The application of 2H2O to measure skeletal muscle protein synthesis

Skeletal muscle protein synthesis has generally been determined by the precursor:product labeling approach using labeled amino acids (e.g., [13C]leucine or [13C]-, [15N]-, or [2H]phenylalanine) as the tracers. Although reliable for determining rates of protein synthesis, this methodological approach requires experiments to be conducted in a controlled environment, and as a result, has limited our understanding of muscle protein renewal under free-living conditions over extended periods of time (i.e., integrative/cumulative assessments). An alternative tracer, 2H2O, has been successfully used to measure rates of muscle protein synthesis in mice, rats, fish and humans. Moreover, perturbations such as feeding and exercise have been included in these measurements without exclusion of common environmental and biological factors. In this review, we discuss the principle behind using 2H2O to measure muscle protein synthesis and highlight recent investigations that have examined the effects of feeding and exercise. The framework provided in this review should assist muscle biologists in designing experiments that advance our understanding of conditions in which anabolism is altered (e.g., exercise, feeding, growth, debilitating and metabolic pathologies)

In-vitro Antimicrobial Activity of Guiera senegalensis Leaves against Uropathogenic Microorganisms

هدفت هذه الدراسة لتقييم النشاط المضاد لمستخلصات اوراق نبات الغبيش المستخلصة بالميثانول والماء والهكسين ضد الميكروبات التي تصيب المسالك البولية. تم قياس النشاط المضاد للميكروبات باستخدام طريقة الانتشار في الاجار باستخدام تقنية تخفيف المستخلصات بتركيزات مختلفة. وايضآ تم تحديد الحد الادني للتركيز المثبط من المستخلص ضد الكائنات الحية باستخدام طريقة التوزيع في الاجار عن طريق اقل تركيز مثبط من المستخلص .اثبتت الدراسة ان كل المستخلصات نشطة بيولوجيا وفعالة ضد ميكروبات المسالك البولية المختبرة وهي الايشريشيا القولونية والمكورات العنقودية المعوية والمكورات العنقودية الذهبية وبكتريا الالتهاب الرئوي والسيدموموناس ايروغينوسا و الزائفة الزنجارية وفطر المبيضات البيضاء(Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebseilla pneumoniae, Proteus vulgaris, Pseudmonas aeruginosa and Candida albicans).. اعطي المستخلص المائي لاوراق نبات الغبيش فعالية تتراوح بين 12 -31.3 مم ومستخلص الميثانول بين 11.5-31.7مم والمستخلصات المتجزاة بالهكسان تتراوح بين 12.3-27.3مم) بين 10.5-27مم ضد الميكروبات المختبرة. معظم المستخلصات اعطت تركيزات مثبطة الحد الادنى يتراوح بين 3.125 -12.5ملجرام /مل والحد الادنى القاتل للبكتيريا يتراوح بين 12.5 - 25 ملجرام/ملل . تحليل المستخلص المائى و مستخلص الميثانول اظهرت وجود المواد الحيوية كالفلافونيدات ، التانيدات، الالكالويدات ، الصابونيات، الجلايكوزيدات و الاستيرويدات تايد قدرة المستخلصات الفاعلة ضد الميكروبات المختبرة.اظهرت مستخلصات الماء والميثانول فعالية اكثر عن المستخلصات المتجزأة وا ختلاف حلقات التثبيط من مستخلص لاخر يؤكد ان فعالية المستخلص تختلف باختلاف المذيب. وجود المواد الحيوية النشطة المثبطة للمكروبات والتركيزات القاتلة للبكتيريا يبرر امكانية استخدام اوراق نبات الغبيش كدواء شعبي او تقليدي. تم التاكد من السمية لهذا المستخلص عن طريق يرقات الجمبري في المختبر واثبتت الدراسة ان المستخلص أمن لذلك يمكن استخدامها كمضادات حيوية ضد بعض الميكربات . من نتائج التجارب نجد انه يمكن استخدام مستخلصات اوراق نبات الغبيش كعلاج بديل للمضادات الحيوية لمعالجة الاصابة بميكروبات المسالك البولية. ختامآ توصي الدراسة باجراء مذيدا من الدراسات والبحوث في النبات في مجالات الاحياء الدقيقة والكيمياء الحيوية والصيدلانية و اختبارات السمية باستخدام حيوانات التجارب لضمان سلامة استخدام النبات فى العلاج

The Effects of Resistance Exercise on In Vivo Cumulative Skeletal Muscle Protein Synthesis

An acute bout of resistance exercise (RE) and dietary protein consumption stimulate muscle protein synthesis (MPS). This anabolic effect is believed to be attenuated with resistance exercise training (RET), however, the mechanism for this plateau" is unknown. In addition, the ideal timing for protein consumption to optimize MPS is not well characterized. The central hypothesis of this research is that RE stimulates cumulative (measured over 24-36 h) MPS in rats and humans. Study one determined whether an acute bout of RE in rats enhances MPS when assessed with the traditional flooding dose (~ 25 min) and 2H2O (4 and 24 h measurements); thus a comparison of the two methodologies was made. An acute session of RE did not result in an elevation in MPS when quantified by either the flooding dose or 2H2O over 4 and 24 h (methods compared qualitatively). Therefore, an acute bout of RE in rats does not appear to be anabolic and adaptation resulting from multiple bouts is likely necessary. Study two determined if RET in rats results in attenuation in MPS (plateau effect) 16 h following the final RE session (peak anabolic window) and if it is due to an increase in 4E-BP1 (a key regulator of mRNA translation initiation) activity; or if the timing in anabolism changes, which could be detected with a cumulative assessment (2H2O). MPS at 16 h was unchanged following RE training. Consistent with this finding, there were no differences in 4E-BP1 activity. Conversely, cumulative MPS was significantly increased with RET, suggesting a temporal shift in anabolism. Study three determined if dietary protein consumed immediately following RE augments cumulative (24 h) MPS in young adult human males when energy and macronutrients are controlled. RE and post-RE protein had no effect on mixed MPS; however, myofibrillar MPS was significantly increased with RE suggesting specific changes within a heterogeneous protein pool. Collectively, these are the first studies to assess changes in cumulative MPS with RE in rats and humans. The long term goals of this research are to understand muscle protein anabolism in "free-living" mammals and the mechanisms that regulate this process

Early structural remodeling and deuterium oxide-derived protein metabolic responses to eccentric and concentric loading in human skeletal muscle

We recently reported that the greatest distinguishing feature between eccentric (ECC) and concentric (CON) muscle loading lays in architectural adaptations: ECC favors increases in fascicle length (Lf), associated with distal vastus lateralis muscle (VL) hypertrophy, and CON increases in pennation angle (PA). Here, we explored the interactions between structural and morphological remodeling, assessed by ultrasound and dual x‐ray absorptiometry (DXA), and long‐term muscle protein synthesis (MPS), evaluated by deuterium oxide (D2O) tracing technique. Ten young males (23 ± 4 years) performed unilateral resistance exercise training (RET) three times/week for 4 weeks; thus, one‐leg trained concentrically while the contralateral performed ECC exercise only at 80% of either CON or ECC one repetition maximum (1RM). Subjects consumed an initial bolus of D2O (150 mL), while a 25‐mL dose was thereafter provided every 8 days. Muscle biopsies from VL midbelly (MID) and distal myotendinous junction (MTJ) were collected at 0 and 4‐weeks. MPS was then quantified via GC–pyrolysis–IRMS over the 4‐week training period. Expectedly, ECC and CON RET resulted in similar increases in VL muscle thickness (MT) (7.5% vs. 8.4%, respectively) and thigh lean mass (DXA) (2.3% vs. 3%, respectively), albeit through distinct remodeling: Lf increasing more after ECC (5%) versus CON (2%) and PA increasing after CON (7% vs. 3%). MPS did not differ between contractile modes or biopsy sites (MID‐ECC: 1.42 vs. MID‐CON: 1.4% day−1; MTJ‐ECC: 1.38 vs. MTJ‐CON: 1.39% day−1). Muscle thickness at MID site increased similarly following ECC and CON RET, reflecting a tendency for a contractile mode‐independent correlation between MPS and MT (P = 0.07; R2 = 0.18). We conclude that, unlike MT, distinct structural remodeling responses to ECC or CON are not reflected in MPS; the molecular mechanisms of distinct protein deposition, and/or the role of protein breakdown in mediating these responses remain to be defined

Over-expression of the vitamin D receptor (VDR) induces skeletal muscle hypertrophy

ObjectiveThe Vitamin D receptor (VDR) has been positively associated with skeletal muscle mass, function and regeneration. Mechanistic studies have focused upon loss of the receptor, with in vivo whole-body knockout models demonstrating reduced myofiber size and function, and impaired muscle development. To understand the mechanistic role upregulation of the VDR elicits in muscle mass/health, we studied the impact of VDR over-expression (OE) in vivo, before exploring the importance of VDR expression upon muscle hypertrophy in humans.MethodsWistar rats underwent in vivo electrotransfer (IVE) to over-express the VDR in Tibialis anterior (TA) muscle for 10 days, before comprehensive physiological and metabolic profiling to characterise the influence of VDR-OE on muscle protein synthesis (MPS), anabolic signalling and satellite cell activity. Stable isotope tracer (D2O) techniques were used to assess sub-fraction protein synthesis, alongside RNA-Seq analysis. Finally, human participants underwent 20-wks resistance exercise training, with body composition and transcriptomic analysis.ResultsMuscle VDR-OE yielded total protein and RNA accretion, manifesting in increased myofibre area i.e. hypertrophy. The observed increases in MPS were associated with enhanced anabolic signalling reflecting translational efficiency (e.g. mTOR-signalling), with no effects upon protein breakdown markers being observed. Additionally, RNA-Seq illustrated marked extracellular matrix (ECM) remodeling, while satellite cell content, markers of proliferation and associated cell-cycled related gene-sets were up-regulated. Finally, induction of VDR mRNA correlated with muscle hypertrophy in humans following long-term resistance exercise type training.ConclusionVDR-OE stimulates muscle hypertrophy ostensibly via heightened protein synthesis, translational efficiency, ribosomal expansion and up-regulation of ECM remodelling related gene-sets. Furthermore, VDR expression is a robust marker of the hypertrophic response to resistance exercise in humans. The VDR is a viable target of muscle maintenance through testable Vitamin D molecules, as active molecules and analogs

Effects of dietary nitrate supplementation on microvascular physiology at 4559 m altitude – a randomised controlled trial (Xtreme Alps)

Native highlanders (e.g. Sherpa) demonstrate remarkable hypoxic tolerance, possibly secondary to higher levels of circulating nitric oxide (NO) and increased microcirculatory blood flow. As part of the Xtreme Alps study (a randomised placebo-controlled trial of dietary nitrate supplementation under field conditions of hypobaric hypoxia), we investigated whether dietary supplementation with nitrate could improve NO availability and microvascular blood flow in lowlanders. Plasma measurements of nitrate, nitrite and nitroso species were performed together with measurements of sublingual (sidestream dark-field camera) and forearm blood flow (venous occlusion plethysmography) in 28 healthy adult volunteers resident at 4559 m for 1 week; half receiving a beetroot-based high-nitrate supplement and half receiving an identically-tasting low nitrate 'placebo'. Dietary supplementation increased plasma nitrate concentrations 4-fold compared to the placebo group, both at sea level (SL; 19.2 vs 76.9 μM) and at day 5 (D5) of high altitude (22.9 vs 84.3 μM,

Cardiometabolic Health in Submariners Returning from a 3-Month Patrol

Confined space, limited exercise equipment, rotating shift work and reduced sleep may affect cardiometabolic health in submariners. To test this hypothesis, 53 male U.S. Submariners (20–39 years) were studied before and after a 3-month routine submarine patrol. Measures included anthropometrics, dietary and physical activity, biomarkers of cardiometabolic health, energy and appetite regulation, and inflammation. Before deployment, 62% of submariners had a body fat % (BF%) ≥ 25% (obesity), and of this group, 30% met the criteria for metabolic syndrome. In obese volunteers, insulin, the homeostatic model assessment of insulin resistance (HOMA-IR), leptin, the leptin/adiponectin ratio, and pro-inflammatory chemokines growth-related oncogene and macrophage-derived chemokine were significantly higher compared to non-obese submariners. Following the patrol, a significant mean reduction in body mass (5%) and fat-mass (11%) occurred in the obese group as a result of reduced energy intake (~2000 kJ) during the patrol; and, independent of group, modest improvements in serum lipids and a mean reduction in interferon γ-induced protein 10 and monocyte chemotactic protein 1 were observed. Since 43% of the submariners remained obese, and 18% continued to meet the criteria for metabolic syndrome following the patrol, the magnitude of weight loss was insufficient to completely abolish metabolic dysfunction. Submergence up to 3-months, however, does not appear to be the cause of obesity, which is similar to that of the general population

Replication Data for: Comparison of Body Composition Assessed by Dual-Energy X-Ray Absorptiometry and BMI in Current and Former U.S. Navy Service Members

Replication Data for: Comparison of Body Composition Assessed by Dual-Energy X-Ray Absorptiometry and BMI in Current and Former U.S. Navy Service Members PLOS ON