Role of Akt/GSK-3beta/beta-catenin transduction pathway in the muscle anti-atrophy action of insulin-like growth factor-I in glucocorticoid-treated rats.

Decrease of muscle IGF-I plays a critical role in muscle atrophy caused by glucocorticoids (GCs) because IGF-I gene electrotransfer prevents muscle atrophy caused by GCs. The goal of the present study was to identify the intracellular mediators responsible for the IGF-I anti-atrophic action in GC-induced muscle atrophy. We first assessed the IGF-I transduction pathway alterations caused by GC administration and their reversibility by local IGF-I overexpression performed by electrotransfer. Muscle atrophy induced by dexamethasone (dexa) administration occurred with a decrease in Akt (-53%; P<0.01) phosphorylation together with a decrease in beta-catenin protein levels (-40%; P<0.001). Prevention of atrophy by IGF-I was associated with restoration of Akt phosphorylation and beta-catenin levels. We then investigated whether muscle overexpression of these intracellular mediators could mimic the IGF-I anti-atrophic effects. Overexpression of a constitutively active form of Akt induced a marked fiber hypertrophy in dexa-treated animals (+175% of cross-sectional area; P<0.001) and prevented dexa-induced atrophy. This hypertrophy was associated with an increase in phosphorylated GSK-3beta (+17%; P<0.05) and in beta-catenin content (+35%; P<0.05). Furthermore, overexpression of a dominant-negative GSK-3beta or a stable form of beta-catenin increased fiber cross-sectional area by, respectively, 23% (P<0.001) and 29% (P<0.001) in dexa-treated rats, preventing completely the atrophic effect of GC. In conclusion, this work indicates that Akt, GSK-3beta, and beta-catenin probably contribute together to the IGF-I anti-atrophic effect in GC-induced muscle atrophy.

Insulin-like growth factor-I gene transfer by electroporation prevents skeletal muscle atrophy in glucocorticoid-treated rats.

Catabolic states caused by injury are characterized by a loss of skeletal muscle. The anabolic action of IGF-I on muscle and the reduction of its muscle content in response to injury suggest that restoration of muscle IGF-I content might prevent skeletal muscle loss caused by injury. We investigated whether local overexpression of IGF-I protein by gene transfer could prevent skeletal muscle atrophy induced by glucocorticoids, a crucial mediator of muscle atrophy in catabolic states. Localized overexpression of IGF-I in tibialis anterior (TA) muscle was performed by injection of IGF-I cDNA followed by electroporation 3 d before starting dexamethasone injections (0.1 mg/kg.d sc). A control plasmid was electroporated in the contralateral TA muscle. Dexamethasone induced atrophy of the TA muscle as illustrated by reduction in muscle mass (403 +/- 11 vs. 461 +/- 19 mg, P < 0.05) and fiber cross-sectional area (1759 +/- 131 vs. 2517 +/- 93 mum(2), P < 0.05). This muscle atrophy was paralleled by a decrease in the IGF-I muscle content (7.2 +/- 0.9 vs. 15.7 +/- 1.4 ng/g of muscle, P < 0.001). As the result of IGF-I gene transfer, the IGF-I muscle content increased 2-fold (15.8 +/- 1.2 vs. 7.2 +/- 0.9 ng/g of muscle, P < 0.001). In addition, the muscle mass (437 +/- 8 vs. 403 +/- 11 mg, P < 0.01) and the fiber cross-sectional area (2269 +/- 129 vs. 1759 +/- 131 mum(2), P < 0.05) were increased in the TA muscle electroporated with IGF-I DNA, compared with the contralateral muscle electroporated with a control plasmid. Our results show therefore that IGF-I gene transfer by electroporation prevents muscle atrophy in glucocorticoid-treated rats. Our observation supports the important role of decreased muscle IGF-I in the muscle atrophy caused by glucocorticoids.

Ordovician chitinozoan biozonation of the Brabant Massif, Belgium.

Chitinozoans from seven Ordovician units (Abbaye de Villers, Tribotte, Rigenée, Ittre, Bornival, and Brutia formations and a new unnamed unit, here provisionally called the Asquempont unit) belonging to the mainly concealed Brabant Massif, Belgium are described herein. Fifty-six samples were taken from rocks cropping out at the south-eastern rim of the massif in the Orneau, Dyle-Thyle and Senne-Sennette valleys. Microfossil preservation is moderate to poor, and the chitinozoans occur in low numbers. Taxonomically, the recovered chitinozoans are distributed into 29 taxa, some placed under open nomenclature. Together with earlier published graptolite and acritarch data, the analysis of the chitinozoan assemblages resulted in an improved chronostratigraphy of the investigated formations. We propose a local chitinozoan biozonation with 11 zones for the Brabant Massif. The oldest investigated units yielded chitinozoans typical for North Gondwana, and younger units (starting in the middle Caradoc), yielded some taxa also common in Baltica. As the Brabant Massif formed part of the microcontinent Avalonia, the chitinozoan assemblages recovered from the massif support the inferred drifting of Avalonia from high latitudes towards middle latitudes in the Ordovician as was suggested earlier.

Chitinozoan faunas from the Rügen Ordovician (Rügen 5/66 and Binz 1/73 wells), NE Germany.

The island of Rügen (NE Germany), situated close to the Trans-European Suture Zone (TESZ), in the southern Baltic Sea is underlain by sedimentary rocks of an Early Palaeozoic age, known only from boreholes. The wells, Rügen 5/66 and Binz 1/73, were investigated for their chitinozoan assemblages to improve the earlier biostratigraphic dating (graptolites and acritarchs) and to facilitate comparisons with other chitinozoan assemblages on both sides of the TESZ. In the lower part of the Rügen 5/66 core (3794.7-3615.8m), Lagenochitina destombesi Elaouad-Debbaj is indicative of an early late Tremadoc age. In the upper part of the same well (3287.3-1709.7m), the observed chitinozoan taxa suggest an age spanning the early Llanvirn to the Caradoc. The entire sampled interval of the Binz 1/73 core (5217.6-5041.8m) is interpreted as belonging to the Siphonochitina formosa Biozone (early-early late Abereiddian, corresponding to the early Llanvirn). The chitinozoan data corroborate the earlier suggested biostratigraphic ages, based on acritarchs and graptolites. The chitinozoans from the Binz 1/73 well point to a high latitude provenance of the investigated host sediments at time of deposition.

Failure of exogenous IGF-I to restore normal growth in rats submitted to dietary zinc deprivation.

Dietary zinc deficiency in rats causes growth retardation associated with decreased circulating IGF-I concentrations. To investigate the potential role of low IGF-I in this condition, we attempted to reverse the growth failure by administration of exogenous IGF-I. Rats were fed for 4 weeks a zinc-deficient diet (ZD, Zn 0 ppm) or were pair-fed a zinc-normal diet (PF, Zn 75 ppm). We compared the anabolic action of recombinant human (rh) IGF-I infused at the dose of 120 microg/day for the last experimental week in ZD, PF and freely fed control (CTRL) rats. Zinc deficiency caused growth stunting (weight gain 47% of PF; P<0.001), decreased circulating IGF-I (52% of PF; P<0.01) and liver IGF-I mRNA (67% of PF; P<0.01). Serum insulin-like growth factor-binding protein-3 (IGFBP-3) assessed by ligand blot was also reduced in ZD rats (65% of PF; P<0. 01). While exogenous IGF-I increased body weight in CTRL (+12 g; P<0. 01) and PF (+7 g; not significant) animals, growth was not stimulated in ZD rats (-1.5 g) in comparison with the corresponding untreated groups. However, circulating IGF-I and IGFBP-3 levels were restored by IGF-I infusion to levels similar to those in untreated CTRL rats. In conclusion, restoration of normal circulating levels of IGF-I and IGFBP-3 by rhIGF-I infusion fails to reverse the growth retardation induced by zinc deficiency. These results suggest that growth retardation related to zinc deficiency is not only caused by low serum IGF-I concentrations, but also by inhibition of the anabolic actions of IGF-I.

Inhibition by interleukin-1 beta and tumor necrosis factor-alpha of the insulin-like growth factor I messenger ribonucleic acid response to growth hormone in rat hepatocyte primary culture.

The cytokines are the putative mediators of the catabolic reaction that accompanies infection and trauma. Evidence suggests that their catabolic actions are indirect and potentially mediated through changes in hormonal axis such as the hypothalamo-pituitary-adrenal axis. Insulin-like growth factor I (IGF-I) is a GH-dependent growth factor that regulates the protein metabolism. To determine whether cytokines can directly inhibit the production of IGF-I by the liver, we investigated the regulation of IGF-I gene expression by interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha (10 ng/ml) in a model of rat primary cultured hepatocytes. Hepatocytes were isolated by liver collagenase perfusion and cultured on Matrigel 48 h before experiments. Each experiment was performed in at least three different animals. In the absence of GH, IL-1 beta and TNF-alpha did not affect the IGF-I messenger RNA (mRNA) basal levels, whereas IL-6 increased it by a factor of 2.5 after 24 h (P < 0.05). GH (500 ng/ml) alone stimulated the IGF-I gene expression markedly (5-to 10-fold increase) after 24 h (P < 0.001). IL-1 beta, and TNF-alpha to a lesser extent, dramatically inhibited the IGF-I mRNA response to GH (IL-1 beta: -82%, P < 0.001 and TNF-alpha: -47%, P < 0.01). The half-maximal inhibition of the IGF-I mRNA response to GH was observed for a concentration of IL-1 beta between 0.1 and 1 ng/ml. Moreover, IL-1 beta abolished the IL-6-induced IGF-I mRNA response. In contrast, IL-6 did not impair the IGF-I mRNA response to GH. To determine the potential role of the GH receptor (GHR) and the GH-binding protein (GHBP) in this GH resistance, we assessed the GHR and GHBP mRNAs response to these cytokines. GH alone did not affect the GHR/GHBP mRNA levels. IL-1 beta markedly decreased the GHR and GHBP mRNA levels (respectively, -68% and -60%, P < 0.05). Neither TNF-alpha nor IL-6 affected the GHR/GHBP gene expression. In conclusion, our results show that IL-1 beta, and TNF-alpha to a lesser extent, blunt the IGF-I mRNA response to GH. The resistance to GH induced by IL-1 beta might be mediated by a decrease of GH receptors, as suggested by the marked reduction of GHR mRNA. These findings suggest that decreased circulating IGF-I, in response to infection and trauma, may be caused by a direct effect of cytokines at the hepatocyte level.