The role of AMPK and CREB-1 in the regulation of mitochondrial biogenesis during muscle differentiation

The aim of the study was to identify main regulators of mitochondrial biogenesis in a skeletal muscle differentiation model. To investigate candidate factors we used the cytochrome c promoter as a representative promoter since cytochrome c levels correlate well with respiratory chain complexes. We chose the AMP activated kinase (AMPK) and cyclic AMP-response element binding protein 1 (CREB-1) as promising candidates, which could control the process of mitochondrial proliferation. To analyze whether AMPK regulates the cytochrome c promoter, a cell line stably expressing a constitutively active AMPKalpha1-CA was generated. Indeed, AMPKalpha1-CA kinase activity was found to be increased in these clones. However, AMPKalpha1-CA and dominant negative AMPKalpha constructs did not change cytochrome c promoter activity in reporter gene assays, total AMPK activity was unchanged during muscle differentiation suggesting that it is not involved in the regulation of mitochondrial biogenesis in this model. In contrast, the constitutively active CREB construct, C2/CREB, elevated cytochrome c promoter activity. This increased promoter activity was diminished in the presence of a dominant negative CREB (A-CREB) construct showing that CREB-1 directly activates the cytochrome c promoter. Western blot analysis revealed two different CREB-1 isoforms: CREB-1alpha and CREB-1delta. Myoblasts contain predominantly CREB-1delta, while myotubes have preferably the CREB-1alpha isoform. The phosphorylation state of these isoforms changed in parallel with their protein levels suggesting a functional isoform switch during differentiation. Two appropriate, alternatively spliced isoforms of CREB-1 mRNA were found but their ratio did not change during differentiation. This suggests that posttranslational modifications and/or recruitment of the CREB-1 isoforms by other factors of the transcriptional complex play a dominant role in determining their cellular levels. To elucidate, which upstream factors regulate the activity of CREB-1, kinase and phosphatase inhibitors were used. A PKA activator and two phosphatase inhibitors increased the phosphorylation of CREB-1 at Ser-133, which is thought to be necessary for its activation. Furthermore, Cyclosporin A not only enhanced phosphorylation of CREB-1, but also the cytochrome c promoter activity. Therefore, it is likely that PP2B (calcineurin) regulates the phosphorylation of CREB-1 during muscle differentiation. Intracellular resting calcium levels were found to be smaller in myotubes compared to myoblasts, thus changes in steady-state calcium levels seem not to be a key regulator of mitochondrial biogenesis in muscle cells. In summary, these data indicate that phosphorylation and an isoform switch of CREB-1 could play a major role in the regulation of cytochrome c promoter and thus mitochondrial biogenesis during muscle differentiation

Seasonal change of the organic carbon content of Lake Balaton during 1972

1. Determinations of organic carbon content obtained by means of the “dry ” (Entz et al., 1963) and “wet” (Walkley and Black, 1934) methods of oxidation are to be used well on the mud of the open water of Lake Balaton. The former method gives higher values above 2 percent organic carbon content th an the latter one. 2. Apart from the Keszthely Bay, the organic carbon content in the mud of the open water of Lake Balaton shows a relatively uniform distribution, it varies between 1.52—1.78 percent. 3. The amount of the organic carbon changes seasonally in the transversal section through the Keszthely Bay. It was 1.83—1.85 percent during summer (June—August) and 1.96—2.27 percent during the spring and autumn. The autumnal increase represents a consequence of the destruction of the macro vegetation

Horizontal distribution of organic carbon content in the upper layer of the bottom deposit in Lake Balaton

Having the organic carbon content of 168 mud-samples taken at 24 transversal sections in Lake Balaton analysed with Walkley’s and Black’s method, th e authors conclude as follows: 1. Compared to other European lakes the organic carbon content of the top 5 cm thick sediment layer of th e lake (varying between 0.27 an d 2.33 per cent) is very low. 2. In the two basins of the south-western region with most of the waters inflowing here, the organic carbon content is relatively higher (over 1.9 per cent). 3. Apart from the central basin the organic carbon is distributed in spots. This phenomenon is due to the typical hydrodynamic conditions of the lake and partly to the effedt of River Zala (Keszthely Bay). 4. The mean organic carbon content of the sections decreases from the south-west end of the lake (section I) to the beginning of the north-east basin (section XVIII) with a slow increase from here. Probably, owing to the currents the mean organic carbon content decreases between the basins

Distribution of organic matter and bacteria in the upper layer of bottom deposit in the open water of Lake Balaton

1. The upper layer of the bottom deposit in the open water of Lake Balaton is poor in organic materials. The values obtained were 3—4% except for the Bay of Keszthely where values of 8—9% were registered. Noteworthy quantitative differences in the organic matter content between the different points of the individual transversal sections were found only in the Bay of Keszthely. 2. Values of Kjeldahl-N content in the different areas of bottom are nearly identical (0,3 —0,5%).3. Amounts of HCl-soluble materials in the Bay of Keszthely and its neighbourhood are 46—51% and in the other areas of the lake over 60%, and proceeding from the southern shore northward the amounts increase in most of the transversal sections. 4. Average bacterial counts and the amounts of the 3 5 µ fractions of particulate organic materials in the bottom are generally higher in the Bay of Keszthely and its neighbourhood than in other areas of the lake. The quantitative distribution of bacteria and particulate organic materials along the transversal sections is heterogeneous