Novel monoclonal antibodies against Pdx1 reveal feedback regulation of Pdx1 protein levels

The aim of this study was to characterize two monoclonal antibodies (F6A11 and F109-D12) generated against Pdx1 (pancreatic and duodenal homeobox-1), a homeodomain transcription factor, which is critical for pancreas formation as well as for normal pancreatic beta cell function. For production of monoclonal antibodies, we immunized Robertsonian POSF (RBF)mice with a GST-Pdx1 fusion protein containing a 68-amino acid C-terminal fragment of rat Pdx1. These monoclonal antibodies detect Pdx1 by western blotting and allow immunohistochemical detection of Pdx1 in both mouse and rat tissue. F6A11 and F109-D12 produce IHC staining patterns indistinguishable from that obtained with highly specific polyclonal Pdx1 antisera raised in rabbits and goats, when applied to embryonic or adult mouse pancreatic tissue. In contrast to previously generated polyclonal anti-Pdx1 antisera, we also demonstrate that F6A11 works for intracellular fluorescence activated cell sorting (FACS) staining of Pdx1. By using F6A11, we characterize the induction of Pdx1 in the Doxycycline (DOX) inducible insulinoma cell line INSrαβ-Pdx1 and follow the reduction of Pdx1 after removing Dox. Finally, we show that induction of exogenous Pdx1 leads to a reduction in endogenous Pdx1 levels, which suggests that a negative feedback loop is involved in maintaining correct levels of Pdx1 in the cell

Contraction and AICAR Stimulate IL-6 Vesicle Depletion From Skeletal Muscle Fibers In Vivo

Recent studies suggest that interleukin 6 (IL-6) is released from contracting skeletal muscles; however, the cellular origin, secretion kinetics, and signaling mechanisms regulating IL-6 secretion are unknown. To address these questions, we developed imaging methodology to study IL-6 in fixed mouse muscle fibers and in live animals in vivo. Using confocal imaging to visualize endogenous IL-6 protein in fixed muscle fibers, we found IL-6 in small vesicle structures distributed throughout the fibers under basal (resting) conditions. To determine the kinetics of IL-6 secretion, intact quadriceps muscles were transfected with enhanced green fluorescent protein (EGFP)-tagged IL-6 (IL-6-EGFP), and 5 days later anesthetized mice were imaged before and after muscle contractions in situ. Contractions decreased IL-6-EGFP–containing vesicles and protein by 62% (P < 0.05), occurring rapidly and progressively over 25 min of contraction. However, contraction-mediated IL-6-EGFP reduction was normal in muscle-specific AMP-activated protein kinase (AMPK) α2-inactive transgenic mice. In contrast, the AMPK activator AICAR decreased IL-6-EGFP vesicles, an effect that was inhibited in the transgenic mice. In conclusion, resting skeletal muscles contain IL-6–positive vesicles that are expressed throughout myofibers. Contractions stimulate the rapid reduction of IL-6 in myofibers, occurring through an AMPKα2-independent mechanism. This novel imaging methodology clearly establishes IL-6 as a contraction-stimulated myokine and can be used to characterize the secretion kinetics of other putative myokines

Enhanced insulin sensitivity associated with provision of mono and polyunsaturated fatty acids in skeletal muscle cells involves counter modulation of PP2A

International audienceAims/Hypothesis: Reduced skeletal muscle insulin sensitivity is a feature associated with sustained exposure to excess saturated fatty acids (SFA), whereas mono and polyunsaturated fatty acids (MUFA and PUFA) not only improve insulin sensitivity but blunt SFA-induced insulin resistance. The mechanisms by which MUFAs and PUFAs institute these favourable changes remain unclear, but may involve stimulating insulin signalling by counter-modulation/repression of protein phosphatase 2A (PP2A). This study investigated the effects of oleic acid (OA; a MUFA), linoleic acid (LOA; a PUFA) and palmitate (PA; a SFA) in cultured myotubes and determined whether changes in insulin signalling can be attributed to PP2A regulation. Principal Findings: We treated cultured skeletal myotubes with unsaturated and saturated fatty acids and evaluated insulin signalling, phosphorylation and methylation status of the catalytic subunit of PP2A. Unlike PA, sustained incubation of rat or human myotubes with OA or LOA significantly enhanced Akt-and ERK1/2-directed insulin signalling. This was not due to heightened upstream IRS1 or PI3K signalling nor to changes in expression of proteins involved in proximal insulin signalling, but was associated with reduced dephosphorylation/inactivation of Akt and ERK1/2. Consistent with this, PA reduced PP2Ac demethylation and tyrosine 307 phosphorylation-events associated with PP2A activation. In contrast, OA and LOA strongly opposed these PA-induced changes in PP2Ac thus exerting a repressive effect on PP2A.Conclusions/Interpretation: Beneficial gains in insulin sensitivity and the ability of unsaturated fatty acids to oppose palmitate-induced insulin resistance in muscle cells may partly be accounted for by counter-modulation of PP2A

Food-dependent, exercise-induced gastrointestinal distress

Among athletes strenuous exercise, dehydration and gastric emptying (GE) delay are the main causes of gastrointestinal (GI) complaints, whereas gut ischemia is the main cause of their nausea, vomiting, abdominal pain and (blood) diarrhea. Additionally any factor that limits sweat evaporation, such as a hot and humid environment and/or body dehydration, has profound effects on muscle glycogen depletion and risk for heat illness. A serious underperfusion of the gut often leads to mucosal damage and enhanced permeability so as to hide blood loss, microbiota invasion (or endotoxemia) and food-born allergen absorption (with anaphylaxis). The goal of exercise rehydration is to intake more fluid orally than what is being lost in sweat. Sports drinks provide the addition of sodium and carbohydrates to assist with intestinal absorption of water and muscle-glycogen replenishment, respectively. However GE is proportionally slowed by carbohydrate-rich (hyperosmolar) solutions. On the other hand, in order to prevent hyponatremia, avoiding overhydration is recommended. Caregiver's responsibility would be to inform athletes about potential dangers of drinking too much water and also advise them to refrain from using hypertonic fluid replacements

Half-Time Strategies to Enhance Second-Half Performance in Team-Sports Players: A Review and Recommendations

The competitive demands of numerous intermittent team sports require that two consecutive periods of play are separated by a half-time break. Typically, half-time allows players to: return to the changing rooms, temporarily relax from the cognitive demands of the first half of match-play, rehydrate, re-fuel, attend to injury or equipment concerns, and to receive tactical instruction and coach feedback in preparation for the second half. These passive practices have been associated with physiological changes which impair physical and cognitive performance in the initial stages of the second half. An increased risk of injury has also been observed following half-time. On the day of competition, modification of half-time practices may therefore provide Sports Scientists and Strength and Conditioning Coaches with an opportunity to optimise second half performance. An overview of strategies that may benefit team sports athletes is presented; specifically, the efficacy of: heat maintenance strategies (including passive and active methods), hormonal priming (through video feedback), post-activation potentiation, and modified hydro-nutritional practices are discussed. A theoretical model of applying these strategies in a manner that compliments current practice is also presented

Examination of the efficacy of acute L-alanyl-L-glutamine ingestion during hydration stress in endurance exercise

<p>Abstract</p> <p>Background</p> <p>The effect of acute L-alanyl-L-glutamine (AG; Sustamine™) ingestion on performance changes and markers of fluid regulation, immune, inflammatory, oxidative stress, and recovery was examined in response to exhaustive endurance exercise, during and in the absence of dehydration.</p> <p>Methods</p> <p>Ten physically active males (20.8 ± 0.6 y; 176.8 ± 7.2 cm; 77.4 ± 10.5 kg; 12.3 ± 4.6% body fat) volunteered to participate in this study. During the first visit (T1) subjects reported to the laboratory in a euhydrated state to provide a baseline (BL) blood draw and perform a maximal exercise test. In the four subsequent randomly ordered trials, subjects dehydrated to -2.5% of their baseline body mass. For T2, subjects achieved their goal weight and were not rehydrated. During T3 - T5, subjects reached their goal weight and then rehydrated to 1.5% of their baseline body mass by drinking either water (T3) or two different doses (T4 and T5) of the AG supplement (0.05 g·kg^-1and 0.2 g·kg^-1, respectively). Subjects then exercised at a workload that elicited 75% of their VO₂max on a cycle ergometer. During T2 - T5 blood draws occurred once goal body mass was achieved (DHY), immediately prior to the exercise stress (RHY), and immediately following the exercise protocol (IP). Resting 24 hour (24P) blood samples were also obtained. Blood samples were analyzed for glutamine, potassium, sodium, aldosterone, arginine vasopressin (AVP), C-reactive protein (CRP), interleukin-6 (IL-6), malondialdehyde (MDA), testosterone, cortisol, ACTH, growth hormone and creatine kinase. Statistical evaluation of performance, hormonal and biochemical changes was accomplished using a repeated measures analysis of variance.</p> <p>Results</p> <p>Glutamine concentrations for T5 were significantly higher at RHY and IP than T2 - T4. When examining performance changes (difference between T2 - T5 and T1), significantly greater times to exhaustion occurred during T4 (130.2 ± 340.2 sec) and T5 (157.4 ± 263.1 sec) compared to T2 (455.6 ± 245.0 sec). Plasma sodium concentrations were greater (p < 0.05) at RHY and IP for T2 than all other trials. Aldosterone concentrations at RHY and IP were significantly lower than that at BL and DHY. AVP was significantly elevated at DHY, RHY and IP compared to BL measures. No significant differences were observed between trials in CRP, IL-6, MDA, or in any of the other hormonal or biochemical measures.</p> <p>Conclusion</p> <p>Results demonstrate that AG supplementation provided a significant ergogenic benefit by increasing time to exhaustion during a mild hydration stress. This ergogenic effect was likely mediated by an enhanced fluid and electrolyte uptake.</p

Impact of inactivity and exercise on the vasculature in humans

The effects of inactivity and exercise training on established and novel cardiovascular risk factors are relatively modest and do not account for the impact of inactivity and exercise on vascular risk. We examine evidence that inactivity and exercise have direct effects on both vasculature function and structure in humans. Physical deconditioning is associated with enhanced vasoconstrictor tone and has profound and rapid effects on arterial remodelling in both large and smaller arteries. Evidence for an effect of deconditioning on vasodilator function is less consistent. Studies of the impact of exercise training suggest that both functional and structural remodelling adaptations occur and that the magnitude and time-course of these changes depends upon training duration and intensity and the vessel beds involved. Inactivity and exercise have direct “vascular deconditioning and conditioning” effects which likely modify cardiovascular risk