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.

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.

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.

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.

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.

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.

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.

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.

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.

Congenital nephropathy and ventriculomegaly: a report of four cases.

Congenital nephrotic syndrome with ventriculomegaly and a normal karyotype is a rare association. We report four cases, three of which were conceived consecutively by one couple. All the cases were associated with elevated maternal serum alpha-fetoprotein. Renal histology in one fetus demonstrated colloid filled cysts distributed in the corticomedullary area. Transmission electron microscopy of the glomeruli showed normally developed foot processes and confirmatory genetic studies excluded Finnish congenital nephrotic syndrome. It is probable that congenital nephropathy in conjunction with ventriculomegaly is the result of an autosomal recessive syndrome.

Phenotypic expression of IGF binding protein transcripts in muscle, in vitro and in vivo.

The actions of the insulin-like growth factors (IGF-I and IGF-II) which are essential components of skeletal muscle growth are mediated via their receptors and modulated by six binding proteins (IGFBPs). We studied IGFBP transcripts in C2C12 cell cultures and in adult control and denervated gastrocnemius muscle. IGFBP-2, -4, -5, and -6 were detected in C2C12 cells. IGFBP-6 mRNA levels remained unchanged, IGFBP-2 levels decreased and IGFBP-4 and -5 increased over 1, 5, and 9 days after serum reduction. In a range of adult muscles studied, IGFBP-4 mRNA levels were similar, IGFBP-5 was present at different levels in slow and fast muscles and IGFBP-6 had the lowest expression in the tibialis anterior. Denervation resulted in dramatic up-regulation of IGFBP-4 and -5 transcripts but there was no change in IGFBP-6. These results suggest that either lack of neural input and/or mechanical loading, both of which contribute to muscle atrophy, affect IGFBP expression.

The LIM-domain protein FHL1 (SLIM 1) exhibits functional regulation in skeletal muscle.

The LIM domain protein FHL1 (SLIM 1) transcript is preferentially expressed in postnatal skeletal muscle but almost nothing is known about its function in this tissue. In this study we have examined the expression of the FHL1 transcript at the cellular level by in situ hybridisation. Muscle fibers exist as a number of discrete subpopulations or "types" which are differentiated by their contractile and metabolic properties. It was observed that the FHL1 transcript was not fiber-type specific but was however more abundant in oxidative fibers. Muscle atrophy induced by disuse caused a significant decline in the expression of the transcript but atrophy induced by short-term denervation did not. Hypertrophy of skeletal muscle induced by passive stretch was associated with an up-regulation of the FHL1 transcript. These data are consistent that FHL1 is involved in synthetic processes within the muscle fibre.

MRF-4 exhibits fiber type- and muscle-specific pattern of expression in postnatal rat muscle.

The crucial role played by the myogenic regulatory factors (MRFs) in the development of skeletal muscle has been well characterized. The continued expression of these factors in skeletal muscle of the postnatal animal has led to the suggestion that they may play a role in the regulation of muscle fiber phenotype. The few studies that have examined the expression of MRF-4 in postnatal muscle have been carried out at the whole muscle level. These studies demonstrated that this factor is expressed at a higher level than any other MRF but suggested that this was not affected by muscle phenotype. In this study, the expression of the MRF-4 transcript has been examined at the cellular level by in situ hybridization. It was observed that in the mixed fiber type muscle the gastrocnemius, MRF-4 was preferentially expressed in slow muscle fibers, but in the slow postural soleus, no fiber type specificity was observed. These observations suggest that MRF-4 may play a role in the regulation of muscle fiber phenotype in the postnatal animal.

The expression of the myogenic regulatory factors in denervated and normal muscles of different phenotypes.

The nerve is known to play a pivotal role in the diversification of muscle fibre types postnatally. Reducing neuronal activity in a slow muscle such as the soleus by denervation, switches on genes associated with a fast muscle phenotype. On the other hand, denervating a fast muscle such as the extensor digitorum longus (EDL) induces the conversion of fast fibres to a 'slower' contractile phenotype. The myogenic regulatory factors (MRFs) are proposed as the regulators of muscle phenotype as MyoD and myogenin have been shown to differentially accumulate in fast and slow muscle upon the induction of fibre type transformation. The denervation model has been used in the present study to induce changes in MRF expression in the muscles of the lower hindlimb which have distinct phenotypic characteristics. The level of MRF expression in pairs of denervated and innervated soleus, EDL, tibialis anterior (TA), plantaris and gastrocnemius muscles has been determined by Northern analysis and compared. The present study has shown that each muscle responds differently to denervation with respect to the increases in MRF expression. Fast muscles responded very quickly to denervation by increasing the level of MRF transcripts while slow muscles did not show significant increases in expression after 48 h denervation. The innervated EDL (fast) and soleus (slow) muscle differed with respect to the level of MRF-4 expressed, MRF-4 being expressed at higher levels in the slow muscle compared to the fast, suggesting that MRF-4 is important in the maintenance of a slow muscle phenotype. Moreover, MRF-4 and myogenin show the greatest fold increases in expression in the fast muscles examined. MyoD and Myf 5 show less dramatic increase in expression in response to denervation but exhibit the greatest fold increases in the fast muscles compared to slow.

The novel sarcomeric protein telethonin exhibits developmental and functional regulation.

We have isolated a cDNA clone from a mouse skeletal muscle library which is preferentially expressed in striated muscle and exhibits a high homology to human telethonin, a sarcomeric protein. The mouse telethonin transcript is developmentally regulated in both cardiac and skeletal muscle in vivo and is down-regulated in response to denervation. In the C2C12 muscle cell-line the mouse telethonin transcript exhibited a pattern of accumulation similar to that observed for a contractile protein and suggests a role in myofibrillar assembly.

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.

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.