Investigating the effects of 5.5 mmoL vs 25 mmoL glucose concentration in culture media on LHCN-M2 cell viability, proliferation, metabolism and differentiation

Introduction: In vitro skeletal muscle cell models are vitally important for investigating the molecular mechanisms of skeletal muscle in metabolic and endocrine diseases, such as obesity and type 2 diabetes. Culture media for skeletal muscle cells can often contain glucose concentrations (GC) five times higher than what’s considered normal in fasting human plasma, thus is not representative of the in vivo environment. Hyperglycaemia in culture media may negatively impact metabolic function, by creating a model of cell toxicity that’s representative of diseases such as diabetes mellitus. The aim of these experiments was to determine the impact of media containing GC of 5.5 mmol (physiological) vs 25 mmol (supraphysiological) on cell viability, proliferation, ATP production and differentiation in human LHCN-m2 myoblasts.Methods: LHCN-m2 myoblasts were cultured in 5.5 mmol or 25 mmol glucose growth media and cell viability, ATP production, and proliferation were determined. Differentiation of LHCN-m2 myoblasts into multinucleated myotubes was induced by reducing levels of human serum within the culture media and analysed by immunofluorescence following 10 days of differentiation.Results: We observed no differences in the viability, proliferation or basal ATP production rates of LHCN-m2 cells grown in 5.5 mmol compared to 25 mmol glucose (P> 0.05 for all). However cells had a trend of higher ATP production rates and faster proliferation in 5.5 mmol compared to 25 mmol. Fluorescence microscopy revealed the formation of multinucleated myotubes differentiated in 5.5 mmol glucose media containing various concentrations of human serum (0.5%, 1% and 2%).Conclusions: Our data demonstrates the ability to differentiate LHCN-m2 cells in 5.5 mmol GC, which allows our in vitro model to be more physiologically-relevant and more comparable to what is observed in vivo in humans. Further work is required to determine the implications of GC on the wider metabolic function in LHCN-m2 myoblasts

Influence of vitamin D supplementation on immune function of healthy aging people:A pilot randomized controlled trial

Objectives: This study aims to investigate the influence of vitamin D supplementation on immune function of healthy older adults. Materials and methods: Designed as a randomized controlled trial, 21 participants (55–85 years) completed the study during May–November 2018 in Coventry, England. The participants were randomized into vitamin D or the control group, stratified by age, gender and body mass index. The vitamin D group (n = 12) took vitamin D3 tablets of 1,000 IU/day for 12 weeks plus vitamin D education leaflet, while the control group (n = 9) were only provided with the leaflet. At baseline, 6 and 12 weeks, plasma 25(OH)D levels and immunological and metabolic parameters including phagocytic activity of granulocytes and monocytes, tumor necrosis factor, interleukin 6, lymphocyte subsets and fasting blood glucose and lipid were measured. Dietary vitamin D intake was analyzed at baseline and week 12. Data were presented as mean ± SD. Two-way repeated measures ANOVA and independent t-test were used to analyze the data. Results: At baseline, 42.9% of the participants were vitamin D deficiency (25(OH)D 50 nmol/L. Overweight/obese participants (n = 9) had significantly lower mean plasma 25(OH)D concentration (22.3 ± 8.7 nmol/L) than normal weight participants (48.1 ± 34.3 nmol/L) (P = 0.043). There was a significant increase in plasma 25(OH)D concentration in vitamin D group compared with that in control group (P = 0.002) during the intervention period. The plasma 25(OH)D concentration in vitamin D group was increased at 6 weeks (from 38.4 ± 37.0 nmol/L at baseline to 51.0 ± 38.2 nmol/L) with little change observed between 6 and 12 weeks (51.8 ± 36.4 nmol/L). The plasma creatinine concentration in vitamin D group was significantly decreased compared with the control group (P = 0.036) (79.8 ± 7.0 μmol/L at baseline vs 75.1 ± 5.4 μmol/L at week 12). No significant effect of vitamin D supplementation was determined on immunological parameters. Conclusion: Vitamin D deficiency is common among the aging population in the UK even during the summertime. Vitamin D supplementation at 1,000 IU/day for 12 weeks significantly increased plasma 25(OH)D concentration but showed no effect on metabolic and immunological parameters except decreased plasma creatinine

Force-time characteristics of repeated bouts of depth jumps and the effects of compression garments

No studies have reported ground-reaction force (GRF) profiles of the repeated depth-jump (DJ) protocols commonly used to study exercise-induced muscle damage (EIMD). Furthermore, whilst compression garments (CG) may accelerate recovery from EIMD, any effects on the repeated-bout effect are unknown. Therefore, we investigated the GRF profiles of two repeated bouts of damage-inducing DJs, and the effects of wearing CG for recovery. Non-resistance trained males randomly received CG (n=9) or placebo (n=8) for 72 h recovery, following 20 x 20 m sprints and 10 x 10 DJs from 0.6 m. Exercise was repeated after 14 days. Using a three-way (set x bout x group) design, changes in GRF were assessed with ANOVA and statistical parametric mapping (SPM). Jump height, reactive strength, peak and mean propulsive forces declined between sets (p<0.001). Vertical stiffness, contact time, force at zero velocity and propulsive duration increased (p<0.05). According to SPM, braking (17–25% of the movement), and propulsive forces (58–81%) declined (p<0.05). During the repeated bout, peak propulsive force and duration increased (p<0.05), whilst mean propulsive force (p<0.05) and GRF from 59–73% declined (p<0.001). A repeated bout of DJs differed in propulsive GRF, without changes to the eccentric phase, or effects from CG

Tiotropium bromide, a long acting muscarinic receptor antagonist triggers intracellular calcium signalling in the heart

Background and purpose: Tiotropium bromide (TB) is a long acting muscarinic receptor antagonist used to manage chronic obstructive pulmonary disease (COPD). Recent meta-analyses suggest an increased risk of cardiovascular events with TB. Ca2+/calmodulin dependent kinase II (CaMKII) and L-type Ca2+ channels regulate Ca2+ concentrations allowing management of Ca2+ across membranes. Pathological increases in Ca2+ are initially slow and progressive, however once the cytosolic concentration rises &gt;1–3 μM from ~100 nM, calcium overload occurs and can lead to cell death. Ipratropium bromide, a short acting muscarinic receptor antagonist has previously been found to induce Ca2+ mediated eryptosis. The aim of this study was to investigate the role of Ca2+ in Tiotropium bromide mediated cardiotoxicity. Experimental approach: Isolated Sprague-Dawley rat hearts were perfused with TB (10–0.1 nM) ± KN-93 (400 nM) or nifedipine (1 nM). Hearts were stained to determine infarct size (%) using triphenyltetrazolium chloride (TTC), or snap frozen to determine p-CaMKII (Thr286) expression. Cardiomyocytes were isolated using a modified Langendorff perfusion and enzymatic dissociation before preparation for Fluo 3-AM staining and flow cytometric analysis. Key results: TB increased infarct size compared to controls by 6.91–8.41%, with no effect on haemodynamic function. KN-93/nifedipine with TB showed a 5.90/7.38% decrease in infarct size compared to TB alone, the combined use of KN-93 with TB also showed a significant increase in left ventricular developed pressure whilst nifedipine with TB showed a significant decrease in coronary flow. TB showed a 42.73% increase in p-CaMKII (Thr286) versus control, and increased Ca2+ fluorescence by 30.63% in cardiomyocytes. Conclusions and implications: To our knowledge, this is the first pre-clinical study to show that Tiotropium bromide induces Ca2+ signalling via CaMKII and L-type Ca2+ channels to result in cell damage. This has significant clinical impact due to long term use of TB in COPD patients, and warrants assessment of cardiac drug safety.</p

Comparative Assessment of the Acute Effects of Whey, Rice and Potato Protein Isolate Intake on Markers of Glycaemic Regulation and Appetite in Healthy Males Using a Randomised Study Design

Global protein consumption has been increasing for decades due to changes in demographics and consumer shifts towards higher protein intake to gain health benefits in performance nutrition and appetite regulation. Plant-derived proteins may provide a more environmentally sustainable alternative to animal-derived proteins. This study, therefore, aimed to investigate, for the first time, the acute effects on glycaemic indices, gut hormones, and subjective appetite ratings of two high-quality, plant-derived protein isolates (potato and rice), in comparison to a whey protein isolate in a single-blind, triple-crossover design study with nine male participants (30.8 ± 9.3 yrs). Following a 12 h overnight fast, participants consumed an equal volume of the three isocaloric protein shakes on different days, with at least a one-week washout period. Glycaemic indices and gut hormones were measured at baseline, then at 30, 60, 120, 180 min at each visit. Subjective palatability and appetite ratings were measured using visual analogue scales (VAS) over the 3 h, at each visit. This data showed significant differences in insulin secretion with an increase in whey (+141.8 ± 35.1 pmol/L; = 0.011) and rice (-64.4 ± 20.9 pmol/L; = 0.046) at 30 min compared to potato protein. A significantly larger total incremental area under the curve (iAUC) was observed with whey versus potato and rice with < 0.001 and = 0.010, respectively. There was no significant difference observed in average appetite perception between the different proteins. In conclusion, this study suggests that both plant-derived proteins had a lower insulinaemic response and improved glucose maintenance compared to whey protein

Risks associated with endotoxins in feed additives produced by fermentation

Acknowledgements We thank Jordi Tarrés Call, who recorded the discussions and offered advice about procedures, and Nicole Reisinger and Gerd Schatzmayr, who provided valuable information about endotoxins in animal feeds. The Rowett Institute of Nutrition and Health is funded by the Rural and Environment Science and Analytical Services Division (RESAS) of the Scottish Government.Peer reviewedPublisher PD

Serum concentration impacts myosin heavy chain expression but not cellular respiration in human LHCN‐M2 myoblasts undergoing differentiation

Abstract The human LHCN‐M2 myoblast cell line has the potential to be used to investigate skeletal muscle development and metabolism. Experiments were performed to determine how different concentrations of human serum affect myogenic differentiation and mitochondrial function of LHCN‐M2 cells. LHCN‐M2 myoblasts were differentiated in serum‐free medium, 0.5% or 2% human serum for 5 and 10 days. Myotube formation was assessed by immunofluorescence staining of myosin heavy chain (MHC) and molecularly by mRNA expression of Myogenic differentiation 1 (MYOD1) and Myoregulatory factor 5 (MYF5). Following differentiation, mitochondrial function was assessed to establish the impact of serum concentration on mitochondrial function. Time in differentiation increased mRNA expression of MYOD1 (day 5, 6.58 ± 1.33‐fold; and day 10, 4.28 ± 1.71‐fold) (P = 0.012), while suppressing the expression of MYF5 (day 5, 0.21 ± 0.11‐fold; and day 10, 0.06 ± 0.03‐fold) (P = 0.001), regardless of the serum concentration. Higher serum concentrations increased MHC area (serum free, 11.92 ± 0.85%; 0.5%, 23.10 ± 5.82%; 2%, 43.94 ± 8.92%) (P = 0.001). Absolute basal respiration approached significance (P = 0.06) with significant differences in baseline oxygen consumption rate (P = 0.025) and proton leak (P = 0.006) when differentiated in 2% human serum, but these were not different between conditions when normalised to total protein. Our findings show that increasing concentrations of serum of LHCN‐M2 skeletal muscle cells into multinucleated myotubes, but this does not affect relative mitochondrial function

Formulated Palmitoylethanolamide Supplementation Improves Parameters of Cognitive Function and BDNF Levels in Young, Healthy Adults:A Randomised Cross-Over Trial

Background: Palmitoylethanolamide (PEA) is an endocannabinoid-like lipid mediator which is naturally produced in the body and found in certain foods. The aim of this study was to assess the effect of a bioavailable formulated form of PEA (Levagen+®) on serum BDNF levels and parameters of cognitive function in healthy adults. Methods: A randomised double-blinded placebo-controlled cross-over trial was implemented to measure the effects of a 6-week 700 mg/day course of formulated PEA supplementation versus a placebo. Participants (n = 39) completed pre- and post-assessments of a lab-based cognitive test. Serum samples were collected to measure BDNF concentrations using an immunoassay. Results: A significant increase in serum BDNF levels was found following PEA supplementation compared with the placebo (p = 0. 0057, d = 0.62). The cognition test battery demonstrated improved memory with PEA supplementation through better first success (p = 0.142, d = 0.54) and fewer errors (p = 0.0287; d = −0.47) on the Paired Associates Learning test. Conclusion: This was the first study to report a direct beneficial effect of Levagen+® PEA supplementation on memory improvement as well as corresponding increases in circulating neurotrophic marker levels. This suggests that formulated PEA holds promise as an innovative and practical intervention for cognitive health enhancement

Age-Related Differences in the Functional Demand Placed on the Lumbar Spine during Walking in Healthy Older versus Younger Men

Age-related declines in the musculoskeletal system may place additional demands on the lumbar spine during everyday activities such as walking. This study aimed to investigate age-related differences in the functional demand (FD) of walking on the lumbar spine in older and younger adults. A motion analysis system with integrated force plates was used to acquire kinematic and kinetic data on 12 older (67.3 ± 6.0 years) and 12 younger (24.7 ± 3.1 years) healthy men during walking at a self-selected speed along a 10 m walkway. Isokinetic dynamometry was used to acquire the maximal joint moment capacity of the lumbar spine. The FD of the lumbar spine was calculated as the muscle moment during key phases of the gait cycle (GC) relative to the maximum moment capacity of the lumbar spine. The difference in FD between age groups was not significant (p = 0.07) and there were no significant differences between the young group (YG) and older group (OG) for any individual phase in the GC. Despite the lack of statistical significance, the results indicate that a practical difference may exist, as walking was approximately 20% more functionally demanding on the lumbar spine in the OG compared to the YG. Therefore, older adults may employ modified gait strategies to reduce mechanical load whilst walking to fall within the limits of their maximal force-producing capacity in the lumbar spine, which may have implications for injury risk