Tabersonine Enhances Olaparib Sensitivity through FHL1-Mediated Epithelial-Mesenchymal Transition in an Ovarian Tumor.

Ovarian cancer (OVC) is one of the most aggressive gynecological malignancies worldwide. Although olaparib treatment has shown favorable outcomes against the treatment of OVC, its effectiveness remains limited in some OVC patients. Investigating new strategies to improve the therapeutic efficacy of olaparib against OVC is imperative. Our study identified tabersonine, a natural indole alkaloid, for its potential to increase the chemosensitivity of olaparib in OVC. The combined treatment of olaparib and tabersonine synergistically inhibited cell proliferation in OVC cells and suppressed tumor growth in A2780 xenografts. The combined treatment effectively suppressed epithelial-mesenchymal transition (EMT) by altering the expression of E-cadherin, N-cadherin, and vimentin and induced DNA damage responses. Integrating quantitative proteomics, FHL1 was identified as a potential regulator to modulate EMT after tabersonine treatment. Increased expression of FHL1 was induced by tabersonine treatment, while downregulation of FHL1 reversed the inhibitory effects of tabersonine on OVC cells by mediating EMT. In vivo findings further reflected that the combined treatment of tabersonine and olaparib significantly inhibited tumor growth and OVC metastasis through upregulation of FHL1. Our findings reveal the role of tabersonine in improving the sensitivity of olaparib in OVC through FHL1-mediated EMT, suggesting that tabersonine holds promise for future application in OVC treatment.

Storax Attenuates Cardiac Fibrosis following Acute Myocardial Infarction in Rats via Suppression of AT1R-Ankrd1-P53 Signaling Pathway.

Myocardial fibrosis following acute myocardial infarction (AMI) seriously affects the prognosis and survival rate of patients. This study explores the role and regulation mechanism of storax, a commonly used traditional Chinese medicine for treatment of cardiovascular diseases, on myocardial fibrosis and cardiac function. The AMI rat model was established by subcutaneous injection of Isoproterenol hydrochloride (ISO). Storax (0.1, 0.2, 0.4 g/kg) was administered by gavage once/d for 7 days. Electrocardiogram, echocardiography, hemodynamic and cardiac enzyme in AMI rats were measured. HE, Masson, immunofluorescence and TUNEL staining were used to observe the degree of pathological damage, fibrosis and cardiomyocyte apoptosis in myocardial tissue, respectively. Expression of AT1R, CARP and their downstream related apoptotic proteins were detected by WB. The results demonstrated that storax could significantly improve cardiac electrophysiology and function, decrease serum cardiac enzyme activity, reduce type I and III collagen contents to improve fibrosis and alleviate myocardial pathological damage and cardiomyocyte apoptosis. It also found that storax can significantly down-regulate expression of AT1R, Ankrd1, P53, P-p53 (ser 15), Bax and cleaved Caspase-3 and up-regulate expression of Mdm2 and Bcl-2. Taken together, these findings indicated that storax effectively protected cardiomyocytes against myocardial fibrosis and cardiac dysfunction by inhibiting the AT1R-Ankrd1-P53 signaling pathway.

Ursolic acid ameliorates hypobaric hypoxia-induced skeletal muscle protein loss via upregulating Akt pathway: An experimental study using rat model.

Hypobaric hypoxic stress leads to oxidative stress, inflammation, and disturbance in protein turnover rate. Aggregately, this imbalance in redox homeostasis is responsible for skeletal muscle protein loss and a decline in physical performance. Hence, an urgent medical need is required to ameliorate skeletal muscle protein loss. The present study investigated the efficacy of ursolic acid (UA), a pentacyclic triterpene acid to ameliorate hypobaric hypoxia (HH)-induced muscle protein loss. UA is a naturally occurring pentacyclic triterpene acid present in several edible herbs and fruits such as apples. It contains skeletal muscle hypertrophy activity; still its potential against HH-induced muscle protein loss is unexplored. To address this issue, an in vivo study was planned to examine the beneficial effect of UA supplementation on HH-induced skeletal muscle loss. Male Sprague Dawley rats were exposed to HH with and without UA supplementation (20 mg/kg; oral) for 3 continuous days. The results described the beneficial role of UA as supplementation of UA with HH exposure attenuated reactive oxygen species production and oxidative protein damage, which indicate the potent antioxidant activity. Furthermore, UA supplementation enhanced Akt, pAkt, and p70S6kinase activity (Akt pathway) and lowered the pro-inflammatory cytokines in HH exposed rats. UA has potent antioxidant and anti-inflammatory activity, and it enhanced the protein content via upregulation of Akt pathway-related proteins against HH exposure. These three biological activities of UA make it a novel candidate for amelioration of HH-induced skeletal muscle damage and protein loss.

Fermented milk retains beneficial effects on skeletal muscle protein anabolism after processing by centrifugation and supernatant removal.

Lactobacillus-fermented milk can stimulate anabolic effects in skeletal muscle. Fermented milk containing Lactobacillus produces aqueous molecules, such as free AA and lactate. This study aimed to investigate how processing fermented milk by centrifugation and removal of supernatant affects AA absorption and postprandial skeletal muscle protein synthesis (MPS) when mice are fed fermented milk. We gavaged male Sprague-Dawley rats with skim milk (S), fermented milk (F), or processed fermented milk (P), and examined the total AA content in portal vein blood (reflecting AA absorption) and plantaris muscle MPS at 30, 60, and 90 min following administration. Relative to fasted rats, at 30 min the total AA concentration in portal vein blood from rats in the P groups was significantly higher, followed by F and S, respectively. The MPS rates were higher for the F or P groups compared with the S group. Phosphorylation levels of p70S6 kinase in the P and F groups were significantly higher than those for the S group 30 min after administration, although the level of Akt phosphorylation was similar among the groups. These results suggested that fermentation improves AA absorption that in turn enhances postprandial MPS via Akt-independent mechanisms, and that processed fermented milk retains these favorable effects on MPS.

Isolated Leucine and Branched-Chain Amino Acid Supplementation for Enhancing Muscular Strength and Hypertrophy: A Narrative Review.

Branched-chain amino acids (BCAA) are one of the most popular sports supplements, marketed under the premise that they enhance muscular adaptations. Despite their prevalent consumption among athletes and the general public, the efficacy of BCAA has been an ongoing source of controversy in the sports nutrition field. Early support for BCAA supplementation was derived from extrapolation of mechanistic data on their role in muscle protein metabolism. Of the three BCAA, leucine has received the most attention because of its ability to stimulate the initial acute anabolic response. However, a substantial body of both acute and longitudinal research has now accumulated on the topic, affording the ability to scrutinize the effects of BCAA and leucine from a practical standpoint. This article aims to critically review the current literature and draw evidence-based conclusions about the putative benefits of BCAA or leucine supplementation on muscle strength and hypertrophy as well as illuminate gaps in the literature that warrant future study.

C/EBPß mediates palmitate-induced musclin expression via the regulation of PERK/ATF4 pathways in myotubes.

Musclin is a muscle-secreted cytokine that disrupts glucose uptake and glycogen synthesis in type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the regulation of musclin gene expression in response to treatment with palmitate. RNA sequencing results showed that biological processes activated by palmitate are mainly enriched in endoplasmic reticulum (ER) stress. The protein kinase RNA-like ER kinase (PERK) signaling pathway is involved in the regulation of musclin expression induced by palmitate. Chromatin immunoprecipitation data showed that activating transcription factor 4 (ATF4)-downstream of PERK-bound to the promoter of the C/EBPß gene. Notably, C/EBPß also contains a binding site in the region -94~-52 of the musclin gene promoter. Knockdown or knockout of PERK and ATF4 using short hairpin RNA or CRISPR-Cas9 decreased the expression of C/EBPß and musclin induced by palmitate. Furthermore, knockdown and knockout of C/EBPß alleviated the high expression of musclin in response to treatment with palmitate. Moreover, CRISPR-Cas9 knockout of the region -94~-52 in which C/EBPß binds to the promoter of musclin abrogated the induction of high musclin expression caused by palmitate. Collectively, these findings suggest that treatment with palmitate activates the PERK/ATF4 signaling pathway, which in turn increases the expression of C/EBPß. C/EBPß binds directly to the promoter of the musclin gene and upregulates its expression.

Sphingosine-1-phosphate protects against brain microvascular endothelial junctional protein disorganization and barrier dysfunction caused by alcohol.

OBJECTIVE: S1P has known endothelial barrier-protective properties, but whether this extends to the BBB is unclear. We hypothesized that alcohol-induced disruption of brain microvascular endothelial barrier function and junctional protein organization can be ameliorated by S1P treatment. METHODS: Cultured primary HBMEC monolayers were used to characterize endothelial-specific mechanisms of BBB regulation. TER and apparent permeability coefficients for albumin, dextran-4 kDa, and sodium fluorescein were used as indices of barrier function. Junctional localization of Claudin-5, VE-cadherin, and ß-catenin was determined by immunofluorescence confocal microscopy. S1P was applied following treatment with alcohol. RESULTS: Alcohol significantly impaired HBMEC TER. Application of S1P after alcohol treatment resulted in a hastened recovery to the baseline HBMEC TER. Alcohol-treated HBMEC had a significantly higher mean permeability than control that was reversed by S1P. Alcohol caused the formation of gaps between cells. Treatment with S1P (after alcohol) increased junctional localization of VE-Cadherin, Claudin-5, and ß-catenin. CONCLUSIONS: Alcohol impairs the barrier function and junctional organization of HBMEC monolayers. S1P enhanced barrier function and restored junctions in the presence of alcohol, and thus may be useful for restoring BBB function during alcohol intoxication.

4-Aminopyridine attenuates muscle atrophy after sciatic nerve crush injury in mice.

INTRODUCTION: We recently demonstrated the beneficial effects of 4-aminopyridine (4-AP), a potassium channel blocker, in enhancing remyelination and recovery of nerve conduction velocity and motor function after sciatic nerve crush injury in mice. Although muscle atrophy occurs very rapidly after nerve injury, the effect of 4-AP on muscle atrophy and intrinsic muscle contractile function is largely unknown. METHODS: Mice were assigned to sciatic nerve crush injury and no-injury groups and were followed for 3, 7, and 14 days with/without 4-AP or saline treatment. Morphological, functional, and transcriptional properties of skeletal muscle were assessed. RESULTS: In addition to improving in vivo function, 4-AP significantly reduced muscle atrophy with increased muscle fiber diameter and contractile force. Reduced muscle atrophy was associated with attenuated expression of atrophy-related genes and increased expression of proliferating stem cells. DISCUSSION: These findings provide new insights into the potential therapeutic benefits of 4-AP against nerve injury-induced muscle atrophy and dysfunction. Muscle Nerve 60: 192-201, 2019.

Arsenic Trioxide Inhibits the Metastasis of Small Cell Lung Cancer by Blocking Calcineurin-Nuclear Factor of Activated T Cells (NFAT) Signaling.

BACKGROUND The inhibitory effect of arsenic trioxide (As2O3) on lung cancer has been reported in some preclinical studies. However, its effect on small cell lung cancer (SCLC) has been poorly explored. Calcineurin and its substrate, nuclear factor of activated T cells (NFAT), mediate the downstream signaling of VEGF, and is critical in the process endothelium activation and tumor metastasis. In this study, we aimed to evaluate whether As2O3 had inhibitory effects on endothelial cells activation and the metastasis of SCLC, and to explore the possible mechanisms. MATERIAL AND METHODS In vitro, human umbilical vein endothelial cells (HUVECs) were used. Cell Counting Kit-8 assay and cell migration assay were performed to determine the effect of As2O3 on HUVECs proliferation and migration. The level of calcineurin, NFAT, downstream factors for Down syndrome candidate region 1 (DSCR1), and the endogenous inhibitor of calcineurin, were evaluated by quantitative PCR and western blotting. In vivo, SCLC metastasis models were established by injecting NCI-H446 cells into tail veins of nude mice. Tumor-bearing mice were treated with As2O3 or calcineurin inhibitor for 10 days, after which tumor metastasis in target organs was evaluated. RESULTS As2O3 significantly inhibited the proliferation and migration of endothelial cells. Also, As2O3 inhibited the expression levels of calcineurin, NFAT, and the downstream target genes CXCR7 and RND1, while it upregulated the level of DSCR1. Both As2O3 and calcineurin inhibitor exhibited notable inhibitory effect on the metastasis of SCLC, without obvious side effects. CONCLUSIONS These findings suggested that As2O3 had remarkable inhibitory effects on the endothelial cell activation and SCLC metastasis, and the mechanism might be related to the blocking of calcineurin-NFAT signaling by upregulating DSCR1.

Ammonia lowering reverses sarcopenia of cirrhosis by restoring skeletal muscle proteostasis.

Sarcopenia or skeletal muscle loss is a frequent, potentially reversible complication in cirrhosis that adversely affects clinical outcomes. Hyperammonemia is a consistent abnormality in cirrhosis that results in impaired skeletal muscle protein synthesis and breakdown (proteostasis). Despite the availability of effective ammonia-lowering therapies, whether lowering ammonia restores proteostasis and increases muscle mass is unknown. Myotube diameter, protein synthesis, and molecular responses in C2C12 murine myotubes to withdrawal of ammonium acetate following 24-hour exposure to 10 mM ammonium acetate were complemented by in vivo studies in the hyperammonemic portacaval anastomosis rat and sham-operated, pair-fed Sprague-Dawley rats treated with ammonia-lowering therapy by l-ornithine l-aspartate and rifaximin orally for 4 weeks. We observed reduced myotube diameter, impaired protein synthesis, and increased autophagy flux in response to hyperammonemia, which were partially reversed following 24-hour and 48-hour withdrawal of ammonium acetate. Consistently, 4 weeks of ammonia-lowering therapy resulted in significant lowering of blood and skeletal muscle ammonia, increase in lean body mass, improved grip strength, higher skeletal muscle mass and diameter, and an increase in type 2 fibers in treated compared to untreated portacaval anastomosis rats. The increased skeletal muscle myostatin expression, reduced mammalian target of rapamycin complex 1 function, and hyperammonemic stress response including autophagy markers normally found in portacaval anastomosis rats were reversed by treatment with ammonia-lowering therapy. Despite significant improvement, molecular and functional readouts were not completely reversed by ammonia-lowering measures. CONCLUSION: Ammonia-lowering therapy results in improvement in skeletal muscle phenotype and function and molecular perturbations of hyperammonemia; these preclinical studies complement previous studies on ammonia-induced skeletal muscle loss and lay the foundation for prolonged ammonia-lowering therapy to reverse sarcopenia of cirrhosis. (Hepatology 2017;65:2045-2058).

Oxaliplatin induces muscle loss and muscle-specific molecular changes in Mice.

INTRODUCTION: Muscle wasting is a frequent, debilitating complication of cancer. The impact of colorectal cancer chemotherapeutic oxaliplatin on the development of muscle loss and associated molecular changes is of clinical importance. METHODS: C57BL/6J male mice were treated with oxaliplatin. Total body weights were measured and behavioral studies performed. Hindlimb muscle weights (gastrocnemius and soleus) were recorded in conjunction with gene and protein expression analysis. RESULTS: Oxaliplatin-treated mice displayed reduced weight gain and behavioral deficits. Mice treated over a shorter course had significantly increased STAT3 phosphorylation in gastrocnemius muscles. Mice receiving extended oxaliplatin treatment demonstrated reduced hindlimb muscle mass with upregulation of myopathy-associated genes Foxo3, MAFbx, and Bnip3. DISCUSSION: The findings suggest that oxaliplatin treatment can directly disrupt skeletal muscle homeostasis and promote muscle loss, which may be clinically relevant in the context of targeting fatigue and weakness in cancer patients. Muscle Nerve 57: 650-658, 2018.

Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies.

Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated (P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control.

Acute high-caffeine exposure increases autophagic flux and reduces protein synthesis in C2C12 skeletal myotubes.

Caffeine is a highly catabolic dietary stimulant. High caffeine concentrations (1-10 mM) have previously been shown to inhibit protein synthesis and increase protein degradation in various mammalian cell lines. The purpose of this study was to examine the effect of short-term caffeine exposure on cell signaling pathways that regulate protein metabolism in mammalian skeletal muscle cells. Fully differentiated C2C12 skeletal myotubes either received vehicle (DMSO) or 5 mM caffeine for 6 h. Our analysis revealed that caffeine promoted a 40% increase in autolysosome formation and a 25% increase in autophagic flux. In contrast, caffeine treatment did not significantly increase the expression of the skeletal muscle specific ubiquitin ligases MAFbx and MuRF1 or 20S proteasome activity. Caffeine treatment significantly reduced mTORC1 signaling, total protein synthesis and myotube diameter in a CaMKKß/AMPK-dependent manner. Further, caffeine promoted a CaMKII-dependent increase in myostatin mRNA expression that did not significantly contribute to the caffeine-dependent reduction in protein synthesis. Our results indicate that short-term caffeine exposure significantly reduced skeletal myotube diameter by increasing autophagic flux and promoting a CaMKKß/AMPK-dependent reduction in protein synthesis.

Influence of biofilm surface layer protein A (BslA) on the gel structure of myofibril protein from chicken breast.

BACKGROUND: Different techniques have been applied to alter myofibril protein (MP) structure, which further promotes protein-protein interactions and influencing the MP gelling characteristics. Influence of BslA from natto food (protein concentration, 30 mg mL-1 ; at 0.001, 0.005, 0.01, 0.05 and 0.1 g kg-1 ) on the characteristics of MP gel of chicken breast was investigated. RESULTS: Results show that cooking loss significantly (P < 0.05) decreased with increased percentage of BslA. Hardness of MP gel did not significantly change at 0.01 g kg-1 BslA. Differential scanning calorimetry disclosed that MP was modified by the addition of BslA. Moreover, BslA produced a high value of storage modulus (G') and low value of phase angle (tan δ) during heating, especially at 0.01 g kg-1 . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis proved the formation of higher-molecular-weight polymers by developing non-disulfide covalent bonds between MP at 0.01 g kg-1 BslA. Surface hydrophobicity of the MP gel was decreased with increased percentage of BslA. Scanning electron microscopy confirmed the increasing number of uniform cavities of MP gel with the increased percentage of BslA. CONCLUSION: Addition of 0.01 g kg-1 BslA significantly improved the water holding capacity and rheological properties of MP by developing non-disulfide covalent bonds. © 2017 Society of Chemical Industry.

Presleep protein ingestion does not compromise the muscle protein synthetic response to protein ingested the following morning.

Protein ingestion before sleep augments postexercise muscle protein synthesis during overnight recovery. It is unknown whether postexercise and presleep protein consumption modulates postprandial protein handling and myofibrillar protein synthetic responses the following morning. Sixteen healthy young (24 ± 1 yr) men performed unilateral resistance-type exercise (contralateral leg acting as a resting control) at 2000. Participants ingested 20 g of protein immediately after exercise plus 60 g of protein presleep (PRO group; n = 8) or equivalent boluses of carbohydrate (CON; n = 8). The subsequent morning participants received primed, continuous infusions of l-[ring-2H5]phenylalanine and l-[1-13C]leucine combined with ingestion of 20 g intrinsically l-[1-13C]phenylalanine- and l-[1-13C]leucine-labeled protein to assess postprandial protein handling and myofibrillar protein synthesis in the rested and exercised leg in CON and PRO. Exercise increased postabsorptive myofibrillar protein synthesis rates the subsequent day (P < 0.001), with no differences between CON and PRO. Protein ingested in the morning increased myofibrillar protein synthesis in both the exercised and rested leg (P < 0.01), with no differences between treatments. Myofibrillar protein bound l-[1-13C]phenylalanine enrichments were greater in the exercised (0.016 ± 0.002 and 0.015 ± 0.002 MPE in CON and PRO, respectively) vs. rested (0.010 ± 0.002 and 0.009 ± 0.002 MPE in CON and PRO, respectively) leg (P < 0.05), with no differences between treatments (P > 0.05). The additive effects of resistance-type exercise and protein ingestion on myofibrillar protein synthesis persist for more than 12 h after exercise and are not modulated by protein consumption during acute postexercise recovery. This work provides evidence of an extended window of opportunity where presleep protein supplementation can be an effective nutrient timing strategy to optimize skeletal muscle reconditioning.

Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.

Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM.

Sodium nitrate co-ingestion with protein does not augment postprandial muscle protein synthesis rates in older, type 2 diabetes patients.

The age-related anabolic resistance to protein ingestion is suggested to be associated with impairments in insulin-mediated capillary recruitment and postprandial muscle tissue perfusion. The present study investigated whether dietary nitrate co-ingestion with protein improves muscle protein synthesis in older, type 2 diabetes patients. Twenty-four men with type 2 diabetes (72 ± 1 yr, 26.7 ± 1.4 m/kg(2) body mass index, 7.3 ± 0.4% HbA1C) received a primed continuous infusion of l-[ring-(2)H5]phenylalanine and l-[1-(13)C]leucine and ingested 20 g of intrinsically l-[1-(13)C]phenylalanine- and l-[1-(13)C]leucine-labeled protein with (PRONO3) or without (PRO) sodium nitrate (0.15 mmol/kg). Blood and muscle samples were collected to assess protein digestion and absorption kinetics and postprandial muscle protein synthesis rates. Upon protein ingestion, exogenous phenylalanine appearance rates increased in both groups (P < 0.001), resulting in 55 ± 2% and 53 ± 2% of dietary protein-derived amino acids becoming available in the circulation over the 5h postprandial period in the PRO and PRONO3 groups, respectively. Postprandial myofibrillar protein synthesis rates based on l-[ring-(2)H5]phenylalanine did not differ between groups (0.025 ± 0.004 and 0.021 ± 0.007%/h over 0-2 h and 0.032 ± 0.004 and 0.030 ± 0.003%/h over 2-5 h in PRO and PRONO3, respectively, P = 0.7). No differences in incorporation of dietary protein-derived l-[1-(13)C]phenylalanine into de novo myofibrillar protein were observed at 5 h (0.016 ± 0.002 and 0.014 ± 0.002 mole percent excess in PRO and PRONO3, respectively, P = 0.8). Dietary nitrate co-ingestion with protein does not modulate protein digestion and absorption kinetics, nor does it further increase postprandial muscle protein synthesis rates or the incorporation of dietary protein-derived amino acids into de novo myofibrillar protein in older, type 2 diabetes patients.

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs.

Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets (n = 11-12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 µmol·kg-1·h-1) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.

High-intensity resistance training attenuates dexamethasone-induced muscle atrophy.

INTRODUCTION: In this study we investigated the effects of high-intensity resistance training (RT) on dexamethasone (DEX)-induced muscle atrophy in flexor hallucis longus (FHL), tibialis anterior (TA), and soleus (SOL) muscles. METHODS: Rats underwent either high-intensity RT or were kept sedentary. In the last 10 days they received either DEX (0.5 mg/kg/day, intraperitoneally) or saline. RESULTS: DEX reduced body weight (-21%), food intake (-28%), FHL and TA muscle mass (-20% and -18%, respectively), and increased muscle-specific ring finger 1 (MuRF-1) protein level (+37% and +45.5%). RT attenuated FHL muscle atrophy through a combination of low increase in MuRF-1 protein level (-3.5%) and significant increases in mammalian target of rapamycin (mTOR) (+63%) and p70S6K (+46% and +49% for control and DEX, respectively) protein levels. CONCLUSION: RT attenuated DEX-induced muscle atrophy through a combination of increases in mTOR and p70S6K protein levels and a low increase in MuRF-1 protein level.

Four-and-a-Half LIM Domains Protein 2 Is a Coactivator of Wnt Signaling in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is a microvascular complication that leads to kidney dysfunction and ESRD, but the underlying mechanisms remain unclear. Podocyte Wnt-pathway activation has been demonstrated to be a trigger mechanism for various proteinuric diseases. Notably, four-and-a-half LIM domains protein 2 (FHL2) is highly expressed in urogenital systems and has been implicated in Wnt/ß-catenin signaling. Here, we used in vitro podocyte culture experiments and a streptozotocin-induced DKD model in FHL2 gene-knockout mice to determine the possible role of FHL2 in DKD and to clarify its association with the Wnt pathway. In human and mouse kidney tissues, FHL2 protein was abundantly expressed in podocytes but not in renal tubular cells. Treatment with high glucose or diabetes-related cytokines, including angiotensin II and TGF-ß1, activated FHL2 protein and Wnt/ß-catenin signaling in cultured podocytes. This activation also upregulated FHL2 expression and promoted FHL2 translocation from cytosol to nucleus. Genetic deletion of the FHL2 gene mitigated the podocyte dedifferentiation caused by activated Wnt/ß-catenin signaling under Wnt-On, but not under Wnt-Off, conditions. Diabetic FHL2(+/+) mice developed markedly increased albuminuria and thickening of the glomerular basement membrane compared with nondiabetic FHL2(+/+) mice. However, FHL2 knockout significantly attenuated these DKD-induced changes. Furthermore, kidney samples from patients with diabetes had a higher degree of FHL2 podocyte nuclear translocation, which was positively associated with albuminuria and progressive renal function deterioration. Therefore, we conclude that FHL2 has both structural and functional protein-protein interactions with ß-catenin in the podocyte nucleus and that FHL2 protein inhibition can mitigate Wnt/ß-catenin-induced podocytopathy.