-treated subjects (p<0.001), whereas mitral inflow E/A had decreased by 18% in controls (p<0.05) and by 21% in Coenzyme Q10-treated subjects (p<0.05). Coenzyme Q10 supplementation did not, therefore, prevent the loss of left ventricular mass or change in diastolic function that occurred following a trek to Everest Base Camp.

Case study: Nutrition and hydration status during 4,254 km of running over 78 consecutive days.

The aims of this study were to assess the dietary intake and monitor self-reported recovery quality and clinical symptomology of a male ultra-endurance runner who completed a multiday ultra-endurance running challenge covering 4,254 km from North Scotland to the Moroccan Sahara desert over 78 consecutive days. Food and fluid intakes were recorded and analyzed through dietary analysis software. Body mass (BM) was determined before and after running each day, and before sleep. Clinical symptomology and perceived recovery quality were recorded each day. Whole blood hemoglobin and serum ferritin were determined before and after the challenge. Total daily energy (mean ± SD: 23.2 ± 3.2 MJ · day(-1)) and macronutrient intake (182 ± 31 g · day(-1) protein, 842 ± 115 g · day(-1) carbohydrate, 159 ± 55 g · day(-1) fat) met consensus nutritional guidelines for endurance performance. Total daily water intake through foods and fluids was 4.8 ± 2.0 L · day(-1). Water and carbohydrate intake rates during running were 239 ± 143 ml · h(-1) and 56 ± 19 g · h(-1), respectively. Immediately after running, carbohydrate and protein intakes were 1.3 ± 1.0 g · kg BM(-1) and 0.4 ± 0.2 g · kg BM(-1), respectively. Daily micronutrient intakes ranged from 109 to 662% of UK RNIs. Prerunning BM was generally maintained throughout. Overall exercise-induced BM loss averaged 0.8 ± 1.0%; although BM losses of ≥ 2% occurred in the latter stages, a reflection of the warmer climate. Varying degrees of self-reported perceived recovery quality and clinical symptomology occurred throughout the challenge. This case study highlights oscillations in dietary habits along 78 consecutive days of ultra-endurance running, dependent on changes in ambient conditions and course topography. Nevertheless, nutrition and hydration status were maintained throughout the challenge. Despite dietary iron intake above RNI and iron supplementation, this alone did not prevent deficiency symptoms.

Short-term consumption of a high-fat diet impairs whole-body efficiency and cognitive function in sedentary men.

We recently showed that a short-term high-fat diet blunted exercise performance in rats, accompanied by increased uncoupling protein levels and greater respiratory uncoupling. In this study, we investigated the effects of a similar diet on physical and cognitive performance in humans. Twenty sedentary men were assessed when consuming a standardized, nutritionally balanced diet (control) and after 7 d of consuming a diet comprising 74% kcal from fat. Efficiency was measured during a standardized exercise task, and cognition was assessed using a computerized assessment battery. Skeletal muscle mitochondrial function was measured using (31)P magnetic resonance spectroscopy. The diet increased mean ± se plasma free fatty acids by 44% (0.32±0.03 vs. 0.46±0.05 mM; P<0.05) and decreased whole-body efficiency by 3% (21±1 vs. 18±1%; P<0.05), although muscle uncoupling protein (UCP3) content and maximal mitochondrial function were unchanged. High-fat diet consumption also increased subjects' simple reaction times (P<0.01) and decreased power of attention (P<0.01). Thus, we have shown that a high-fat diet blunts whole-body efficiency and cognition in sedentary men. We suggest that this effect may be due to increased respiratory uncoupling.

A ketone ester diet increases brain malonyl-CoA and Uncoupling proteins 4 and 5 while decreasing food intake in the normal Wistar Rat.

Three groups of male Wistar rats were pair fed NIH-31 diets for 14 days to which were added 30% of calories as corn starch, palm oil, or R-3-hydroxybutyrate-R-1,3-butanediol monoester (3HB-BD ester). On the 14th day, animal brains were removed by freeze-blowing, and brain metabolites measured. Animals fed the ketone ester diet had elevated mean blood ketone bodies of 3.5 mm and lowered plasma glucose, insulin, and leptin. Despite the decreased plasma leptin, feeding the ketone ester diet ad lib decreased voluntary food intake 2-fold for 6 days while brain malonyl-CoA was increased by about 25% in ketone-fed group but not in the palm oil fed group. Unlike the acute effects of ketone body metabolism in the perfused working heart, there was no increased reduction in brain free mitochondrial [NAD(+)]/[NADH] ratio nor in the free energy of ATP hydrolysis, which was compatible with the observed 1.5-fold increase in brain uncoupling proteins 4 and 5. Feeding ketone ester or palm oil supplemented diets decreased brain L-glutamate by 15-20% and GABA by about 34% supporting the view that fatty acids as well as ketone bodies can be metabolized by the brain.

Transformational leadership, transnational culture and political competence in globalizing health care services: a case study of Jordan's King Hussein Cancer Center.

BACKGROUND: Following the demise of Jordan's King Hussein bin Talal to cancer in 1999, the country's Al-Amal Center was transformed from a poorly perceived and ineffectual cancer care institution into a Western-style comprehensive cancer center. Renamed King Hussein Cancer Center (KHCC), it achieved improved levels of quality, expanded cancer care services and achieved Joint Commission International accreditation under new leadership over a three-year period (2002-2005). METHODS: An exploratory case research method was used to explain the rapid change to international standards. Sources including personal interviews, document review and on-site observations were combined to conduct a robust examination of KHCC's rapid changes. RESULTS: The changes which occurred at the KHCC during its formation and leading up to its Joint Commission International (JCI) accreditation can be understood within the conceptual frame of the transformational leadership model. Interviewees and other sources for the case study suggest the use of inspirational motivation, idealized influence, individualized consideration and intellectual stimulation, four factors in the transformational leadership model, had significant impact upon the attitudes and motivation of staff within KHCC. Changes in the institution were achieved through increased motivation and positive attitudes toward the use of JCI continuous improvement processes as well as increased professional training. The case study suggests the role of culture and political sensitivity needs re-definition and expansion within the transformational leadership model to adequately explain leadership in the context of globalizing health care services, specifically when governments are involved in the change initiative. CONCLUSION: The KHCC case underscores the utility of the transformational leadership model in an international health care context. To understand leadership in globalizing health care services, KHCC suggests culture is broader than organizational or societal culture to include an informal global network of medical professionals and Western technologies which facilitate global interaction. Additionally, political competencies among leaders may be particularly relevant in globalizing health care services where the goal is achieving international standards of care. Western communication technologies facilitate cross-border interaction, but social and political capital possessed by the leaders may be necessary for transactions across national borders to occur thus gaining access to specialized information and global thought leaders in a medical sub-specialty such as oncology.