Effects of leucine and its metabolite ß-hydroxy-ß-methylbutyrate on human skeletal muscle protein metabolism.

Maintenance of skeletal muscle mass is contingent upon the dynamic equilibrium (fasted losses-fed gains) in protein turnover. Of all nutrients, the single amino acid leucine (Leu) possesses the most marked anabolic characteristics in acting as a trigger element for the initiation of protein synthesis. While the mechanisms by which Leu is 'sensed' have been the subject of great scrutiny, as a branched-chain amino acid, Leu can be catabolized within muscle, thus posing the possibility that metabolites of Leu could be involved in mediating the anabolic effect(s) of Leu. Our objective was to measure muscle protein anabolism in response to Leu and its metabolite HMB. Using [1,2-(13)C2]Leu and [(2)H5]phenylalanine tracers, and GC-MS/GC-C-IRMS we studied the effect of HMB or Leu alone on MPS (by tracer incorporation into myofibrils), and for HMB we also measured muscle proteolysis (by arteriovenous (A-V) dilution). Orally consumed 3.42 g free-acid (FA-HMB) HMB (providing 2.42 g of pure HMB) exhibited rapid bioavailability in plasma and muscle and, similarly to 3.42 g Leu, stimulated muscle protein synthesis (MPS; HMB +70% vs. Leu +110%). While HMB and Leu both increased anabolic signalling (mechanistic target of rapamycin; mTOR), this was more pronounced with Leu (i.e. p70S6K1 signalling 90 min vs. 30 min for HMB). HMB consumption also attenuated muscle protein breakdown (MPB; -57%) in an insulin-independent manner. We conclude that exogenous HMB induces acute muscle anabolism (increased MPS and reduced MPB) albeit perhaps via distinct, and/or additional mechanism(s) to Leu.

Endotoxin transiently inhibits protein synthesis through Akt and MAPK mediating pathways in C2C12 myotubes.

In this study, the effect of lipopolysaccharide (LPS) on protein synthesis (PS) and intracellular signaling factors that regulate it have been investigated in C2C12 murine-derived myotubes. In particular, the role of Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinases (MAPKs) [p38 and extracelluar regulated protein kinase (ERK1/2)] have been examined. The direct effect of LPS on PS was measured at 3 and 18 h. LPS significantly decreased PS at 3 h but not at the 18-h time point. This effect was preceded by decreased Akt phosphorylation at 5 and 30 min after LPS administration. The mTOR phosphorylation exhibited a long time dose-dependent increase at all the time points. Similarly, the activity-related phosphorylation of p38 and ERK1/2 significantly increased in a time- and dose-dependent manner at all the time points. Polymyxin B abolished the LPS-induced decrease in PS rate. The phosphatidylinositol 3-kinase inhibitor LY-0294002 in combination with LPS significantly decreased the rate of PS by 81% and alone by 66%, respectively, for the 3- and 18-h time points, whereas p38 and ERK inhibitors in combination with LPS significantly decreased the rate PS rate at the 18-h time point by 41% and 59%, respectively, compared with control cells. In conclusion, LPS alone transiently decreased the rate of PS by 50% at 3 h; this effect is most likely mediated via the Toll-like receptor 4 (TLR4)-Akt/mTOR pathway, and both p38 and ERK when inhibited in the presence of LPS at 3 h have a similar effect in preventing the LPS-induced reduction in PS.

PIERS proteinuria: relationship with adverse maternal and perinatal outcome.

OBJECTIVE: To examine the ability of three different proteinuria assessment methods (urinary dipstick, spot urine protein:creatinine ratio [Pr/Cr], and 24-hour urine collection) to predict adverse pregnancy outcomes. METHODS: We performed a prospective multicentre cohort study, PIERS (Preeclampsia Integrated Estimate of RiSk), in seven academic tertiary maternity centres practising expectant management of preeclampsia remote from term in Canada, New Zealand, and Australia. Eligible women were those admitted with preeclampsia who had at least one antenatal proteinuria assessment by urinary dipstick, spot urine Pr/Cr ratio, and/or 24-hour urine collection. Proteinuria assessment was done either visually at the bedside (by dipstick) or by hospital clinical laboratories for spot urine Pr/Cr and 24-hour urine collection. We calculated receiver operating characteristic area under the curve (95% CI) for each proteinuria method and each of the combined adverse maternal outcomes (within 48 hours) or adverse perinatal outcomes (at any time). Models with AUC ≥ 0.70 were considered of interest. Analyses were run for all women who had each type of proteinuria assessment and for a cohort of women ("ALL measures") who had all three proteinuria assessments. RESULTS: More women were proteinuric by urinary dipstick (≥ 2+, 61.4%) than by spot urine Pr/Cr (≥ 30 g/mol, 50.4%) or 24-hour urine collection (≥ 0.3g/d, 34.7%). Each proteinuria measure evaluated had some discriminative power, and dipstick proteinuria (categorical) performed as well as other methods. No single method was predictive of adverse perinatal outcome. CONCLUSION: The measured amount of proteinuria should not be used in isolation for decision-making in women with preeclampsia. Dipstick proteinuria performs as well as other methods of assessing proteinuria for prediction of adverse events.

Cyclic stretch reduces myofibrillar protein synthesis despite increases in FAK and anabolic signalling in L6 cells.

Muscle protein synthesis is increased after exercise, but evidence is now accruing that during muscular activity it is suppressed. In life, muscles are subjected to shortening forces due to contraction, but may also be subject to stretching forces during lengthening. It would be biologically inefficient if contraction and stretch have different effects on muscle protein turnover, but little is known about the metabolic effects of stretch. To investigate this, we assessed myofibrillar and sarcoplasmic protein synthesis (MPS, SPS, respectively) by incorporation of [1-13C]proline (using gas chromatography-mass spectrometry) and anabolic signalling (by phospho-immunoblotting and kinase assays) in cultured L6 skeletal muscle cells during 30 min of cyclic stretch and over 30 min intervals for up to 120 min afterwards. SPS was unaffected, whereas MPS was suppressed by 40 +/- 0.03% during stretch, before returning to basal rates by 90-20 min afterwards. Paradoxically, stretch stimulated anabolic signalling with peak values after 2-30 min: e.g. focal adhesion kinase (FAK Tyr576/577; +28 +/- 6%), protein kinase B activity (Akt; +113 +/- 31%), p70S6K1 (ribosomal S6 kinase Thr389; 25 +/- 5%), 4E binding protein 1 (4EBP1 Thr37/46; 14 +/- 3%), eukaryotic elongation factor 2 (eEF2 Thr56; -47 +/- 4%), extracellular regulated protein kinase 1/2 (ERK1/2 Tyr202/204; +65% +/- 9%), eukaryotic initiation factor 2alpha (eIF2alpha Ser51; -20 +/- 5%, P < 0.05) and eukaryotic initiation factor 4E (eIF4E Ser209; +33 +/- 10%, P < 0.05). After stretch, except for Akt activity, stimulatory phosphorylations were sustained: e.g. FAK (+26 +/- 11%) for > or =30 min, eEF2 for > or =60 min (peak -45 +/- 4%), 4EBP1 for > or =90 min (+33 +/- 5%), and p70S6K1 remained elevated throughout (peak +64 +/- 7%). Adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was unchanged throughout. We report for the first time that acute cyclic stretch specifically suppresses MPS, despite increases in activity/phosphorylation of elements thought to increase anabolism.

Temperature acclimation: improved sustained swimming performance in carp at low temperatures.

At low temperatures, the reduction in mechanical power output of the aerobic muscle forces cold-blooded animals, such as carp, to recruit their rapidly fatiguing anaerobic fibers at relatively slow swimming speeds. Previous experimental data have suggested that changes in the biochemistry and morphology of the aerobic muscle during cold acclimation might increase its output of mechanical power. The present experiments show that, because of these changes, carp can swim faster at low temperature using only their aerobic muscle, which results in an increase in their sustainable swimming speed. By modifying their musculature, cold-blooded animals can achieve some independence from the effects of seasonal changes in environmental temperature.

Elevated placental expression of the imprinted PHLDA2 gene is associated with low birth weight.

The identification of genes that regulate fetal growth will help establish the reasons for intrauterine growth restriction. Most autosomal genes are expressed biallelically, but some are imprinted, expressed only from one parental allele. Imprinted genes are associated with fetal growth and development. The growth of the fetus in utero relies on effective nutrient transfer from the mother to the fetus via the placenta. Some current research on the genetic control of fetal growth has focused on genes that display imprinted expression in utero. The expression levels of four imprinted genes, the paternally expressed insulin growth factor 2 (IGF2), the mesoderm-specific transcript isoform 1 (MEST); the maternally expressed pleckstrin homology-like domain, family A, member 2 (PHLDA2); and the polymorphically imprinted insulin-like growth factor 2 (IGF2R) gene are all known to have roles in fetal growth and were studied in the placentae of 200 white European, normal term babies. The quantitative expression analysis with real-time PCR showed the maternally expressing PHLDA2 but not the paternally expressing IGF2 and MEST, nor the polymorphic maternally expressing IGF2R placental levels to have a statistically significant effect on birth weight. PHLDA2 expression levels are negatively correlated with size at birth. These data implicate PHLDA2 as an imprinted gene important in fetal growth and also as a potential marker of fetal growth.

Hypertension, fetal growth restriction and obstructive sleep apnoea in pregnancy.

OBJECTIVE: To test the hypothesis that obstructive sleep apnoea (OSA) is more common in pregnancies complicated by hypertensive disease and fetal growth restriction. STUDY DESIGN: An observational study comparing pregnant women with these two complications with normal pregnant women and non-pregnant women in two UK maternity hospitals. Each participant completed a sleep apnoea questionnaire and underwent nocturnal oxygen saturation monitoring. RESULTS: Using a strict definition of obstructive sleep apnoea confirmed by oxygen saturation monitoring only two mild cases were seen, 0/50 non-pregnant women, 1/69 of normal pregnant women, 0/48 women with various types of hypertensive disease, and 1/33 women carrying fetuses affected with fetal growth restriction. Even using less strict definitions and self-reported sleepiness scores there was no relation between sleep apnoea and either fetal growth restriction or hypertensive diseases. CONCLUSION: Obstructive sleep apnoea is at most a rare cause of either growth restriction or hypertensive disease in pregnancy.

Work overload induced changes in fast and slow skeletal muscle myosin heavy chain gene expression.

Work induced hypertrophy of the slow postural soleus and the fast phasic plantaris muscles was produced by tenotomy of the synergistic gastrocnemius muscle. Increases in weight of both muscles were associated with proportionately even larger increases in total RNA and mRNA levels. Alterations in levels of specific myosin heavy chain (MHC) isoform mRNAs were measured using the slot blot procedure with radioactively labelled oligonucleotides as probes. Type 1 MHC gene expression was unaffected in both muscles by work overload, whereas type 2a was deinduced in the soleus and type 2b was deinduced in the plantaris. The neonatal MHC gene was transiently reinduced in the plantaris.

Two myogenic regulatory factor transcripts exhibit muscle-specific responses to disuse and passive stretch in adult rats.

Levels of myogenic regulatory factor (MRF) transcripts are altered in a muscle-specific manner in response to hind limb immobilisation of adult male rats, for a 2 day period, in either a lengthened or shortened position which result in passive stretch or disuse atrophy respectively. Myogenin transcript levels were dramatically elevated in the stretched plantaris but not soleus, whereas the MRF4 transcript was significantly elevated in soleus but not plantaris. Levels of myogenin mRNA were unaffected by disuse in either muscle and MRF4 was markedly lower in plantaris in response to disuse.

Interactions between growth hormone and nutrition in hypophysectomized rats: body composition and production of insulin-like growth factor-I.

Hypophysectomy of adult rats results in a loss of body growth which can be reversed by treatment with GH. The increased growth caused by administration of GH is accompanied by an increase in food consumption. The effects of GH and interactions with nutrition were investigated by treating hypophysectomized rats with GH and either providing unrestricted food or preventing the increased food consumption by pair-feeding with the same intake as that of the hypophysectomized animals. Over the 7-day experimental period, the GH-treated animals grew significantly when food was available ad libitum but did not gain body weight when an increase in food intake was prevented. However, there was a significant interaction between GH and nutrition on body composition; GH significantly decreased body fat and increased the protein: fat ratio only in the animals with the restricted intake. Gastrocnemius muscle weight was increased by GH regardless of food intake, but heart weight did not increase and liver weight was actually decreased by GH treatment when food intake was restricted. Serum concentrations of insulin and insulin-like growth factor-I (IGF-I) were increased by GH in the rats with food available ad libitum but not in the pair-fed rats. However, the liver concentration of IGF-I and its mRNA were increased by GH although the increase in IGF-I mRNA was modulated by the restricted food intake. The decreased weight of the liver in the pair-fed GH-treated rats, despite the increase in IGF-I mRNA, suggests that IGF-I does not influence liver growth. In the gastrocnemius muscle, however, GH increased IGF-I mRNA concentration similarly in both rats with food available ad libitum and in pair-fed rats. Decreased nutrition therefore modulated the action of GH but emphasized its nutrient partitioning effect, thus increasing the anabolic drive towards skeletal muscle growth; this appeared to be mediated by the local production of IGF-I within the muscle.

Effect of inactivity and passive stretch on protein turnover in phasic and postural rat muscles.

A state of hypokinesia and hypodynamia has been induced in the hindlimb muscles of the rat (100 g) using a suspension model. The ensuing muscle atrophy was assessed by reference to muscles in fully mobile control animals, which were either fed ad libitum or fed the same lower food intake of the suspended animals. Over a total of 7 days of suspension the slow-twitch postural soleus muscle underwent a much greater atrophy than the fast-twitch phasic extensor digitorum longus. Changes with respect to the position of the suspended foot, and hence muscle length, necessitate caution in comparing the extent of the atrophy between different muscle types. After 3 days of inactivity the atrophy of the soleus muscle was explained by a 21% decrease in the fractional rate of synthesis (measured in vivo) and a 100% increase in the rate of protein breakdown. The reduction in the synthetic rate was associated with a net loss (23%) of RNA and hence muscle ribosomes. In contrast when this inactive soleus muscle was permanently stretched the RNA content (44%) and protein synthetic rate increased (59%) markedly above control values. Although protein breakdown remained elevated in this stretched muscle, the extent of the atrophy in response to hypokinesia and hypodynamia was greatly reduced.

Effects of hypokinesia and hypodynamia upon protein turnover in hindlimb muscles of the rat.

Hypokinesia/hypodynamia was induced in the hindlimb muscles of the rat using a suspension technique. This caused differing degrees of atrophy in different muscles, however, this atrophy was reduced in muscles held in a lengthened position. The greatest degree of wasting was observed in the unstretched soleus, a slow postural muscle, where both Type 1 and Type 2a fibers atrophied to the same degree. However, wasting of the gastrocnemius muscle was associated with a reduction in the size of the Type 2b fibers. In both slow postural and fast phasic hindlimb muscles, atrophy was brought about by a reduction in the rate of protein synthesis in conjunction with an elevation in the rate of protein degradation. When inactive muscles were passively stretched, both protein synthesis and degradation were dramatically elevated. Even periods of stretch of as little as 0.5 h.d-1 were found to significantly decrease atrophy in inactive muscles.

OS102. Continuing pathology following a hypertensive pregnancy and the risk of future disease.

INTRODUCTION: New onset hypertension in pregnancy affects up to 6-8% of all pregnancies. For most women, hypertension and proteinuria settle following delivery. The National Institute for Health and Clinical Excellence (NICE) Hypertension in Pregnancy guideline recommends that this group of patients are reviewed by a medical professional postnatally [3]. However, studies have shown that blood pressure and urinalysis are often not checked in the postpartum period [4]. Women with a history of hypertension in pregnancy have a higher risk of future hypertension and cardiovascular disease (CVD) than women who have uncomplicated pregnancies [2]. Risk scores are available for assessing an individual's risk of CVD although they are not validated in women under 30. In UK, the most appropriate is QRISK2 score [1]. OBJECTIVES: To determine the frequency of ongoing problems following a new onset hypertensive pregnancy and assess the risk of future cardiovascular disease. METHODS: 351 women with new onset hypertension in pregnancy were reviewed 6 weeks postnatally. They were assessed for ongoing disease and cardiovascular risk. 10 year QRISK2 scores and heart age (the age at which a matched person has that score) were calculated. RESULTS: 211 women with pre-eclampsia (PE) and 140 with gestational hypertension (GH) were reviewed. 9% and 11% of women with previous PE and GH respectively still required antihypertensive agents at follow-up. Only 1 woman required more than one antihypertensive medication (PE group). 19 women with PE (9%) had ongoing proteinuria (PCR>30). 5% had an estimated GFR <60ml/min. In addition to those with a strong family history of hypertension, 23 patients (6.5%) required investigation for ongoing problems. Risk factors for CVD were common 6 weeks after delivery: Although the overall risk of CVD was low (median 10 year QRISK2 score 0.3, median relative risk 1.0), with 41% of women having the lowest possible heart age, 22% of women had a significantly elevated risk of CVD (QRISK2 heart age ⩾age+10). CONCLUSION: 16% of women had ongoing hypertension or proteinuria, evidence supporting the NICE guidance that all women with hypertension in pregnancy need follow-up after delivery. The overall risk of future CVD in women with previous hypertension in pregnancy is low but about one-fifth of women are at very high risk. A program of risk assessment is required to allow preventative measures to be implemented.

No correlation between ultrasound placental grading at 31-34 weeks of gestation and a surrogate estimate of organ function at term obtained by stereological analysis.

We test the experimental hypothesis that early changes in the ultrasound appearance of the placenta reflect poor or reduced placental function. The sonographic (Grannum) grade of placental maturity was compared to placental function as expressed by the morphometric oxygen diffusive conductance of the villous membrane. Ultrasonography was used to assess the Grannum grade of 32 placentas at 31-34 weeks of gestation. Indications for the scans included a history of previous fetal abnormalities, previous fetal growth problems or suspicion of IUGR. Placentas were classified from grade 0 (most immature) to grade III (most mature). We did not exclude smokers or complicated pregnancies as we aimed to correlate the early appearance of mature placentas with placental function. After delivery, microscopical fields on formalin-fixed, trichrome-stained histological sections of each placenta were obtained by multistage systematic uniform random sampling. Using design-based stereological methods, the exchange surface areas of peripheral (terminal and intermediate) villi and their fetal capillaries and the arithmetic and harmonic mean thicknesses of the villous membrane (maternal surface of villous trophoblast to adluminal surface of vascular endothelium) were estimated. An index of the variability in thickness of this membrane, and an estimate of its oxygen diffusive conductance, were derived secondarily as were estimates of the mean diameters and total lengths of villi and fetal capillaries. Group comparisons were drawn using analysis of variance. We found no significant differences in placental volume or composition or in the dimensions or diffusive conductances of the villous membrane. Subsequent exclusion of smokers did not alter these main findings. Grannum grades at 31-34 weeks of gestation appear not to provide reliable predictors of the functional capacity of the term placenta as expressed by the surrogate measure, morphometric diffusive conductance.

Cyclosporin-A inhibits stretch-induced changes in myosin heavy chain expression in C2C12 skeletal muscle cells.

Studies in vivo, have shown that passive stretch of skeletal muscle induces changes in contractile protein expression. In the present study the effects of passive stretch upon myosin heavy chain (MyHC) expression were examined in C2C12 cell myotubes. Passive stretch induced an upregulation of adult fast and slow MyHCs, which was prevented by cyclosporin A (CsA), an inhibitor of calcineurin. Calcineurin has been shown to act via the dephosphorylation of NFAT and MEF2 transcriptional factors. In this study no significant change in the phosphorylation state of these factors was observed. In contrast stretch induced an alteration in the levels of the myogenic regulatory factors (MRFs) MyoD, myogenin and myf5. The modulation in the level of these MRFs was also inhibited by CsA. These data indicate that changes in muscle phenotype in C2C12 can be modulated by passive stretch and some of these changes are calcineurin dependent.

Electrical stimulation modulates IGF binding protein transcript levels in C2C12 myotubes.

Electrical stimulation (ES) of skeletal muscle can produce changes in metabolic enzyme and contractile protein gene expression resulting in fast-to-slow phenotypic changes. The molecular mechanism by which ES induces changes in phenotype is not entirely understood but recent reports have demonstrated that the calcineurin/NF-AT signalling pathway is involved. IGF-1 is also capable of inducing changes in phenotype through the same calcineurin/NF-AT pathway but little is known of the direct effect of ES on the IGF system. In this study, we examined the effects of ES on the expression of igf-1, igf-2 and the six igfbp genes in the C2C12 muscle cell line. Results showed that ES induced a change in phenotype that was accompanied by downregulation of igf-2 and upregulation of igfbp-4 mRNA levels. However, ES did not significantly alter the transcription of igf-1, igfbp-2, igfbp-5 and igfbp-6 genes. This study demonstrates that ES of muscle cells in vitro not only directly modulates the gene expression of contractile proteins but also modulates proteins that are part of the IGF regulatory system, in particular IGFBP-4.

Alterations in the mRNA levels of two metabolic enzymes in rat skeletal muscle during stretch-induced hypertrophy and disuse atrophy.

Changes in carbonic anhydrase III (CAIII: normally predominant in slow type 1 fibres) and phosphoglucoisomerase (PGI: normally predominant in fast type 2 fibres) mRNAs were studied in rat slow postural soleus and fast phasic plantaris muscles which had been immobilised in shortened (disuse) and lengthened (passive stretch) positions for 2 and 5 days. Our results provide evidence that limb immobilisation in both positions affects the expression of these metabolic enzymes. Muscle disuse resulted in considerable loss of CAIII mRNA in soleus but not in plantaris, whereas, PGI mRNA levels were unaffected in soleus but declined in plantaris after 2 days. Passive stretch caused an increase in CAIII mRNA in soleus muscles after 2 days, although this was not maintained after 5 days when a decrease was observed, and an increase in plantaris muscles after 5 days. In contrast, PGI mRNA declined in both muscles. These results indicate that immobilisation of muscles in the shortened and lengthened positions affects the levels of transcripts of these soluble enzymes in different ways and these effects are muscle specific.

Passive stretch modulates denervation induced alterations in skeletal muscle myosin heavy chain mRNA levels.

The effect of denervation and denervation combined with immobilisation in either the shortened or lengthened position (passive stretch) upon myosin heavy chain (MyHC) mRNA levels was examined in three rat hind-limb muscles with differing phenotypes. Denervation alone caused a reduction in type I and type IIa MyHC transcripts in all three muscles. In contrast denervation caused a 72% increase in type IIb in the slow postural soleus muscle only which was prevented by immobilisation in the lengthened position. In the same muscle passive stretch also significantly retarded the effects of denervation upon the type I transcript (from 38% below control levels to 24% below) and type IIa transcript (from 59% to 32% below control levels). The levels of both type I and IIa transcripts, in the fast phasic plantaris muscle, were both unaffected by stretch combined with denervation when compared to denervation alone. In the mixed gastrocnemius muscle stretch affected the level of the type I but not the type IIa transcript. These data suggest that passive stretch can modulate MyHC gene expression independently of innervation but that it does so in a muscle-specific manner.

Interactions between growth hormone and nutrition in hypophysectomised rats: skeletal muscle myosin heavy chain mRNA levels.

The aim of this study was to examine the role of growth hormone (GH) in regulating muscle phenotype and to determine how this is modulated by altered nutrition. Total RNA was extracted from gastrocnemius muscles of hypophysectomised rats treated with saline, GH or GH but fed a restricted intake. Type 1, 2A, 2B, embryonic and neonatal myosin heavy chain mRNA levels were estimated by slot blot hybridization. Hypophysectomy reduced the concentrations of types 1, 2A and embryonic mRNAs and dramatically elevated types 2B and neonatal compared to control levels, but this was time-dependent. All MHC mRNA levels were partially restored to control levels in the GH-treated rats except for type 1; the level of this transcript was only elevated by GH in the restricted intake group. Restricted food intake modulated the effects of GH administration for all other MHC mRNA concentrations.