Despina Constantin

The involvement of the ubiquitin proteasome system in human skeletal muscle remodelling and atrophy

Skeletal muscle exhibits great plasticity in response to altered activity levels, ultimately resulting in tissue remodelling and substantial changes in mass. Animal research would suggest that the ubiquitin proteasome system, in particular the ubiquitin ligases MAFbx/atrogin-1 and MuRF1, are instrumental to the processes underlying these changes. This review article therefore examines the role of proteasomal-mediated protein degradation in human skeletal muscle in health and disease. Specifically, the effects of exercise, disuse and inflammatory disease states on the ubiquitin proteasome system in human skeletal muscle are examined. The article also identifies several inconsistencies between published human studies and data obtained from animal models of muscle atrophy, highlighting the need for a more comprehensive examination of the molecular events responsible for modulating muscle mass in humans.

Progesterone suppresses the inflammatory response and nitric oxide synthase-2 expression following cerebral ischemia.

Gender differences in outcome following cerebral ischemia have frequently been observed and attributed to the actions of steroid hormones. Progesterone has been shown to possess neuroprotective properties following transient ischemia, with respect to decreasing lesion volume and improving functional recovery. The present study was designed to determine the mechanisms of progesterone neuroprotection, and whether these relate to the inflammatory response. Male mice underwent either 60 min or permanent middle cerebral artery occlusion (MCAO) and received progesterone (8 mg/kg ip) or vehicle 1 h, 6 h and 24 h post-MCAO. Forty-eight hours following transient MCAO, structural magnetic resonance imaging revealed a significant decrease in the amount of edematous tissue present in progesterone-treated mice as compared with vehicle. Using real-time PCR we found that progesterone treatment significantly suppressed the injury-induced upregulation of interleukin (IL)-1beta, transforming growth factor (TGF)beta2, and nitric oxide synthase (NOS)-2 mRNAs in the ipsilateral hemisphere while having no effect on tumor necrosis factor (TNF)-alpha mRNA expression. Progesterone treatment following permanent MCAO also resulted in a significant decrease in lesion volume. This was not apparent in mice lacking a functional NOS-2 gene. Thus, progesterone is neuroprotective in both permanent and transient ischemia, and this effect is related to the suppression of specific aspects of the inflammatory response.

A potential role for Akt/FOXO signalling in both protein loss and the impairment of muscle carbohydrate oxidation during sepsis in rodent skeletal muscle

Sepsis causes muscle atrophy and insulin resistance, but the underlying mechanisms are unclear. Therefore, the present study examined the effects of lipopolysaccharide (LPS)‐induced endotoxaemia on the expression of Akt, Forkhead Box O (FOXO) and its downstream targets, to identify any associations between changes in FOXO‐dependent processes influencing muscle atrophy and insulin resistance during sepsis. Chronically instrumented male Sprague–Dawley rats received a continuous intravenous infusion of LPS (15 μg kg−1 h−1) or saline for 24 h at 0.4 ml h−1. Animals were terminally anaesthetized and the extensor digitorum longus muscles from both hindlimbs were removed and snap‐frozen. Measurements were made of mRNA and protein expression of selected signalling molecules associated with pathways regulating protein synthesis and degradation and carbohydrate metabolism. LPS infusion induced increases in muscle tumour necrosis factor‐α (8.9‐fold, P < 0.001) and interleukin‐6 (8.4‐fold, P < 0.01), paralleled by reduced insulin receptor substrate‐1 mRNA expression (−0.7‐fold, P < 0.01), and decreased Akt1 protein and cytosolic FOXO1 and FOXO3 phosphorylation. These changes were accompanied by significant increases in muscle atrophy F‐box mRNA (5.5‐fold, P < 0.001) and protein (2‐fold, P < 0.05) expression, and pyruvate dehydrogenase kinase 4 mRNA (15‐fold, P < 0.001) and protein (1.6‐fold, P < 0.05) expression. There was a 29% reduction in the muscle protein: DNA ratio, a 56% reduction in pyruvate dehydrogenase complex (PDC) activity (P < 0.05), and increased glycogen degradation and lactate accumulation. The findings of this study suggest a potential role for Akt/FOXO in the simultaneous impairment of carbohydrate oxidation, at the level of PDC, and up‐regulation of muscle protein degradation, in LPS‐induced endotoxaemia.

Novel events in the molecular regulation of muscle mass in critically ill patients

Non‐technical summary A clinical trait of critically ill patients following trauma, surgical complications and/or sepsis is the presence of a marked skeletal muscle wasting, which severely compromises muscle function, especially the respiratory muscles, and clinical outcomes such as morbis, mortality and the length of hospitalization. However, the molecular mechanisms responsible for this are largely unresolved. In this paper we provide novel and interesting translational data that show how the muscle mass is regulated during critical illness. The present observations also show that the muscle signalling pathways are not only important for the size of muscle mass, but could also play a significant role in the whole body glucose control. This is extremely important and relevant to the clinical setting as uncontrolled blood glucose concentrations in critically ill patients impact on clinical outcome. From a clinical perspective, the present data suggest therapeutic strategies to preserve muscle mass and metabolic function in critical illness.

PPARδ agonism induces a change in fuel metabolism and activation of an atrophy programme, but does not impair mitochondrial function in rat skeletal muscle

PPARα agonism impairs mitochondrial function, but the effect of PPARδ agonism on mitochondrial function is equivocal. Furthermore, PPARα and δ agonism increases muscle fatty acid oxidation, potentially via activation of FOXO1 signalling and PDK4 transcription. Since FOXO1 activation has also been suggested to increase transcription of MAFbx and MuRF‐1, and thereby the activation of ubiquitin–proteasome mediated muscle proteolysis, this raises the possibility that muscle fuel selection and the induction of a muscle atrophy programme could be regulated by a single common signalling pathway. We therefore investigated the effect of PPARδ (delta) agonist, GW610742, administration on muscle mitochondrial function, fuel regulation, and atrophy and growth related signalling pathways in vivo. Twenty‐four male Wistar rats received vehicle or GW610742 (5 and 100 mg per kg body mass (bm)) orally for 6 days. Soleus muscle was used to determine maximal rates of ATP production (MRATP) in isolated mitochondria, gene and protein expression, and enzyme activities. MRATP were unchanged by GW610742.  Muscle PDK2 and PDK4 mRNA expression increased with GW610742 (100 mg (kg bm)−1) compared to vehicle (P < 0.05), and was paralleled by a twofold increase in PDK4 protein expression (P < 0.05). The activity of β‐hydroxyacyl‐CoA dehydrogenase increased with GW610742 (P < 0.05). Muscle MuRF1 and MAFbx mRNA expression was increased by GW610742 (100 mg (kg bm)−1) compared to vehicle (P < 0.05), and was matched by increased protein expression (P < 0.001), whilst Akt1 protein declined (P < 0.05). There was no effect of GW610742 on 20S proteasome activity and mRNA expression, or the muscle DNA: protein ratio. GW610742 switched muscle fuel metabolism towards decreased carbohydrate use and enhanced lipid utilization, but did not induce mitochondrial dysfunction. Furthermore, GW610742 initiated a muscle atrophy programme, possibly via changes in the Akt1/FOXO/MAFbx and MuRF1 signalling pathway.

The neuroprotective effect of progesterone after traumatic brain injury in male mice is independent of both the inflammatory response and growth factor expression

Previous studies suggest that progesterone may possess neuroprotective properties after traumatic insult but, with the exception of reduced formation of cerebral oedema, limited experimental evidence has been presented to support this claim. In the present study we focused on the effect of progesterone treatment on structural and functional deficits in an experimental model of traumatic brain injury. Female mice exhibited significantly (P = 0.0445) reduced lesion volumes compared with males after aseptic cryogenic cerebral injury (ACI), suggesting that female sex steroids provide protection against this injury. In male mice, progesterone treatment after injury (three intraperitoneal doses of 8 mg/kg) reduced lesion volume (P = 0.0429) and improved performance in a spatial cognitive task (Morris water maze; P = 0.0014). However, progesterone had no demonstrable effect on the formation of oedema as measured using T2‐weighted magnetic resonance imaging, nor did it affect brain water content. The pro‐inflammatory cytokines TNF‐α and IL‐1β, and growth factors BDNF and G‐CSF, were all strongly transcriptionally activated after ACI. However, progesterone administration did not affect expression of these genes. This study provides strong evidence that progesterone possesses neuroprotective properties in a mouse model of traumatic brain injury, but suggests that the steroid achieves this effect through mechanism(s) independent of the inflammatory response or growth factor up‐regulation.

Cellular mechanisms underlying the protective effects of preoperative feeding: a randomized study investigating muscle and liver glycogen content, mitochondrial function, gene and protein expression.

Objective:To investigate the effects of preoperative feeding with a carbohydrate-based drink that also contained glutamine and antioxidants (oral nutritional supplement [ONS], Fresenuis Kabi, Germany) on glycogen reserves, mitochondrial function, and the expression of key metabolic genes and proteins. Summary Background Data:Preoperative carbohydrate loading attenuates the decline in postoperative insulin sensitivity but the cellular mechanisms underlying this remain unclear. Methods:Two groups of 20 patients undergoing laparoscopic cholecystectomy participated in this randomized placebo-controlled double-blind study. Patients received either 600 mL of ONS or placebo the evening before surgery, and again 300 mL 3 to 4 hours before anesthesia. A 300-mL aliquot of ONS contained 50 g of carbohydrate, 15 g of glutamine and antioxidants. Blood was sampled before ingestion of the evening drink, after induction of anesthesia, and on postoperative day 1 for measurement of concentrations of glucose, glutamine, and antioxidants. Rectus abdominis muscle and liver biopsies were performed intraoperatively to determine glycogen and glutamine concentrations, mitochondrial function, pyruvate dehydrogenase kinase (PDK4), forkhead transcription factor 1 (FOXO1), and metallothionein 1A (Mt1A) expression. Results:There were no drink-related complications. ONS ingestion led to increased intraoperative liver glycogen reserves (44%, P < 0.001) and plasma glutamine and antioxidant concentrations, the latter 2 remaining elevated up to the first postoperative day. Muscle PDK4 mRNA, PDK4 protein expression, and Mt1A mRNA expression were 4-fold (P < 0.001), 44% (P < 0.05), and 1.5-fold (P < 0.001), respectively, lower in the ONS group. There were no differences in FOXO1 mRNA and protein expression. Conclusions:The changes in muscle PDK4 may explain the mechanism by which preoperative feeding with carbohydrate-based drinks attenuates the development of postoperative insulin resistance.

Skeletal muscle molecular responses to resistance training and dietary supplementation in COPD

Background Skeletal muscle dysfunction is a systemic feature of chronic obstructive pulmonary disease (COPD), contributing to morbidity and mortality. Physical training improves muscle mass and function in COPD, but the molecular regulation therein is poorly understood. Methods Candidate genes and proteins regulating muscle protein breakdown (ubiquitin proteasome pathway), muscle protein synthesis (phosphatidylinositol 3 kinase/Akt/mammalian target of rapamycin pathway), myogenesis (MyoD, myogenin and myostatin) and transcription (FOXO1, FOXO3 and RUNX1) were determined in quadriceps muscle samples taken at four time points over 8 weeks of knee extensor resistance training (RT) in patients with COPD and healthy controls (HCs). Patients with COPD were randomly allocated to receive protein/carbohydrate or placebo supplements during RT. Results 59 patients with COPD (mean (SD) age 68.0 (9.3) years, forced expiratory volume in 1 s (FEV1) 46.9 (17.8) % predicted) and 21 HCs (66.1 (4.8) years, 105.0 (21.6) % predicted) were enrolled. RT increased lean mass (∼5%) and strength (∼20%) in all groups. Absolute work done during RT was lower throughout in patients with COPD compared with HCs. RT resulted in increases (from basal) in catabolic, anabolic, myogenic and transcription factor protein expression at 24 h, 4 weeks and 8 weeks of exercise in HCs. This response was blunted in patients with COPD, except for myogenic signalling, which was similar. Nutritional supplementation did not augment functional or molecular responses to RT. Conclusions The potential for muscle rehabilitation in response to RT is preserved in COPD. Except for markers of myogenesis, molecular responses to RT are not tightly coupled to lean mass gains but reflect the lower work done during RT, suggesting some caution when identifying molecular targets for intervention. Increasing post-exercise protein and carbohydrate intake is not a prerequisite for a normal training response in COPD.

The Role of FOXO and PPAR Transcription Factors in Diet-Mediated Inhibition of PDC Activation and Carbohydrate Oxidation During Exercise in Humans and the Role of Pharmacological Activation of PDC in Overriding These Changes

High-fat feeding inhibits pyruvate dehydrogenase complex (PDC)–controlled carbohydrate (CHO) oxidation, which contributes to muscle insulin resistance. We aimed to reveal molecular changes underpinning this process in resting and exercising humans. We also tested whether pharmacological activation of PDC overrides these diet-induced changes. Healthy males consumed a control diet (CD) and on two further occasions an isocaloric high-fat diet (HFD). After each diet, subjects cycled for 60 min after intravenous infusion with saline (CD and HFD) or dichloroacetate (HFD+DCA). Quadriceps muscle biopsies obtained before and after 10 and 60 min of exercise were used to estimate CHO use, PDC activation, and mRNAs associated with insulin, fat, and CHO signaling. Compared with CD, HFD increased resting pyruvate dehydrogenase kinase 2 (PDK2), PDK4, forkhead box class O transcription factor 1 (FOXO1), and peroxisome proliferator–activated receptor transcription factor α (PPARα) mRNA and reduced PDC activation. Exercise increased PDC activation and whole-body CHO use in HFD, but to a lower extent than in CD. Meanwhile PDK4 and FOXO1, but not PPARα or PDK2, mRNA remained elevated. HFD+DCA activated PDC throughout and restored whole-body CHO use during exercise. FOXO1 appears to play a role in HFD-mediated muscle PDK4 upregulation and inhibition of PDC and CHO oxidation in humans. Also, pharmacological activation of PDC restores HFD-mediated inhibition of CHO oxidation during exercise.

Neisseria meningitidis-induced death of cerebrovascular endothelium: mechanisms triggering transcriptional activation of inducible nitric oxide synthase.

The intense host response to meningococcus reflects marked functional and morphological alterations in blood–brain barriers. We showed previously that mouse‐derived cerebrovascular endothelium responded to meningococcal lysates with a robust nitric oxide (NO) response, resulting in the loss of cell viability. To understand how the NO synthase‐2 gene in endothelium is activated by meningococcus, we investigated upstream roles for specific protein kinases. Using known kinase inhibitors, and measuring both mRNA expression and nitrite release, we found MAPK/ERK kinase (MEK)2, p38 kinase and phosphoinositide 3‐kinase (but not MEK1 or phospholipase C) to be implicated in the NO synthase‐2 response. Recruitment of these kinases by meningococcus did not depend on the prior release of the proinflammatory cytokines tumour necrosis factor α or interleukin‐1β from endothelium. These endothelial cells were found to express toll‐like receptors (TLR) 2, 4 and 9 and antibodies directed against TLR 2 and 4 (but not TLR 9) blocked the NO synthase‐2 response to meningococcus. Both meningococcus‐induced translocation of nuclear factor‐kB (NF‐kB) and endothelial cell death were blocked by a known inhibitor of p38 kinase. Calpain inhibitor‐1 blocked the NO synthase‐2 response to meningococcus, which is further evidence of a role for NF‐kB.