NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - but not palmitate-induced toxicity

The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

Stabilizing and destabilizing protein surfactant-based foams in the presence of a chemical surfactant: Effect of adsorption kinetics.

Stimuli-responsive protein surfactants promise alternative foaming materials that can be made from renewable sources. However, the cost of protein surfactants is still higher than their chemical counterparts. In order to reduce the required amount of protein surfactant for foaming, we investigated the foaming and adsorption properties of the protein surfactant, DAMP4, with addition of low concentrations of the chemical surfactant sodium dodecylsulfate (SDS). The results show that the small addition of SDS can enhance foaming functions of DAMP4 at a lowered protein concentration. Dynamic surface tension measurements suggest that there is a synergy between DAMP4 and SDS which enhances adsorption kinetics of DAMP4 at the initial stage of adsorption (first 60s), which in turn stabilizes protein foams. Further interfacial properties were revealed by X-ray reflectometry measurements, showing that there is a re-arrangement of adsorbed protein-surfactant layer over a long period of 1h. Importantly, the foaming switchability of DAMP4 by metal ions is not affected by the presence of SDS, and foams can be switched off by the addition of zinc ions at permissive pH. This work provides fundamental knowledge to guide formulation using a mixture of protein and chemical surfactants towards a high performance of foaming at a low cost.

Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1alpha

The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1alpha therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1alpha and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1alpha and VHL plasmids. Combination therapy resulted in increased losses of HIF-1alpha, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1alpha, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.

Graphene quantum dots induce apoptosis, autophagy, and inflammatory response via p38 mitogen-activated protein kinase and nuclear factor-κB mediated signaling pathways in activated THP-1 macrophages.

The biomedical application of graphene quantum dots (GQDs) is a new emerging area. However, their safety data are still in scarcity to date. Particularly, the effect of GQDs on the immune system remains unknown. This study aimed to elucidate the interaction of GQDs with macrophages and the underlying mechanisms. Our results showed that GQDs slightly affected the cell viability and membrane integrity of macrophages, whereas GQDs significantly increased reactive oxygen species (ROS) generation and apoptotic and autophagic cell death with an increase in the expression level of Bax, Bad, caspase 3, caspase 9, beclin 1, and LC3-I/II and a decrease in that of Bcl-2. Furthermore, low concentrations of GQDs significantly increased the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-8, whereas high concentrations of GQDs elicited opposite effects on the cytokines production. SB202190, a selective inhibitor of p38 mitogen-activated protein kinase (MAPK), abolished the cytokine-inducing effect of GQDs in macrophages. Moreover, GQDs significantly increased the phosphorylation of p38 MAPK and p65, and promoted the nuclear translocation of nuclear factor-κB (NF-κB). Taken together, these results show that GQDs induce ROS generation, apoptosis, autophagy, and inflammatory response via p38MAPK and NF-κB mediated signaling pathways in THP-1 activated macrophages.

Growth of Australian shortfin eel (Anguilla Australis) elvers given different dietary protein and lipid levels

The Australian shortfin eel, Anguilla australis is a potential candidate for intensive aquaculture. The present study was undertaken to evaluate the growth of elvers (5.4 g ± 0.1 initial weight) fed with diets of varying protein and lipid content, and to assess the potential of using soya-bean meal as a dietary ingredient. A 10 week experiment was conducted at 24 (±1.0) °C by rearing fish, in 60 L conical fibre glass tanks using a closed recirculation system. Diets having protein concentrations of 40 or 50% (by dry weight) were tested at three lipid levels (15, 20, 25%); diets being designated P₄₀L₁₅, P₄₀L₂₀, P₄₀L₂₅, P₅₀L₁₅, etc. All these diets contained 5% soya-bean meal. In addition P₅₀L₂₀ diets were formulated to contain 10 and 20% soya-bean meal in the diet (Diets S1 & S2). Shortfin eel grew best on the P₅₀L₁₅ diet, with an average specific growth rate of 2.26%. Food conservation ratio (FCR) and Protein efficiency ratio (PER) ranged from 1.21 (P₅₀L₁₅) to 2.12 (P₄₀L₂₅), and 0.92 (P₅₀L₂₅) to 1.65 (P₅₀L₁₅), respectively. Based on all criteria the best growth performance of shortfin eel was on the P₅₀L₁₅ diet, followed by P₄₀L₂₀ and P₄₀L₁₅. At both protein levels fish reared on diets with 25% lipid performed poorly. The performance of shortfin eel was not affected by the amount of soya-bean meal in the diet, up to a maximum of 20% dietary inclusion. No significant differences in muscle protein were evident in shortfin eel reared on different dietary treatments, nor was the lipid content of muscle related to dietary lipid level.

Performance of juvenile Murray cod, Maccullochella peelii peelii (Mitchell), fed with diets of different protein to energy ratio

The results of a 56-day experiment on juvenile Murray cod, Maccullochella peelii peelii, an Australian native fish with a high aquaculture potential, of mean weight 14.9 ± 0.04 g, fed with five experimental diets, one a series of 40% protein content and lipid levels of 10, 17 and 24% (P40L10, P40L17 and P40L24), and another of 50% protein and 17 and 24% (P50L17 and P50L24) lipid are presented. The specific growth rate (SGR) (% day−1) of fish maintained on different diets ranged from 1.18 to 1.41, and was not significantly different between dietary treatments, except P40L10 and the rest. However, there was a general tendency for SGR to increase with increasing dietary lipid content at both protein levels. The food conversion ratio (FCR) for the 40% protein series diets were poorer compared with those of the 50% protein diets, and the best FCR of 1.14 was observed with the P50L17 diet. The protein efficiency ratio (PER), however, was better in fish reared on low protein diets. The net protein utilization (NPU) also did not differ significantly (P > 0.05) in relation to dietary treatment. As in the case of PER the highest NPU was observed in Murray cod reared on diet P40L24 and the lowest in fish fed with diet P50L24. The carcass lipid content reflected that of the diets, when significant increases in the lipid content was observed in relation to dietary lipid content at both protein levels. However, body muscle lipid content did not increase with increasing dietary lipid content, and was significantly lower than in the whole body. The fatty acids found in highest concentration amongst the saturates, monoenes and polyunsaturates (PUFAs) were 16 : 0, 18 : 1n-9 and 22 : 6n-3, respectively, and each of these accounted for more than 60% of each of the group's total. The muscle fatty acid content was affected by the dietary lipid content; for example the total amount (in μg mg−1 lipid) of monoenes ranged from 72 ± 5.1 (P40L10) to 112 ± 10 (P40L24) and 112 ± 2.8 (P50L17) to 132 ± 11.8 (P50L24) and the n-6 series fatty acids increased with increasing dietary lipid content, although not always significant. Most notably, 18 : 2n-6 increased with the dietary lipid level in both series of diets.

Identification of an integral plasma membrane pool of protein kinase C in mammalian tissues and cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases that are pivotal in cellular regulation. Since its discovery in 1977, PKCs have been known as cytosolic and peripheral membrane proteins. However, there are reports that PKC can insert into phospholipids vesicles in vitro. Given the intimate relationship between the plasma membrane and the activation of PKC, it is important to determine whether such “membrane-inserted” form of PKC exists in mammalian cells or tissues. Here, we report the identification of an integral plasma membrane pool for all the 10 PKC isozymes in vivo by their ability to partition into the detergent-rich phase in Triton X-114 phase partitioning, and by their resistance to extractions with 0.2 M sodium carbonate (pH 11.5), 2 M urea and 2 M sodium chloride. The endogenous integral membrane pool of PKC in mouse fibroblasts is found to be acutely regulated by phorbol ester or diacylglycerol, suggesting that this pool of PKC may participate in cellular processes known to be regulated by PKC. At least for PKCα, the C2–V3 region at the regulatory domain of the kinase is responsible for membrane integration. Further exploration of the function of this novel integral plasma membrane pool of PKC will not only shed new light on molecular mechanisms underlying its cellular functions but also provide new strategies for pharmaceutical modulation of this important group of kinases.

Pituitary adenylate cyclase-activating polypeptide induces translocation of its G-protein-coupled receptor into caveolin-enriched membrane microdomains, leading to enhanced cyclic AMP generation and neurite outgrowth in PC12 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family expressed throughout the nervous system, binds to the PACAP-specific G-protein-coupled receptor family members to promote both neuronal differentiation and survival. Although the PACAP receptor is known to activate its effector protein, adenylate cyclase (AC), and thus enhance cAMP generation, the molecular mechanism utilized by the receptor to activate AC is lacking. Here, we show that PACAP induces neurite outgrowth in PC12 cells by induction of translocation of the PACAP type 1 receptor (PAC1R) into caveolin-enriched Triton X-100-insoluble microdomains, leading to stronger PAC1R-AC interaction and elevated cAMP production. Moreover, we demonstrate that translocation of PAC1R is blocked by various treatments that selectively disrupt caveolae. As a result, intracellular cAMP level is decreased and consequently the PACAP-induced neurite outgrowth retarded. In contrast, addition of exogenous ganglioside GM1 to the cells shows the opposite effects. These results therefore identify the PACAP-induced translocation of its G-protein-coupled receptor into caveolae, where both AC and the regulating G-proteins reside, as the key molecular event in activating AC and inducing cAMP-mediated differentiation of PC12 cells.

Prolonged submaximal exercise induces isoform-specific Na+-K+-ATPase mRNA and protein responses in human skeletal muscle

This study investigated effects of prolonged submaximal exercise on Na⁺-K⁺-ATPase mRNA and protein expression, maximal activity, and content in human skeletal muscle. We also investigated the effects on mRNA expression of the transcription initiator gene, RNA polymerase II (RNAP II), and key genes involved in protein translation, eukaryotic initiation factor-4E (eIF-4E) and 4E-binding protein 1 (4E-BP1). Eleven subjects (6 men, 5 women) cycled at 75.5% (SD 4.8%) peak O₂ uptake and continued until fatigue. A vastus lateralis muscle biopsy was taken at rest, fatigue, and 3 and 24 h postexercise. We analyzed muscle for Na⁺-K⁺-ATPase α1, α2, α3, β1, β2, and β3, as well for RNAP II, eIF-4E, and 4E-BP1 mRNA expression by real-time RT-PCR and Na⁺-K⁺-ATPase isoform protein abundance using immunoblotting. Muscle homogenate maximal Na⁺-K⁺-ATPase activity was determined by 3-O-methylfluorescein phosphatase activity and Na⁺-K⁺-ATPase content by [³H]ouabain binding. Cycling to fatigue [54.5 (SD 20.6) min] immediately increased {alpha}3 (P = 0.044) and {beta}2 mRNA (P = 0.042) by 2.2- and 1.9-fold, respectively, whereas {alpha}1 mRNA was elevated by 2.0-fold at 24 h postexercise (P = 0.036). A significant time main effect was found for α3 protein abundance (P = 0.046). Exercise transiently depressed maximal Na⁺-K⁺-ATPase activity (P = 0.004), but Na⁺-K⁺-ATPase content was unaltered throughout recovery. Exercise immediately increased RNAP II mRNA by 2.6-fold (P = 0.011) but had no effect on eIF-4E and 4E-BP1 mRNA. Thus a single bout of prolonged submaximal exercise induced isoform-specific Na⁺-K⁺-ATPase responses, increasing α1, α3, and β2 mRNA but only α3 protein expression. Exercise also increased mRNA expression of RNAP II, a gene initiating transcription, but not of eIF-4E and 4E-BP1, key genes initiating protein translation.

Expression of uncoupling protein-3 subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its moduclation by fasting

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.

Molecular characterization of Osh6p, an oxysterol binding protein homolog in the yeast Saccharomyces cerevisiae

Oxysterol binding protein (OSBP) and its homologs have been shown to regulate lipid metabolism and vesicular transport. However, the exact molecular function of individual OSBP homologs remains uncharacterized. Here we demonstrate that the yeast OSBP homolog, Osh6p, bound phosphatidic acid and phosphoinositides via its N-terminal half containing the conserved OSBP-related domain (ORD). Using a green fluorescent protein fusion chimera, Osh6p was found to localize to the cytosol and patch-like or punctate structures in the vicinity of the plasma membrane. Further examination by domain mapping demonstrated that the N-terminal half was associated with FM4-64 positive membrane compartments; however, the C-terminal half containing a putative coiled-coil was localized to the nucleoplasm. Functional analysis showed that the deletion of OSH6 led to a significant increase in total cellular ergosterols, whereas OSH6 overexpression caused both a significant decrease in ergosterol levels and resistance to nystatin. Oleate incorporation into sterol esters was affected in OSH6 overexpressing cells. However, Lucifer yellow internalization, and FM4-64 uptake and transport were unaffected in both OSH6 deletion and overexpressing cells. Furthermore, osh6Δ exhibited no defect in carboxypeptidase Y transport and maturation. Lastly, we demonstrated that both the conserved ORD and the putative coiled-coil motif were indispensable for the in vivo function of Osh6p. These data suggest that Osh6p plays a role primarily in regulating cellular sterol metabolism, possibly stero transport.

Reduced Skeletal Muscle Uncoupling Protein-3 Content in Prediabetic Subjects and Type 2 Diabetic Patients: Restoration by Rosiglitazone Treatment

Context: The mitochondrial uncoupling protein-3 (UCP3) has been implicated in the protection of the mitochondrial matrix against lipid-induced mitochondrial damage. Recent evidence points toward mitochondrial aberrations as a major contributor to the development of insulin resistance and diabetes, and UCP3 is reduced in diabetes.
Objective: We compared skeletal muscle UCP3 protein levels in prediabetic subjects [i.e. impaired glucose tolerance (IGT)], diabetic patients, and healthy controls and examined whether rosiglitazone treatment was able to restore UCP3.
Patients, Design, Intervention: Ten middle-aged obese men with type 2 diabetes mellitus [age, 61.4 ± 3.1 yr; body mass index (BMI), 29.8 ± 2.9 kg/m2], nine IGT subjects (age, 59.0 ± 6.6 yr; BMI, 29.7 ± 3.0 kg/m2), and 10 age- and BMI-matched healthy controls (age, 57.3 ± 7.4 yr; BMI, 30.1 ± 3.9 kg/m2) participated in this study. After baseline comparisons, diabetic patients received rosiglitazone (2 x 4 mg/d) for 8 wk.
Main Outcome Measures: Muscle biopsies were sampled to determine UCP3 and mitochondrial protein (complex I–V) content.
Results: UCP3 protein content was significantly lower in prediabetic IGT subjects and in diabetic patients compared with healthy controls (39.0 ± 28.5, 47.2 ± 24.7, and 72.0 ± 23.7 arbitrary units, respectively; P < 0.05), whereas the levels of the mitochondrial protein complex I–V were similar between groups. Rosiglitazone treatment for 8 wk significantly increased insulin sensitivity and muscle UCP3 content (from 53.2 ± 29.9 to 66.3 ± 30.9 arbitrary units; P < 0.05).
Conclusion: We show that UCP3 protein content is reduced in prediabetic subjects and type 2 diabetic patients. Eight weeks of rosiglitazone treatment restores skeletal muscle UCP3 protein in diabetic patients.

Global and targeted gene expression and protein content in skeletal muscle of young men following short-term creatine monohydrate supplementation

Creatine monohydrate (CrM) supplementation has been shown to increase fat-free mass and muscle power output possibly via cell swelling. Little is known about the cellular response to CrM. We investigated the effect of short-term CrM supplementation on global and targeted mRNA expression and protein content in human skeletal muscle. In a randomized, placebo-controlled, crossover, double-blind design, 12 young, healthy, nonobese men were supplemented with either a placebo (PL) or CrM (loading phase, 20 g/day x 3 days; maintenance phase, 5 g/day x 7 days) for 10 days. Following a 28-day washout period, subjects were put on the alternate supplementation for 10 days. Muscle biopsies of the vastus lateralis were obtained and were assessed for mRNA expression (cDNA microarrays + real-time PCR) and protein content (Kinetworks KPKS 1.0 Protein Kinase screen). CrM supplementation significantly increased fat-free mass, total body water, and body weight of the participants (P < 0.05). Also, CrM supplementation significantly upregulated (1.3- to 5.0-fold) the mRNA content of genes and protein content of kinases involved in osmosensing and signal transduction, cytoskeleton remodeling, protein and glycogen synthesis regulation, satellite cell proliferation and differentiation, DNA replication and repair, RNA transcription control, and cell survival. We are the first to report this large-scale gene expression in the skeletal muscle with short-term CrM supplementation, a response that suggests changes in cellular osmolarity.