Effects of creatine supplementation on muscle wasting and glucose homeostasis in rats treated with dexamethasone

We aimed to investigate the possible role of creatine (CR) supplementation in counteracting dexamethasone-induced muscle wasting and insulin resistance in rats. Also, we examined whether CR intake would modulate molecular pathways involved in muscle remodeling and insulin signaling. Animals were randomly divided into four groups: (1) dexamethasone (DEX); (2) control pair-fed (CON-PF); (3) dexamethasone plus CR (DEX-CR); and (4) CR pair-fed (CR-PF). Dexamethasone (5 mg/kg/day) and CR (5 g/kg/day) were given via drinking water for 7 days. Plantaris and extensor digitorum longus (EDL) muscles were removed for analysis. Plantaris and EDL muscle mass were significantly reduced in the DEX-CR and DEX groups when compared with the CON-PF and CR-PF groups (P < 0.05). Dexamethasone significantly decreased phospho-Ser(473)-Akt protein levels compared to the CON-PF group (P < 0.05) and CR supplementation aggravated this response (P < 0.001). Serum glucose was significantly increased in the DEX group when compared with the CON-PF group (DEX 7.8 +/- A 0.6 vs. CON-PF 5.2 +/- A 0.5 mmol/l; P < 0.05). CR supplementation significantly exacerbated hyperglycemia in the dexamethasone-treated animals (DEX-CR 15.1 +/- A 2.4 mmol/l; P < 0.05 vs. others). Dexamethasone reduced GLUT-4 translocation when compared with the CON-PF and CR-PF (P < 0.05) groups and this response was aggravated by CR supplementation (P < 0.05 vs. others). In conclusion, supplementation with CR resulted in increased insulin resistance and did not attenuate muscle wasting in rats treated with dexamethasone. Given the contrast with the results of human studies that have shown benefits of CR supplementation on muscle atrophy and insulin sensitivity, we suggest caution when extrapolating this animal data to human subjects.

Increased hepcidin expression in multibacillary leprosy

Iron is essential for all organisms and its availability can control the growth of microorganisms; therefore, we examined the role of iron metabolism in multibacillary (MB) leprosy, focusing on the involvement of hepcidin. Erythrograms, iron metabolism parameters, pro-inflammatory cytokines and urinary hepcidin levels were evaluated in patients with MB and matched control subjects. Hepcidin expression in MB lesions was evaluated by quantitative polymerase chain reaction. The expression of ferroportin and hepcidin was evaluated by immunofluorescence in paucibacillary and MB lesions. Analysis of hepcidin protein levels in urine and of hepcidin mRNA and protein levels in leprosy lesions and skin biopsies from healthy control subjects showed elevated hepcidin levels in MB patients. Decreases in haematologic parameters and total iron binding capacity were observed in patients with MB leprosy. Moreover, interleukin-1 beta, ferritin, soluble transferrin receptor and soluble transferrin receptor/log ferritin index values were increased in leprosy patients. Hepcidin was elevated in lepromatous lesions, whereas ferroportin was more abundant in tuberculoid lesions. In addition, hepcidin and ferroportin were not colocalised in the biopsies from leprosy lesions. Anaemia was not commonly observed in patients with MB; however, the observed changes in haematologic parameters indicating altered iron metabolism appeared to result from a mixture of anaemia of inflammation and iron deficiency. Thus, iron sequestration inside host cells might play a role in leprosy by providing an optimal environment for the bacillus.

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

In this study, we evaluated the effects of obesity and insulin resistance induced by a high-fat diet on prostate morphophysiology, focusing on cell proliferation, expression of androgen (AR) and estrogen receptors (ER) and proteins of the insulin signaling pathway. Adult male Wistar rats were fed a high-fat diet (20% fat) for 15 weeks, whereas control animals received a balanced diet (4% fat). Both groups were then divided and treated for 2 weeks with 1 mg/kg body weight/day of the aromatase inhibitor letrozole or vehicle only. The ventral prostate was analyzed with immunohistochemical, histopathological, stereological, and Western blotting methods. Obese rats showed insulin resistance, hyperinsulinemia, and reduced plasma testosterone levels. The incidence of prostatic intraepithelial neoplasia (PIN) was 2.7 times higher in obese rats and affected 0.4% of the gland compared with 0.1% PIN areas found in control rats. Obesity doubled cell proliferation in both prostate epithelium and stroma. AR content decreased in the prostate of obese rats and estrogen receptor beta (ER beta) increased in this group. Protein levels of insulin receptor substrate 1 and protein kinase B diminished in the obese group, whereas phosphatidylinositol 3-kinase (PI3K) increased significantly. Most structural changes observed in the prostate of obese rats normalized after letrozole treatment, except for increased stromal cell proliferation and ER beta expression, which might be associated with insulin resistance. This experimental model of obesity and insulin resistance induced by a high-fat diet increases cell proliferation in rat prostate. Such alterations are associated with decreased levels of AR and increased ER beta and PI3K proteins. This change can facilitate the establishment of proliferative lesions in rat prostate.

Heterogeneous time-dependent response of adipose tissue during the development of cancer cachexia

Cancer cachexia induces loss of fat mass that accounts for a large part of the dramatic weight loss observed both in humans and in animal models; however, the literature does not provide consistent information regarding the set point of weight loss and how the different visceral adipose tissue depots contribute to this symptom. To evaluate that, 8-week-old male Wistar rats were subcutaneously inoculated with 1 ml (2 x 10(7)) of tumour cells (Walker 256). Samples of different visceral white adipose tissue (WAT) depots were collected at days 0, 4, 7 and 14 and stored at -80 degrees C (seven to ten animals/each day per group). Mesenteric and retroperitoneal depot mass was decreased to the greatest extent on day 14 compared with day 0. Gene and protein expression of PPAR gamma(2) (PPARG) fell significantly following tumour implantation in all three adipose tissue depots while C/EBP alpha (CEBPA) and SREBP-1c (SREBF1) expression decreased over time only in epididymal and retroperitoneal depots. Decreased adipogenic gene expression and morphological disruption of visceral WAT are further supported by the dramatic reduction in mRNA and protein levels of perilipin. Classical markers of inflammation and macrophage infiltration (f4/80, CD68 and MIF-1 alpha) in WAT were significantly increased in the later stage of cachexia (although showing a incremental pattern along the course of cachexia) and presented a depot-specific regulation. These results indicate that impairment in the lipid-storing function of adipose tissue occurs at different times and that the mesenteric adipose tissue is more resistant to the 'fat-reducing effect' than the other visceral depots during cancer cachexia progression. Journal of Endocrinology (2012) 215, 363-373

Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1

ADAM17, which is also known as TNF alpha-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.

Ethanol Consumption Alters the Expression and Reactivity of Adrenomedullin in the Rat Mesenteric Arterial Bed

Aims: Adrenomedullin (AM) is a peptide that displays cardiovascular protective activity. We investigated the effects of chronic ethanol consumption on arterial blood pressure, vascular reactivity to AM and the expression of AM system components in the rat mesenteric arterial bed (MAB). Methods: Male Wistar rats were treated with ethanol (20% vol/vol) for 6 weeks. Systolic, diastolic and mean arterial blood pressure were monitored in conscious rats. Vascular reactivity experiments were performed on isolated rat MAB. Matrix metalloproteinase-2 (MMP-2) levels were determined by gelatin zymography. Nitrite and nitrate generation were measured by chemiluminescence. Protein and mRNA levels of pre-pro-AM, CRLR (calcitonin receptor-like receptor) and RAMP1, 2 and 3 (receptor activity-modifying proteins) were assessed by western blot and quantitative real-time polymerase chain reaction, respectively. Results: Ethanol consumption induced hypertension and decreased the relaxation induced by AM and acetylcholine in endothelium-intact rat MAB. Phenylephrine-induced contraction was increased in endothelium-intact MAB from ethanol-treated rats. Ethanol consumption did not alter basal levels of nitrate and nitrite, nor did it affect the expression of MMP-2 or the net MMP activity in the rat MAB. Ethanol consumption increased mRNA levels of pre-pro-AM and protein levels of AM in the rat MAB. Finally, no differences in protein levels or mRNA of CRLR and RAMP1, 2 and 3 were observed after treatment with ethanol. Conclusion: Our study demonstrates that ethanol consumption increases blood pressure and the expression of AM in the vasculature and reduces the relaxation induced by this peptide in the rat MAB.

Endurance training stimulates growth and survival pathways and the redox balance in rat pancreatic islets

Calegari VC, Abrantes JL, Silveira LR, Paula FM, Costa JM Jr, Rafacho A, Velloso LA, Carneiro EM, Bosqueiro JR, Boschero AC, Zoppi CC. Endurance training stimulates growth and survival pathways and the redox balance in rat pancreatic islets. J Appl Physiol 112: 711-718, 2012. First published December 15, 2011; doi:10.1152/japplphysiol.00318.2011.-Endurance training has been shown to increase pancreatic beta-cell function and mass. However, whether exercise modulates beta-cell growth and survival pathways signaling is not completely understood. This study investigated the effects of exercise on growth and apoptotic markers levels in rat pancreatic islets. Male Wistar rats were randomly assigned to 8-wk endurance training or to a sedentary control group. After that, pancreatic islets were isolated; gene expression and the total content and phosphorylation of several proteins related to growth and apoptotic pathways as well as the main antioxidant enzymes were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Reactive oxygen species (ROS) production was measured by fluorescence. Endurance training increased the time to reach fatigue by 50%. Endurance training resulted in increased protein phosphorylation content of AKT (75%), AKT substrate (AS160; 100%), mTOR (60%), p70s6k (90%), and ERK1/2 (50%), compared with islets from control group. Catalase protein content was 50% higher, whereas ROS production was 49 and 77% lower in islets from trained rats under basal and stimulating glucose conditions, respectively. Bcl-2 mRNA and protein levels increased by 46 and 100%, respectively. Bax and cleaved caspase-3 protein contents were reduced by 25 and 50% in islets from trained rats, respectively. In conclusion, these results demonstrate that endurance training favors the beta-cell growth and survival by activating AKT and ERK1/2 pathways, enhancing antioxidant capacity, and reducing ROS production and apoptotic proteins content.

Fibronectin expression is decreased in metastatic renal cell carcinoma following endostatin gene therapy

Tumor cells induce the disruption of homeostasis between cellular and extracellular compartments to favor tumor progression. The expression of fibronectin (FN), a matrix glycoprotein, is increased in several carcinoma cell types, including renal cell carcinoma (RCC). RCC are highly vascularized tumors and are often amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the modulation of FN gene expression by ES gene therapy in a murine metastatic renal cell carcinoma (mRCC) model. Balb/C mice bearing Renca cells were treated with NIH/3T3-LXSN cells or NIH/3T3-LendSN cells. At the end of the experiment, the ES serum levels were measured, and the FN gene expression was assessed using real-time PCR. The tissue FN was evaluated by western blotting and by immunofluorescence analysis. The ES serum levels in treated mice were higher than those in the control group (P < 0.05). ES treatment led to significant decreases at the FN mRNA (P < 0.001) and protein levels (P < 0.01). Here, we demonstrate the ES antitumor effect that is mediated by down-regulation of FN expression in mRCC. (C) 2012 Elsevier Masson SAS. All rights reserved.

Mannose-binding lectin and MBL-associated serine protease-2 gene polymorphisms in a Brazilian population from Rio de Janeiro

Mannose-binding lectin (MBL) is a protein able to bind to carbohydrate patterns on pathogen membranes; upon MBL binding, its associated serine protease MBL-associated serine protease type 2 (MASP2) is autoactivated, promoting the activation of complement via the lectin pathway. For both MBL2 and MASP2 genes, the frequencies of polymorphisms are extremely variable between different ethnicities, and this aspect has to be carefully considered when performing genetic studies. While polymorphisms in the MBL-encoding gene (MBL2) have been associated, depending upon ethnicity, with several diseases in different populations, little is known about the distribution of MASP2 gene polymorphisms in human populations. The aim of our study was thus to determine the frequencies of MBL2 (exon 1 and promoter) and MASP2 (p.D371Y) polymorphisms in a Brazilian population from Rio de Janeiro. A total of 294 blood donor samples were genotyped for 27 polymorphisms in the MBL2 gene by direct sequencing of a region spanning from the promoter polymorphism H/L rs11003125 to the rs1800451 polymorphism (at codon 57 in the first exon of the gene). Genotyping for MASP2 p.D371Y was carried out using fluorogenic probes. To our knowledge, this is the first study reporting the prevalence of the MASP2 p.D371Y polymorphism in a Brazilian population. The C allele frequency 39% is something intermediate between the reported 14% in Europeans and 90% in Sub-Saharan Africans. MBL2 polymorphisms frequencies were quite comparable to those previously reported for admixed Brazilians. Both MBL2 and MASP2 polymorphisms frequencies reported in our study for the admixed Brazilian population are somehow intermediate between those reported in Europeans and Africans, reflecting the ethnic composition of the southern Brazilian population, estimated to derive from an admixture of Caucasian (31%), African (34%) and Native American (33%) populations. In conclusion, our population genetic study describes the frequencies of MBL2 and MASP2 functional SNPs in a population from Rio de Janeiro, with the aim of adding new information concerning the distribution of these SNPs in a previously unanalysed Brazilian population, thus providing a new genetic tool for the evaluation of the association of MBL2 and MASP2 functional SNPs with diseases in Brazil, with particular emphasis on the state of Rio de Janeiro.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

Vectorial signalling mechanism required for cell-cell communication during sporulation in Bacillus subtilis

Spore formation in Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of cellcell communication. One pathway, which couples the proteolytic activation of the mother cell transcription factor sE to the action of a forespore synthesized signal molecule, SpoIIR, has remained enigmatic. Signalling by SpoIIR requires the protein to be exported to the intermembrane space between forespore and mother cell, where it will interact with and activate the integral membrane protease SpoIIGA. Here we show that SpoIIR signal activity as well as the cleavage of its N-terminal extension is strictly dependent on the prespore fatty acid biosynthetic machinery. We also report that a conserved threonine residue (T27) in SpoIIR is required for processing, suggesting that signalling of SpoIIR is dependent on fatty acid synthesis probably because of acylation of T27. In addition, SpoIIR localization in the forespore septal membrane depends on the presence of SpoIIGA. The orchestration of sE activation in the intercellular space by an acylated signal protein provides a new paradigm to ensure local transmission of a weak signal across the bilayer to control cellcell communication during development.

New insight into the mechanisms associated with the rapid effect of T-3 on AT1R expression

The angiotensin II type 1 receptor (AT1R) is involved in the development of cardiac hypertrophy promoted by thyroid hormone. Recently, we demonstrated that triiodothyronine (T-3) rapidly increases AT1R mRNA and protein levels in cardiomyocyte cultures. However, the molecular mechanisms responsible for these rapid events are not yet known. In this study, we investigated the T-3 effect on AT1R mRNA polyadenylation in cultured cardiomyocytes as well as on the expression of microRNA-350 (miR-350), which targets AT1R mRNA. The transcriptional and translational actions mediated by T-3 on AT1R levels were also assessed. The total content of ubiquitinated proteins in cardiomyocytes treated with T-3 was investigated. Our data confirmed that T-3 rapidly raised AT1R mRNA and protein levels, as assessed by real-time PCR and western blotting respectively. The use of inhibitors of mRNA and protein synthesis prevented the rapid increase in AT1R protein levels mediated by T-3. In addition, T-3 rapidly increased the poly-A tail length of the AT1R mRNA, as determined by rapid amplification of cDNA ends poly-A test, and decreased the content of ubiquitinated proteins in cardiomyocytes. On the other hand, T-3 treatment increased miR-350 expression. In parallel with its transcriptional and translational effects on the AT1R, T-3 exerted a rapid posttranscriptional action on AT1R mRNA polyadenylation, which might be contributing to increase transcript stability, as well as on translational efficiency, resulting to the rapid increase in AT1R mRNA expression and protein levels. Finally, these results show, for the first time, that T-3 rapidly triggers distinct mechanisms, which might contribute to the regulation of AT1R levels in cardiomyocytes. Journal of Molecular Endocrinology (2012) 49, 11-20

Systematic Analysis of FKBP Inducible Degradation Domain Tagging Strategies for the Human Malaria Parasite Plasmodium falciparum

Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD) tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP) and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the C-terminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA) at its C-terminus. The tagged protein demonstrated an important modulation of its expression.

alpha 1-Acid Glycoprotein Decreases Neutrophil Migration and Increases Susceptibility to Sepsis in Diabetic Mice

The mechanisms underlying immune deficiency in diabetes are largely unknown. In the present study, we demonstrate that diabetic mice are highly susceptible to polymicrobial sepsis due to reduction in rolling, adhesion, and migration of leukocytes to the focus of infection. In addition, after sepsis induction, CXCR2 was strongly downregulated in neutrophils from diabetic mice compared with nondiabetic mice. Furthermore, CXCR2 downregulation was associated with increased G-protein coupled receptor kinase 2 (GRK2) expression in these cells. Different from nondiabetic mice, diabetic animals submitted to mild sepsis displayed a significant augment in alpha 1-acid glycoprotein (AGP) hepatic mRNA expression and serum protein levels. Administration of AGP in nondiabetic mice subjected to mild sepsis inhibited the neutrophil migration to the focus of infection, as well as induced t-selectin shedding and rise in CD11b of blood neutrophils. Insulin treatment of diabetic mice reduced mortality rate, prevented the failure of neutrophil migration, impaired GRK2-mediated CXCR2 downregulation, and decreased the generation of AGP. Finally, administration of AGP abolished the effect of insulin treatment in diabetic mice. Together, these data suggest that AGP may be involved in reduction of neutrophil migration and increased susceptibility to sepsis in diabetic mice. Diabetes 61:1584-1591, 2012

Are antibiotics necessary after lower third molar removal?

Objective. Patients (n = 110) free of antibiotics, operated on by 3 surgeons ranging in clinical experiences, were evaluated for infection. Study Design. In the preoperative period and during the second and seventh postoperative days, the following parameters were analyzed: pain, infection, swelling, trismus, body temperature, C-reactive protein levels (CRP), and salivary neutrophil counts (SNC). During surgery, the following parameters were analyzed: systolic, diastolic, and mean arterial pressure; oximetry; heart rate; anesthesia quality; local anesthetic amount; bleeding; surgery difficulty; and surgery duration. Results. There were some differences in the surgery duration, local anesthetic amount, anesthesia quality, bleeding, pain experienced, trismus, CRP, and SNC, and no changes in hemodynamic parameters, rescue analgesic medication, wound healing, swelling, body temperature, confirmed case of dry socket, or any other type of local infection. Particularly, no systemic infections were found after lower third molar removal (LTMR). Conclusions. This study suggests that antibiotic prescriptions are unnecessary after LTMR when preoperative infections are absent. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;114(suppl 5):S199-S208)