Effect of short-time treatment with TNF-α on stem cell activity and barrier function in enteroids

Although tumor necrosis factor-α (TNF-α) is a known major inflammatory mediator in inflammatory bowel disease (IBD) and has various effects on intestinal epithelial cell (IEC) homeostasis, the changes in IECs in the early inflammatory state induced during short-time treatment (24 h) with TNF-α remain unclear. In this study, we investigated TNF-α-induced alterations in IECs in the early inflammatory state using mouse jejunal organoids (enteroids). Of the inflammatory cytokines, i.e., TNF-α, IL-1β, IL-6, and IL-17, only TNF-α markedly increased the mRNA level of macrophage inflammatory protein 2 (MIP-2; the mouse homologue of interleukin-8), which is induced in the early stages of inflammation. TNF-α stimulation (3 h and 6 h) decreased the mRNA level of the stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and polycomb group ring finger 4 and the progenitor cell marker prominin-1, which is also known as CD133. In addition, TNF-α treatment (24 h) decreased the number of Lgr5-positive cells and enteroid proliferation. TNF-α stimulation at 3 h and 6 h also decreased the mRNA level of chromogranin A and mucin 2, which are respective markers of enteroendocrine and goblet cells. Moreover, enteroids treated with TNF-α (24 h) not only decreased the integrity of tight junctions and cytoskeletal components but also increased intercellular permeability in an influx test with fluorescent dextran, indicating disrupted intestinal barrier function. Taken together, our findings indicate that short-time treatment with TNF-α promotes the inflammatory response and decreases intestinal stem cell activity and barrier function.

     

Exercise training changes IL-10/TNF-α ratio in the skeletal muscle of post-MI rats

Heart failure (HF) is associated with changes in the skeletal muscle (SM) which might be a consequence of the unbalanced local expression of pro- (TNF-α) and anti- (IL-10) inflammatory cytokines, leading to inflammation-induced myopathy, and SM wasting. This local effect of HF on SM may, on the other hand, contribute to systemic inflammation, as this tissue actively secretes cytokines. Since increasing evidence points out to an anti-inflammatory effect of exercise training, the goal of the present study was to investigate its effect in rats with HF after post-myocardial infarction (MI), with special regard to the expression of TNF-α and IL-10 in the soleus and extensor digitorum longus (EDL), muscles with different fiber composition. Wistar rats underwent left thoracotomy with ligation of the left coronary artery, and were randomly assigned to either a sedentary (Sham-operated and MI sedentary) or trained (Sham-operated and MI trained) group. Animals in the trained groups ran on a treadmill (0% grade at 13–20 m/min) for 60 min/day, 5 days/week, for 8–10 weeks. The training protocol was able to reverse the changes induced by MI, decreasing TNF-α protein (26%, P < 0.05) and mRNA (58%, P < 0.05) levels in the soleus, when compared with the sedentary MI group. Training also increased soleus IL-10 expression (2.6-fold, P < 0.001) in post-MI HF rats. As a consequence, the IL-10/TNF-α ratio was increased. This “anti-inflammatory effect” was more pronounced in the soleus than in the EDL, suggesting a fiber composition dependent response.     

Effect of prostaglandin F2α on anterior pituitary function in man

Five healthy adult men received iv PGF_2α at dosages of 0.05, 0.20 and 2.0 μg/kg/min for 30 min. There were no significant changes in serum FSH, LH or TSH levels. Serum GH and cortisol levels were slightly increased at the highest dosage. These responses were associated with, and presumably a result of, stressful side effects. Thus, PGF_2α cannot be used as a provocative test of pituitary hormone reserve.Prostaglandins (PG's) have recently been implicated in the release of a number of hormones from the anterior pituitary gland. The stimulation of GH release by PG's of the E series from incubated rat pituitary slices has been demonstrated. $\text{In vivo}$ stimulation by PGE₁ of ACTH in rats and of GH release in man has also been shown.The present study was undertaken in order to examine the efficacy of iv administration of PGF_2α as a provocative test of anterior pituitary hormone reserve in man. The responses in circulating levels of gonadotropins, TSH, GH, and cortisol (as an index of ACTH) were measured.     

Transgenic overexpression of LARGE induces α-dystroglycan hyperglycosylation in skeletal and cardiac muscle

Background

LARGE is one of seven putative or demonstrated glycosyltransferase enzymes defective in a common group of muscular dystrophies with reduced glycosylation of α-dystroglycan. Overexpression of LARGE induces hyperglycosylation of α-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, restoring functional glycosylation. Viral delivery of LARGE to skeletal muscle in animal models of dystroglycanopathy has identical effects in vivo, suggesting that the restoration of functional glycosylation could have therapeutic applications in these disorders. Pharmacological strategies to upregulate Large expression are also being explored.

Methodology/Principal Findings

In order to asses the safety and efficacy of long term LARGE over-expression in vivo, we have generated four mouse lines expressing a human LARGE transgene. On observation, LARGE transgenic mice were indistinguishable from the wild type littermates. Tissue analysis from young mice of all four lines showed a variable pattern of transgene expression: highest in skeletal and cardiac muscles, and lower in brain, kidney and liver. Transgene expression in striated muscles correlated with α-dystroglycan hyperglycosylation, as determined by immunoreactivity to antibody IIH6 and increased laminin binding on an overlay assay. Other components of the dystroglycan complex and extracellular matrix ligands were normally expressed, and general muscle histology was indistinguishable from wild type controls. Further detailed muscle physiological analysis demonstrated a loss of force in response to eccentric exercise in the older, but not in the younger mice, suggesting this deficit developed over time. However this remained a subclinical feature as no pathology was observed in older mice in any muscles including the diaphragm, which is sensitive to mechanical load-induced damage.

Conclusions/Significance

This work shows that potential therapies in the dystroglycanopathies based on LARGE upregulation and α-dystroglycan hyperglycosylation in muscle should be safe.     

Effects of long-term taurine supplementation on age-related changes in skeletal muscle function of Sprague–Dawley rats

Taurine (2-aminoethanesulfonic acid) is a free amino acid found abundantly in mammalian tissues. Increasing evidence suggests that taurine plays a role in the maintenance of skeletal muscle function and increase of exercise capacity. Most energy drinks contain this amino acid; however, there is insufficient research on the effects of long-term, low-dose supplementation of taurine. In this study, we investigated the effects of long-term administration of taurine at low doses on aging in rodents. In Experiment 1, we examined age-related changes in aging Sprague–Dawley (SD) rats (32–92 weeks old) that O₂ consumption and spontaneous activity decreased significantly with aging. In Experiment 2, we examined the effects of long-term (21-week) administration of taurine on healthy aging SD rats. SD rats were stabilized for 32–34 weeks and divided into three groups, administrated water (control), 0.5% taurine (25 mg/kg  body weight (BW)/day), or 1% taurine (50 mg/kg  BW/day) from age 34 to 56 weeks (5 days/week, 5 mL/kg BW). Our findings suggest that long-term administration of taurine at relatively low dose could attenuate the age-related decline in O₂ consumption and spontaneous locomotor activity. Upon intestinal absorption, taurine might modulate age-related changes in respiratory metabolism and skeletal muscle function via peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), succinate dehydrogenase (SDH), cytochrome c (Cycs), myocyte enhancer factor 2A (MEF2A), glucose transporter 4 (GLUT4), and myoglobin, which are regulated by the activation of AMP-activated protein kinase (AMPK). This article examines the mechanism underlying the effects of taurine on age-related changes, which may have potential clinical implications.

     

Effect of 1,25 dihydroxyvitamin D3 on intracellular IFN-γ and TNF-α positive T cell subsets in pulmonary tuberculosis

We studied the immunomodulatory effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α cytokines in T cell subsets of pulmonary tuberculosis (PTB) patients (n = 22) and normal healthy subjects (n = 22). Peripheral blood mononuclear cells (PBMCs) were cultured with live Mycobacterium tuberculosis (MTB) with or without 1,25(OH)₂D₃ (10^?7 M) for 48 h. T cell subsets positive for IFN-γ and TNF-α were enumerated by flow cytometry and the culture supernatants were assayed for both the cytokines using ELISA. In both NHS and PTB patients, a significantly reduced percentage of IFN-γ and TNF-α expressing CD3+, CD3+CD4+ and CD3+CD8+ T cells were observed in cultures stimulated with live MTB and treated with 1,25(OH)₂D₃ compared to cultures without 1,25(OH)₂D₃ (NHS; CD3+ IFN-γ+: p < 0.0001; CD3+TNF-α +: p = 0.0292 and PTB; CD3+ IFN-γ+: p = 0.0292; CD3+ TNF-α +: p = 0.0028). The levels of IFN-γ and TNF-α in the culture supernatants of 1,25(OH)₂D₃ treated cultures were also found to be significantly decreased in both groups (NHS; IFN-γ: p = 0.0001; TNF-α: p < 0.0001) and (PTB; IFN-γ: p < 0.0001; TNF-α: p < 0.0001). A positive correlation was observed between IFN-γ and TNF-α expressing CD3+CD8+ T cells in MTB stimulated cultures treated with or without 1,25(OH)₂D₃ in NHS (p = 0.0001; p = 0.001, respectively) and PTB patients (p = 0.002; p = 0.005, respectively). The present study revealed the suppressive effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α by CD3+CD4+ and CD3+CD8+ T cells in pulmonary tuberculosis. This suppressive effect of 1,25(OH)₂D₃ on proinflammatory and Th1 cytokine positive cells might have a role in reducing inflammation at the site of infection.     

The role of PGC-1α in the regulation of skeletal muscle metabolism

The purpose of this review was to provide an understanding of the role of PGC-1α in the regulation of skeletal muscle metabolism and to describe the results of studies on the association of the polymorphism gene PPARGC1A with human muscle performance.     

Effect of exercise intensity and AICAR on isoform-specific expressions of murine skeletal muscle PGC-1α mRNA: a role of β₂-adrenergic receptor activation

There are three isoforms of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) mRNA, which promotes mitochondrial biogenesis in skeletal muscles. Compared with PGC-1α-a mRNA, PGC-1α-b or PGC-1α-c mRNA is transcribed by a different exon 1 of the PGC-1α gene. In this study, effects of exercise intensity and 5-aminoimidazole-4-carboxamide-1β-d-ribofuranoside (AICAR) on isoform-specific expressions of PGC-1α were investigated. All isoforms were increased in proportion to exercise intensity of treadmill running (10-30 m/min for 30 min). Preinjection of β?-adrenergic receptor (AR) antagonist (ICI 118551) inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs, but not the increase in PGC-1α-a mRNA, in response to high-intensity exercise. Although high-intensity exercise activated α2-AMP-activated protein kinase (α2-AMPK) in skeletal muscles, inactivation of α2-AMPK activity did not affect high-intensity exercise-induced mRNA expression of all PGC-1α isoforms, suggesting that activation of α2-AMPK is not mandatory for an increase in PGC-1α mRNA by high-intensity exercise. A single injection in mice of AICAR, an AMPK activator, increased mRNAs of all PGC-1α isoforms. AICAR increased blood catecholamine concentrations, and preinjection of β?-AR antagonist inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs but not the increase in PGC-1α-a mRNA. Direct exposure of epitrochlearis muscle to AICAR increased PGC-1α-a but not the -b isoform. These data indicate that exercise-induced PGC-1α expression was dependent on the intensity of exercise. Exercise or AICAR injection increased PGC-1α-b and PGC-1α-c mRNAs via β?-AR activation, whereas high-intensity exercise increased PGC-1α-a expression by a multiple mechanism in which α2-AMPK is one of the signaling pathways.     

The effect of SIRT1 protein knock down on PGC-1α acetylation during skeletal muscle contraction

[Purpose]

The purpose of this study was to investigate the effect of Sirtuin 1 (SIRT1) and General control nonderepressible 5 (GCN5) knock down on peroxisome proliferator- activated receptor gamma coactivator 1-alpha (PGC-1α) deacetylation during electrical stimulated skeletal muscle contraction.

[Methods]

Skeletal muscle primary cell were isolated from C57BL/6 mice gastrocnemius and transfected lentiviral SIRT1 and GCN5 shRNA. Knock downed muscle cell were stimulated by electrical stimulation (1Hz, 3min) and collected for PGC-1α deceatylation assays. Immunoprecipitation performed for PGC-1α deacetylation, acetyl-lysine level was measured.

[Results]

Our resulted showed SIRT1 knock down not influenced to PGC-1α deacetylation during electrical stimulation induced muscle contraction while GCN5 knock down decreased PGC-1α deacetylation significantly (p<0.05).

[Conclusion]

This study can be concluded that GCN5 is a critical factor for muscle contraction induced PGC-1α deacetylation.     

Effect ofγ-irradiated carbohydrates on isolated synaptic membranes

The effect of-irradiated solutions of carbohydrates, mainly glucose, upon Na⁺, K⁺-ATPase and lipid peroxidation in rat brain synaptosomal membranes was studied. The membrane damage by irradiated glucose was enhanced in the presence of Fe²⁺ and was diminished when a free-radical scavenger (BHT) or metal chelators (EDTA, EGTA) were present. It is suggested that a key element in the free-radical membrane damage by irradiated carbohydrates is an Fe²⁺-complex of some species of the radiolysis products. Participation of radiotoxins of carbohydrate origin in radiobiological effects is discussed.     

Rescue of dystrophic skeletal muscle by PGC-1α involves a fast to slow fiber type shift in the mdx mouse

Increased utrophin expression is known to reduce pathology in dystrophin-deficient skeletal muscles. Transgenic over-expression of PGC-1α has been shown to increase levels of utrophin mRNA and improve the histology of mdx muscles. Other reports have shown that PGC-1α signaling can lead to increased oxidative capacity and a fast to slow fiber type shift. Given that it has been shown that slow fibers produce and maintain more utrophin than fast skeletal muscle fibers, we hypothesized that over-expression of PGC-1α in post-natal mdx mice would increase utrophin levels via a fiber type shift, resulting in more slow, oxidative fibers that are also more resistant to contraction-induced damage. To test this hypothesis, neonatal mdx mice were injected with recombinant adeno-associated virus (AAV) driving expression of PGC-1α. PGC-1α over-expression resulted in increased utrophin and type I myosin heavy chain expression as well as elevated mitochondrial protein expression. Muscles were shown to be more resistant to contraction-induced damage and more fatigue resistant. Sirt-1 was increased while p38 activation and NRF-1 were reduced in PGC-1α over-expressing muscle when compared to control. We also evaluated if the use a pharmacological PGC-1α pathway activator, resveratrol, could drive the same physiological changes. Resveratrol administration (100 mg/kg/day) resulted in improved fatigue resistance, but did not achieve significant increases in utrophin expression. These data suggest that the PGC-1α pathway is a potential target for therapeutic intervention in dystrophic skeletal muscle.     

Vanadate as an activator of ATP — sensitive potassium channels in mouse skeletal muscle

The inside-out mode of the patch-clamp technique was used to study adenosine-5-triphosphate (ATP)-sensitive K⁺ channels in mammalian skeletal muscle. Vanadate, applied to the cytoplasmic face of excised patches, was a potent activator of ATP-sensitive K⁺ channels. Divalent cations (Mg²⁺, Ca²⁺) were a prerequisite for the activating process. The maximal effect was achieved using 1 mM vanadate dissolved in Ringer, increasing the open-state probability about ninefold. The active 5 + redox form of vanadate which stimulates ATP-sensitive K⁺ channels is likely to be decavanadate V₁₀O _inf28 ^sup6– . ATP concentration-response curves have Hill coefficients near three in internal Na⁺-rich Ringer and between one and two in internal KCl solutions. Half maximal channel blockage was observed at ATP concentrations of 4 and 8 M in Ringer and KCl solutions, respectively. Internal vanadate shifted the curves towards higher ATP concentrations without affecting their slopes. Thus 50% channel blockage occurred at 65 M ATP in internal Ringer containing 0.5 mM vanadate. The results indicate that the affinity and stoichiometry of ATP binding to ATP-sensitive K⁺ channels are strongly modulated by internal cations and that the ATP sensitivity is weakened by vanadate. Offprint requests to: B. Neumcke     

The α-subunit of the skeletal muscle sodium channel is encoded proximal to Tk-1 on mouse Chromosome 11

Recent evidence suggests that the human neuromuscular disorders, hyperkalemic periodic paralysis and paramyotonia congenita, are both caused by genetic defects in the -subunit of the adult skeletal muscle sodium channel, which maps near the growth hormone cluster (GH) on Chromosome (Chr) 17q. In view of the extensive homology between this human chromosome and mouse Chr 11, we typed an interspecies backcross to determine whether the murine homolog (Scn4a) of this sodium channel gene mapped within the conserved chromosomal segment. The cytosolic thymidine kinase gene, Tk-1, was also positioned on the genetic map of Chr 11. Both Scn4a and Tk-1 showed clear linkage to mouse Chr 11 loci previously typed in this backcross, yielding the map order: Tr ^J-(Re, Hox-2, Krt-1)-Scn4a-Tk-1. No mouse mutant that could be considered a model of either hyperkalemic periodic paralysis or paramyotonia congenita has been mapped to the appropriate region of mouse Chr 11. These data incorporate an additional locus into the already considerable degree of homology observed for these human and mouse chromosomes. These data are also consistent with the view that the conserved segment region may extend to the telomere on mouse Chr 11 and on human 17q.     

Ectopic overexpression of swine PPARγ2 upregulated adipocyte genes expression and triacylglycerol in skeletal muscle of mice

Peroxisome proliferator-activated receptor ??2 (PPAR??2) is a key regulator of adipocyte differentiation, fatty acid uptake and storage in mammals. The primary goal of the present study was to investigate the consequences of PPAR??2 overexpression in the muscle. A swine muscle creatine kinase promoter was used to drive swine PPAR??2 (sPPAR??2) overexpression in the muscle of a transgenic mice model. The results showed that the mRNA of multiple adipocyte genes was increased in the skeletal muscle, as evidenced by the up-regulation of fatty acid synthase (2.11-fold, P?<?0.05), lipoprotein lipase (2.08-fold, P?<?0.01), fatty acid-binding protein 4 (14.30-fold, P?<?0.01), and CD36 antigen (5.50-fold, P?<?0.01). Meanwhile, skeletal muscle triacylglycerol was increased (P?<?0.01) and the fatty acid profile of muscle fat was changed in that more polyunsaturated fats acid were augmented. The present study may further serve to develop transgenic pigs with higher intramuscular fat content and improved pork quality.     

Effects of (−)-epicatechin on molecular modulators of skeletal muscle growth and differentiation

Sarcopenia is a notable and debilitating age-associated condition. Flavonoids are known for their healthy effects and limited toxicity. The flavanol (−)-epicatechin (Epi) enhances exercise capacity in mice, and Epi-rich cocoa improves skeletal muscle structure in heart failure patients. (−)-Epicatechin may thus hold promise as treatment for sarcopenia.We examined changes in protein levels of molecular modulators of growth and differentiation in young vs. old, human and mouse skeletal muscle. We report the effects of Epi in mice and the results of an initial proof-of-concept trial in humans, where muscle strength and levels of modulators of muscle growth were measured. In mice, myostatin and senescence-associated β-galactosidase levels increase with aging, while those of follistatin and Myf5 decrease. (−)-Epicatechin decreases myostatin and β-galactosidase and increases levels of markers of muscle growth. In humans, myostatin and β-galactosidase increase with aging while follistatin, MyoD and myogenin decrease. Treatment for 7 days with (−)-epicatechin increases hand grip strength and the ratio of plasma follistatin/myostatin.In conclusion, aging has deleterious effects on modulators of muscle growth/differentiation, and the consumption of modest amounts of the flavanol (−)-epicatechin can partially reverse these changes. This flavanol warrants its comprehensive evaluation for the treatment of sarcopenia.     

Pathways of Ca²⁺ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle

Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.     

Elevated concentration of TNF-α induces trophoblast differentiation in mouse blastocyst outgrowths

Elevated expression of tumour necrosis factor- (TNF-) is associated with adverse pregnancy outcome. This study has examined the expression of TNF- and its receptors (TNF-Rs) by mouse blastocysts and blastocyst outgrowths from day 4 to 9.5 of pregnancy and investigated the effects of elevated TNF- on the inner cell mass (ICM) and trophoblast cells of blastocyst outgrowths. RT-PCR demonstrated TNF- mRNA expression from day 7.5 to 9.5, TNF-R1 from day 6.5 to 9.5 and TNF-R2 from day 5.5 to 7.5 of pregnancy, and in situ hybridisation revealed the trophoblast giant cells (TGCs) of the early placenta as the site of TNF- expression. Day 4 blastocysts were cultured in a physiologically high concentration of TNF- (100 ng/ml) for 72 h to the outgrowth stage and then compared to blastocysts cultured in media alone. TNF--treated blastocyst outgrowths exhibited a significant reduction in ICM cells (mean ± SD 23.90±10.42 vs 9.37±7.45, t-test, P<0.0001) with no significant change in the numbers of trophoblast cells (19.97±8.14 vs 21.73±7.79, t-test, P=0.39). Within the trophoblast cell population, the TNF--treated outgrowths exhibited a significant increase in multinucleated cells (14.10±5.53 vs 6.37±5.80, t-test, P<0.0001) and a corresponding significant decrease in mononucleated cells (5.87±3.60 vs 15.37±5.87, t-test, P<0.0001). In summary, this study describes the expression of TNF- and its receptors during the peri-implantation period in the mouse. It also reports that elevated TNF- restricts ICM proliferation in the blastocyst and changes the ratio of mononucleated to multinucleated trophoblast cells. These findings suggest a mechanism by which increased expression of TNF- during trophoblast differentiation may be detrimental to pregnancy.This work was supported by the National Health and Medical Research Council of Australia     

The effect of transcutaneous application of carbon dioxide (CO₂) on skeletal muscle

Quasispecies is a remarkable characteristic of hepatitis C virus (HCV) and has profound roles in HCV biology and clinical practice. The understanding of HCV quasispecies behavior, in particular in acute HCV infection, is valuable for vaccine development and therapeutic interference. However, acute HCV infection is seldom encountered in clinic practice due to its silent onset. In the present study, we reported a unique case of de novo HCV infection associated with the transplantation of bone marrow from a HCV-positive donor. HCV quasispecies diversity was determined in both the donor and the recipient over a 4-year follow-up, accompanied with simultaneous measurement of HCV neutralizing antibody. Detailed genetic and phylogenetic analyses revealed a divergent quasispecies evolution, which was not related to dynamic changes of HCV neutralizing antibody. Instead, our data suggested an essential role of the fitness adaptation of founder viral population in driving such an evolutionary pattern.