Nitric oxide synthase inhibition prevents activity-induced calcineurin-NFATc1 signalling and fast-to-slow skeletal muscle fibre type conversions.

The calcineurin­NFAT (nuclear factor of activated T-cells) signalling pathway is involved in the regulation of activity-dependent skeletal muscle myosin heavy chain (MHC) isoform type expression. Emerging evidence indicates that nitric oxide (NO) may play a critical role in this regulatory pathway. Thus, the purpose of this study was to investigate the role of NO in activity-induced calcineurin­NFATc1 signalling leading to skeletal muscle faster-to-slower fibre type transformations in vivo. Endogenous NO production was blocked by administering L-NAME (0.75 mg ml(−1)) in drinking water throughout 0, 1, 2, 5 or 10 days of chronic low-frequency stimulation (CLFS; 10 Hz, 12 h day(−1)) of rat fast-twitch muscles (L+Stim; n = 30) and outcomes were compared with control rats receiving only CLFS (Stim; n = 30). Western blot and immunofluorescence analyses revealed that CLFS induced an increase in NFATc1 dephosphorylation and nuclear localisation, sustained by glycogen synthase kinase (GSK)-3ß phosphorylation in Stim, which were all abolished in L+Stim. Moreover, real-time RT-PCR revealed that CLFS induced an increased expression of MHC-I, -IIa and -IId(x) mRNAs in Stim that was abolished in L+Stim. SDS-PAGE and immunohistochemical analyses revealed that CLFS induced faster-to-slower MHC protein and fibre type transformations, respectively, within the fast fibre population of both Stim and L+Stim groups. The final fast type IIA to slow type I transformation, however, was prevented in L+Stim. It is concluded that NO regulates activity-induced MHC-based faster-to-slower fibre type transformations at the transcriptional level via inhibitory GSK-3ß-induced facilitation of calcineurin­NFATc1 nuclear accumulation in vivo, whereas transformations within the fast fibre population may also involve translational control mechanisms independent of NO signalling.

Satellite cell ablation attenuates short-term fast-to-slow fibre type transformations in rat fast-twitch skeletal muscle.

The purpose of this time-course study was to determine whether satellite cell ablation within rat tibialis anterior (TA) muscles exposed to short-term chronic low-frequency stimulation (CLFS) would limit fast-to-slow fibre type transformations. Satellite cells of the left TA were ablated by exposure to gamma-irradiation before 1, 2, 5 or 10 days of CLFS and 1 week later where required. Control groups received only CLFS or a sham operation. Continuous infusion of 5-bromo-2'-deoxyuridine revealed that CLFS first induced an increase in satellite cell proliferation at 1 day, up to a maximum at 10 days over control (mean +/- SEM, 5.7 +/- 0.7 and 20.4 +/- 1.0 versus 1.5 +/- 0.2 mm(-2), respectively, P < 0.007) that was abolished by gamma-irradiation. Myosin heavy chain mRNA, immunohistochemical and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses revealed CLFS-induced fast-to-slow fibre type transformation began at 5 days and continued at 10 days; in those muscles that were also exposed to gamma-irradiation, attenuation occurred within the fast fibre population, and the final fast-twitch to slow-twitch adaptation did not occur. These findings indicate satellite cells play active and obligatory roles early on in the time course during skeletal muscle fibre type adaptations to CLFS.

Temporal regulation of BMP2, BMP6, BMP15, GDF9, BMPR1A, BMPR1B, BMPR2 and TGFBR1 mRNA expression in the oocyte, granulosa and theca cells of developing preovulatory follicles in the pig.

This study aimed to describe the abundance and localization of BMP2, BMP6, BMP15, GDF9, BMPR1A, BMPR1B, BMPR2 and TGFBR1 mRNA during pig preovulatory follicular development and to evaluate their implication in improving follicular maturity in the preovulatory period preceding the second versus first post-weaning oestrus. Oocytes, granulosa (GC) and theca cells (TC) were recovered from antral follicles of primiparous sows at day 1, 2 and 4 after weaning and at day 14, 16 and 20 of their subsequent oestrous cycle. Real-time PCR analysis revealed that with the exception of BMP6 mRNA, which was absent in GC, all genes were expressed in every cell type. Although BMP6, BMP15 and GDF9 mRNA were most abundant in the oocyte, their expression remained relatively constant during follicular development. By contrast, receptor BMPR1B and TGFBR1 expressions in the GC and TC were temporally regulated. BMPR1B mRNA abundance was positively correlated with plasma oestradiol (E2) suggesting that its regulation by oestrogen may be implicated in normal folliculogenesis. Interestingly, the increase in BMPR1B mRNA and protein abundance during the periovulatory period in GC and TC suggests a role for bone morphogenetic protein (BMP) 15 in the ovulatory process. Finally, expression of these ligands and receptors was not associated with potential differences in follicle maturity observed during the second versus first post-weaning preovulatory follicular wave. In conclusion, our results clearly demonstrate the presence of a complex signalling system within the pig follicle involving the transforming growth factor-beta superfamily and their receptors, and provide evidence to support a role for BMP15 and BMPR1B during ovulation.

Proteomics of dark cutting longissimus thoracis muscle from heifer and steer carcasses.

Studies have suggested that the phenomenon of dark cutting (Canada B4) beef may also be related to muscle glycolytic proteins. The objective of this study, therefore, was to analyze longissimus thoracis (LT; n=23), from Canada AA (n=8), atypical (AB4; pH<5.9, n=8) and typical (TB4; pH>5.9, n=7) B4 heifer and steer carcasses, for sarcoplasmic and myofibrillar proteins using 2-D gel electrophoresis and LC-MS/MS mass spectrometry. Results indicated that AB4 LT had intramuscular pH and lactate concentration similar to Canada AA but lower (P<0.05) L* and b*. Moreover, AB4 LT were tougher than Canada AA even at 21days post-mortem, unlike TB4. Canada AB4 LT had reduced (P<0.05) levels of creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and glycerol-3-phosphate dehydrogenase [NAD(+)], indicating a compromised glycolytic capacity in AB4. Canada AB4 LT had increased (P<0.05) abundances of phosphatidylethanolamine-binding protein 1 and small heat shock proteins.

Transcriptomic and Epigenetic Profiling of the Lung of Influenza-Infected Pigs: A Comparison of Different Birth Weight and Susceptibility Groups.

Influenza viruses are a common cause of respiratory disease in swine. Infections range in severity from asymptomatic to causing significant morbidity. The main objective of this study was to compare lung transcriptomic and epigenetic responses to influenza infection in pigs from high or low birth weight litters. The latter is a potential indicator of intrauterine growth restriction, a significant risk factor for prenatal programming effects. Individual pigs from high (HBW) or low birth weight (LBW) litters (n = 17) were inoculated with influenza A virus and euthanized 48 hours later. Lesion severity and viral loads were assessed as previously described. The transcriptional response to infection in LBW and HBW groups (n = 16) was assessed by microarray. A separate analysis of pigs classified as 'Resilient' (RES) or 'Susceptible' (SUS) (n = 6) on the basis of severity of lung pathology was also conducted. Eight genes were confirmed as differentially expressed for the birth weight comparison, including three antiviral genes with lower expression in LBW: ISG15, OAS1, and OAS2 (P<0.05). The promoter region methylation status of these three genes was assessed for each birth weight group, and no differences were found. These expression data are consistent with our previous finding that LBW pigs had less severe lesion scores and a trend towards lower viral titres in lung than the HBW cohort. The SUS v RES comparison identified 91 differentially expressed genes (FDR<0.05) that were enriched with functional annotation terms and pathways associated with inflammation. The cytokine genes IL6, IL8, and CCL2 were all upregulated in SUS pigs, and may have driven disease severity in these animals. In conclusion, this study found no evidence that the transcriptional immune response to influenza was adversely affected by low litter birth weight, but did identify several candidate genes for driving disease pathology.

Creatine loading elevates the intracellular phosphorylation potential and alters adaptive responses of rat fast-twitch muscle to chronic low-frequency stimulation.

This study tested the hypothesis that elevating the intracellular phosphorylation potential (IPP = [ATP]/[ADP]free) within rat fast-twitch tibialis anterior muscles by creatine (Cr) loading would prevent fast-to-slow fibre transitions induced by chronic low-frequency electrical stimulation (CLFS, 10 Hz, 12 h/day). Creatine-control and creatine-CLFS groups drank a solution of 1% Cr + 5% dextrose, ad libitum, for 10 days before and during 10 days of CLFS; dextrose-control and dextrose-CLFS groups drank 5% dextrose. Cr loading increased total Cr (P < 0.025), phosphocreatine (PCr) (P < 0.003), and the IPP (P < 0.0008) by 34%, 45%, and 64%, respectively. PCr and IPP were 46% (P < 0.002) and 76% (P < 0.02) greater in creatine-CLFS than in dextrose-CLFS. Higher IPP was confirmed by a 58% reduction in phospho-AMP-activated protein kinase α (Thr172) (P < 0.006). In dextrose-CLFS, myosin heavy chain (MyHC) I and IIa transcripts increased 32- and 38-fold (P < 0.006), respectively, whereas MyHC-IIb mRNA decreased by 75% (P < 0.03); the corresponding MyHC-I and MyHC-IIa protein contents increased by 2.0- (P < 0.03) and 2.7-fold (P < 0.05), respectively, and MyHC-IIb decreased by 30% (P < 0.03). In contrast, within creatine-CLFS, MyHC-I and MyHC-IIa mRNA were unchanged and MyHC-IIb mRNA decreased by 75% (P < 0.003); the corresponding MyHC isoform contents were not altered. Oxidative reference enzymes were similarly elevated (P < 0.01) in dextrose-CLFS and creatine-CLFS, but reciprocal reductions in glycolytic reference enzymes occurred only in dextrose-CLFS (P < 0.02). Preservation of the glycolytic potential and greater SERCA2 and parvalbumin contents in creatine-CLFS coincided with prolonged time to peak tension and half-rise time (P < 0.01). These results highlight the IPP as an important physiological regulator of muscle fibre plasticity and demonstrate that training-induced changes typically associated with improvements in muscular endurance or increased power output are not mutually exclusive in Cr-loaded muscles.

Impact of feed processing and mixed ruminal culture on the fate of recombinant EPSP synthase and endogenous canola plant DNA.

The impact of feed processing and in vitro ruminal cultures on the persistence of recombinant and canola-specific endogenous DNA was studied using various canola substrates (whole seed, cracked seed, meal and diet). For both, parental and genetically modified substrates, ribulose-1,5-bisphosphate carboxylase/oxygenase gene was amplifiable up to varying time points. Persistence of recombinant DNA, encoding 5-enolpyruvylshikimate-3-phosphate synthase (1,363 bp) was detected up to 8 h for meal and 4 h for mixed diet. Upon processing of canola, DNA large enough to contain intact plant genes remains. In an in vitro environment, plant DNA was rapidly degraded upon its release into rumen fluid.

Short-chain fatty acids and total parenteral nutrition affect intestinal gene expression.

BACKGROUND: The supplementation of total parenteral nutrition (TPN) formulas with short-chain fatty acids (SCFAs) increases glucose uptake and the expression of glucose transporters in parenterally fed animals. Several signals may be involved in intestinal adaptation; however, increased messenger RNA (mRNA) levels for proglucagon and several early-response genes, including c-myc and c-fos, are seen in animals receiving SCFA-supplemented TPN. Although the effects of a mixture of SCFAs are well documented, the relative contribution of individual SCFAs is unknown. Butyrate is a preferred fuel of colonocytes, with documented effects on cellular proliferation and gene expression. Accordingly, this study was undertaken to determine the relative role of butyrate in initiating an adaptive response in nonresected rats receiving TPN. METHODS: Animals received standard TPN for 66 hours, followed by 6 hours of either standard TPN, TPN supplemented with a mixture of SCFAs (acetate, propionate, and butyrate, 60 mmol/L total), or TPN supplemented with butyrate alone (9 mmol/L). An oral control group was fed an elemental diet, similar in macronutrient content to the TPN, so that all animals received the same amount of energy daily. RESULTS: SCFAs increased ileal glucose transporter 2 (GLUT2) mRNA expression compared with the orally fed group. SCFAs also increased proglucagon mRNA expression compared with the TPN group. No changes in Na+K(+)-adenosine triphosphatase or early-response gene expression were found in this study. CONCLUSIONS: In a rat model of TPN, the use of 9 mmol/L butyrate did not have the same effect on GLUT2 and proglucagon expression as a 60-mmol/L mixture of SCFAs. This suggests that the effect of a mixture of SCFAs on intestinal gene expression is not butyrate specific.

Characterization of the altered gene expression profile in early porcine embryos generated from parthenogenesis and somatic cell chromatin transfer.

The in vitro production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from in vitro Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than in vivo-derived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of in vivo (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The "eIF2 signalling", "mitochondrial dysfunction", "regulation of eIF4 and p70S6K signalling", "protein ubiquitination", and "mTOR signalling" pathways were down-regulated in PA HB. Dysregulation of notch signalling-associated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (NANOG, GATA2, KRT8, LGMN, and DPP4) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.

Nitric oxide synthase inhibition delays low-frequency stimulation-induced satellite cell activation in rat fast-twitch muscle.

This study examined the effect of nitric oxide synthase (NOS) inhibition via N(ω)-nitro-l-arginine methyl ester (l-NAME) administration on low-frequency stimulation-induced satellite cell (SC) activation in rat skeletal muscle. l-NAME only delayed stimulation-induced increases in SC activity. Also, stimulation-induced increases in hepatocyte growth factor (HGF) mRNA and protein expression were only abrogated at the mRNA level in l-NAME-treated animals. Therefore, early stimulation-induced SC activation appears to be NOS-dependent, while continued activation may involve NOS-independent HGF translational control mechanisms.

Lysine α-ketoglutarate reductase, but not saccharopine dehydrogenase, is subject to substrate inhibition in pig liver.

The activity of lysine α-ketoglutarate reductase (LKR), the initial enzyme in the principal pathway of lysine catabolism, is a primary determinant of whole-body lysine status. Past research indicated that LKR activity was predominantly hepatic; recent in vivo data suggest that other tissues can also catabolize lysine. The hypothesis of this investigation was that lysine catabolism takes place in extrahepatic tissues in pigs and that the enzymes involved may be subject to inhibition or activation. Using mitochondria from various tissues of market-age pigs, the activities of LKR and saccharopine dehydrogenase were measured. Liver mitochondria had the highest LKR activity, and the enzyme was subject to substrate inhibition. Mitochondria from the muscle, kidney, heart, and intestinal epithelial cells all had measurable LKR activity. The LKR activity was significantly inhibited by a variety of compounds including saccharopine, α-aminoadipate, α-ketoadipate, 5-hydroxy-l-lysine, and several metals. Oxidation of (14)C-lysine to (14)CO(2) was demonstrated in mitochondria isolated from the liver, muscle, and intestinal epithelial cells. Western blotting confirmed the presence of the α-aminoadipate δ-semialdehyde synthase protein in some extrahepatic tissues. These data show a significant capacity for lysine degradation in these extrahepatic tissues, most notably in cells of the intestine and muscle. These tissues should be considered important contributors to whole-body lysine catabolism.

Seminal plasma proteins as potential markers of relative fertility in boars.

This study investigated whether specific proteins from distinct seminal plasma fractions of boars could be related to in vivo fertility. Nine boars with acceptable sperm motility and morphology for use in artificial insemination demonstrated major differences in total number born and pregnancy rate when low sperm doses (1.5 billion sperm) were used to breed a minimum of 50 gilts per boar. The 2 lowest-fertility and 2 highest-fertility boars were chosen for evaluation of specific seminal plasma proteins. On 4 occasions, semen was collected and separated into 3 fractions based on sperm concentration (Sperm-Peak, Sperm-Rich, and Sperm-Free), and the fractions were analyzed for total protein concentration and abundance of major seminal plasma glycoprotein (PSP-I), AWN-1, and osteopontin protein using Western blotting techniques. The concentrations of these seminal plasma proteins were lower in the Sperm-Peak fractions compared with the Sperm-Free fractions (P < .05). Seminal plasma from the pooled Sperm-Rich fraction used for artificial insemination was also subjected to two-dimensional gel electrophoresis to investigate novel protein markers related to in vivo fertility. Total piglets born (r = -0.76, P = .01) and sperm motility at day 7 (r = -0.74, P = .037) were again negatively correlated with a 22-kDa protein identified by mass spectrometry as PSP-I. However, fertility index and farrowing rate tended to be positively correlated (P < .10) with a 25-kDa protein, identified as glutathione peroxidase (GPX5), an antioxidant enzyme that may protect sperm membranes from oxidative damage. These candidate proteins merit further investigation as markers of fertility in boars.

Pig preovulatory oocytes modulate cumulus cell protein and gene expression in vitro.

This study investigated the changes in protein and gene expression in oocytectomized cumulus cells (OOX) of medium-sized follicles from gilts, cultured with or without denuded oocytes isolated from large oestrogenic sow follicles. Proteomic analysis identified 14 proteins that were differentially expressed in OOX, of which the protein 14-3-3 eta, a signal transduction pathway modulator, was down-regulated in the presence of oocytes. Oocyte co-culture also down-regulated FSHR mRNA expression in OOX, as measured by real-time PCR, and FSHR and 14-3-3 eta mRNA abundance were positively correlated. The oocyte also up-regulated HSD3B mRNA, suggesting an effect on cumulus cell progesterone synthesis. Together with data on gene expression in granulosa cells during the follicular phase of the sow oestrous cycle, this study suggests that modulation of the expression of steroidogenesis related proteins and genes in cumulus cells by the porcine preovulatory oocyte reflects the specific physiological requirements of the preovulatory follicle.

Effect of satellite cell ablation on low-frequency-stimulated fast-to-slow fibre-type transitions in rat skeletal muscle.

The purpose of this study was to determine whether satellite cell ablation within rat fast-twitch muscles exposed to chronic low-frequency stimulation (CLFS) would limit fast-to-slow fibre-type transitions. Twenty-nine male Wistar rats were randomly assigned to one of three groups. Satellite cells of the left tibialis anterior were ablated by weekly exposure to a 25 Gy dose of gamma-irradiation during 21 days of CLFS (IRR-Stim), whilst a second group received only 21 days of CLFS (Stim). A third group received weekly doses of gamma-irradiation (IRR). Non-irradiated right legs served as internal controls. Continuous infusion of 5-bromo-2'-deoxyuridine (BrdU) revealed that CLFS induced an 8.0-fold increase in satellite cell proliferation over control (mean +/-s.e.m.: 23.9 +/- 1.7 versus 3.0 +/- 0.5 mm(-2), P < 0.0001) that was abolished by gamma-irradiation. M-cadherin and myogenin staining were also elevated 7.7- and 3.8-fold (P < 0.0001), respectively, in Stim compared with control, indicating increases in quiescent and terminally differentiating satellite cells; these increases were abolished by gamma-irradiation. Myonuclear content was elevated 3.3-fold (P < 0.0001) in Stim, but remained unchanged in IRR-Stim. Immunohistochemical analyses revealed attenuation of fast-to-slow fibre-type transitions in IRR-Stim compared with Stim. Comparable changes were observed at the protein level by SDS-PAGE. It is concluded that although considerable adaptive potential exists within myonuclei, satellite cells play a role in facilitating fast-to-slow fibre-type transitions.

Cell adhesion-mediated transformation of a human SCLC cell line is associated with the development of a normal phenotype.

Small cell lung carcinoma (SCLC) is a highly metastatic disease with a poor prognosis due to its resistance to current modes of therapy. SCLC cells appear to arise by oncogenic transformation of self-renewing pulmonary neuroendocrine cells, which have the potential to differentiate into a variety of lung epithelial cell lineages. Epithelial-mesenchymal conversion involved in such cell type transitions leads to the acquisition of an invasive and metastatic phenotype and may be critical for neoplastic progression and its eventual resistance to therapy. In order to investigate mechanisms involved in such transitions, a SCLC cell line was exposed to 5-bromodeoxyuridine. This treatment induced a dramatic conversion from non-substrate-adherent aggregates to monolayers of cells exhibiting an epithelioid phenotype. The phenotypic transition was concomitant with downregulation of vimentin, upregulation of cytokeratins, and cell-cell and cell-matrix adhesion molecules as well as redistribution of the actin cytoskeleton. The changes in the levels and organization of cell-cell and cell-matrix adhesion molecules were correlated with an in vivo loss of tumorigenicity.

Effects of chronic AICAR administration on the metabolic and contractile phenotypes of rat slow- and fast-twitch skeletal muscles.

The present study examined the effects of chronic activation of 5'-AMP-activated protein kinase (AMPK) on the oxidative capacity and myosin heavy chain (MHC) based fibre phenotype of rodent fast- and slow-twitch muscles. Sprague-Dawley rats received daily injections for 4 weeks of the known AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) or vehicle (control). The AICAR group displayed increases in hexokinase-II (HXK-II) activity, expression, and phosphorylation in fast-twitch muscles (P<0.001) but not in the slow-twitch soleus (SOL). In the AICAR group, citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35) were elevated 1.6- and 2.1-fold (P<0.05), respectively, in fast-twitch medial gastrocnemius (MG), and by 1.2- and 1.4-fold (P<0.05) in the slower-twitch plantaris (PLANT). No changes were observed in the slow-twitch SOL. In contrast, the activity of glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12) remained unchanged in all muscles. AICAR treatment did not alter the MHC-based fibre type composition in fast- or slow-twitch muscles, as determined by immunohistochemical and electrophoretic analytical methods or by RT-PCR. We conclude that chronic activation of AMPK mimics the metabolic changes associated with chronic exercise training (increased oxidative capacity) in the fast-twitch MG and PLANT, but does not coordinately alter MHC isoform content or mRNA expression.

Regulation of IGF-I and porcine oviductal secretory protein (pOSP) secretion into the pig oviduct in the peri-ovulatory period, and effects of previous nutrition.

The mechanisms regulating oviduct function were investigated. In Experiment 1, porcine oviductal secretory protein (pOSP) mRNA, and pOSP and insulin-like growth factor (IGF-I) in oviductal flushings, decreased through the peri-ovulatory period. In Experiment 2, higher plasma steroids in oviductal veins, ipsilateral (INT), rather than contralateral (OVX), to the remaining ovary in unilaterally ovariectomized gilts, were associated with higher pOSP in INT oviductal flushings. In Experiment 3, oviduct function was assessed as part of a collaborative study in cyclic gilts. Feed restriction in the late, compared to the early, luteal phase reduced estradiol concentrations in oviductal plasma, pOSP mRNA in oviductal tissue, and IGF-I concentrations and pOSP abundance in oviduct flushings. Previous insulin treatment differentially affected oviduct function. These data provide the first direct evidence for effects of previous feed restriction and insulin treatment on the oviduct environment in the peri-ovulatory period, which may contribute to nutritional effects on embryonic survival.

Expression and activity of matrix metallo proteinases 2 and 9 and their inhibitors in rat lungs during the perinatal period and in diaphragmatic hernia.

During lung development, the extracellular matrix undergoes dynamic remodeling. Matrix metalloproteinases (MMPs), and tissue inhibitors of matrix metalloproteinases (TIMPs), are important enzymes that participate in regulating tissue remodeling. There is an abnormal balance of the synthesis and degradation of collagen and elastin in perinatal lung associated with congenital diaphragmatic hernia (CDH). This study was designed to (1) determine the expression and gelatinolytic activity patterns of MMPs 2 and 9 and TIMPs 1 and 2 in rat lungs during the perinatal period, and (2) to test the hypothesis that they are abnormal in nitrofen-induced CDH. Measurements were made using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and zymography. The mRNA expression and activity of MMP 2 did not change significantly from embryonic day 16 to postnatal day 14. The most striking feature found was the rapid increase in the expression of MMP 9 soon after birth. Measurements were repeated on lung tissue isolated from embryonic rats with nitrofen-induced CDH. The expression and activity of MMPs and TIMPs were similar to control values and thus we conclude that these proteins appear not to be responsible for the altered extracellular matrix and morphological abnormalities noted in CDH lungs at birth.

AMPK activation increases uncoupling protein-3 expression and mitochondrial enzyme activities in rat muscle without fibre type transitions.

The present study examined the effect of chronic activation of 5'-AMP-activated protein kinase (AMPK) on the metabolic profile, including uncoupling protein-3 (UCP-3) and myosin heavy chain (MHC)-based fibre phenotype of rodent fast-twitch tibialis anterior muscle. Sprague-Dawley rats were given daily injections of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), a known activator of AMPK, or vehicle (control) for 28 days. After AICAR treatment, UCP-3 expression at the mRNA level was elevated 1.6 +/- 0.1-fold (P < 0.006) and corresponded to a 3.3 +/- 0.2-fold increase in UCP-3 protein content (P < 0.0001). In addition, the activities of the mitochondrial reference enzymes citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35), which are known to increase in proportion to mitochondrial volume density, were elevated 1.6-fold (P < 0.006), while the activity of lactate dehydrogenase (EC 1.1.1.27) was reduced to 80 % of control (P < 0.02). No differences were detected after AICAR treatment in the activities of the glycolytic reference enzymes glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) or phosphofructokinase (EC 2.7.1.11), nor were MHC-based fibre-type transitions observed, using immunohistochemical or electrophoretic analytical methods. These changes could not be attributed to variations in inter-organ signalling by metabolic substrates or insulin. We conclude that an AMPK-dependent pathway of signal transduction does mimic some of the metabolic changes associated with chronic exercise training, but does not affect expression of the MHC-based structural phenotype. Thus, the metabolic and MHC-based fibre types do not appear to be regulated in a co-ordinated way, but may be independently modified by different signalling pathways.

Chronic low-frequency stimulation upregulates uncoupling protein-3 in transforming rat fast-twitch skeletal muscle.

The purpose of this investigation was to examine the temporal changes in uncoupling protein (UCP)-3 expression, as well as related adaptive changes in mitochondrial density and fast-to-slow fiber type transitions during chronically enhanced contractile activity. We examined the effects of 1-42 days of chronic low-frequency electrical stimulation (CLFS), applied to rat tibialis anterior (TA) for 10 h/day, on the expression of UCP-3 and concomitant changes in myosin heavy chain (MHC) protein expression and increases in oxidative capacity. UCP-3 protein content increased from 1 to 12 days, reaching 1.5-fold over control (P < 0.0005); it remained elevated for up to 42 days. In contrast, UCP-3 mRNA decreased in response to CLFS, reaching a level that was threefold lower than control (P < 0.0007). The activities of the mitochondrial reference enzymes citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35), which are known to increase in proportion to mitochondrial density, progressively increased up to an average of 2.3-fold (P < 0.00001). These changes were accompanied by fast-to-slow fiber type transitions, characterized by a shift in the pattern of MHC expression (P <0.0002): MHCI and MHCIIa expression increased by 1.7- and 4-fold, whereas MHCIIb displayed a 2.4-fold reduction. We conclude that absolute increases in UCP-3 protein content in the early adaptive phase were associated with the genesis of mitochondria containing a normal complement of UCP-3. However, during exposure to long-term CLFS, mitochondria were generated with a lower complement of UCP-3 and coincided with the emergence of a growing population of oxidative type IIA fibers.