The cAMP-dependent protein kinase (cAPK) plays an essential role during differentiation and fruit morphogenesis in Dictyostelium discoideum. The presence of an open reading frame on the gene, pkaC (previously named either Dd PK2 or Dd PK3 by different groups), predicts a 73-kDa polypeptide with 54% similarity to the catalytic subunits of cAPKs from other organisms. Using anti-peptide antibodies, we show that the pkaC gene product, PkaC, is a 73-kDa polypeptide. Despite the fact that PkaC is about twice the size of its mammalian counterparts, it possesses all of the properties required of a catalytic subunit. It is physically associated with the regulatory subunit, and this association results in an inhibition of the catalytic activity which is reverted by cAMP. PkaC copurifies with cAPK activity, and an increased cAPK activity is observed in cells overexpressing PkaC. We conclude that PkaC is a catalytic subunit of the Dictyostelium discoideum cAPK and discuss the unusual features of this protein with the highest molecular weight of known cAPKs.
Low calorie diets are designed to reduce body weight and fat mass, but they also lead to a detrimental loss of lean body mass, which is an important problem for overweight people trying to lose weight. In this context, a specific dietary intervention that preserves muscle mass in people following a slimming regime would be of great benefit. Leucine (LEU) and Citrulline (CIT) are known to stimulate muscle protein synthesis (MPS) in post-prandial and post-absorptive state, respectively. This makes them interesting bioactive components to test in the context of dietary restriction. We tested the concept of combining LEU and CIT in adult female rats. We postulated that the sequential administration of LEU (mixed in chow) and CIT (given in drinking water before a rest period) could be beneficial for preservation of muscle function during food restriction. Sixty female rats (22 weeks old) were randomized into six groups: one group fed ad libitum with a standard diet (C) and five food-restricted groups (60 % of spontaneous intake for 2 weeks) receiving a standard diet (R group), a CIT-supplemented diet (0.2 or 1 g/kg/day, CIT0.2 group and CIT1 group, respectively), a LEU-supplemented diet (1.0 g/kg/day) or a CIT + LEU-supplemented diet (CIT + LEU 1.0 g/kg/day each). At the end of the experiment, body composition, muscle contractile properties and muscle protein synthesis (MPS) rate were studied in the tibialis anterior muscle. Dietary restriction tended to decrease MPS (R: 2.5 ± 0.2 vs. C: 3.4 ± 0.4 %/day, p = 0.06) and decrease muscle strength (R: 3,045 ± 663 vs. C: 5,650 ± 661 A.U., p = 0.03). Only CIT administration (1 g/kg) was able to restore MPS (CIT1: 3.4 ± 0.3 vs. R: 2.5 ± 0.2 %/day, p = 0.05) and increase muscle maximum tetanic force (CIT1: 441 ± 15 vs. R: 392 ± 22 g, p = 0.05) and muscle strength (CIT1: 4,259 ± 478 vs. R: 3,045 ± 663 A.U., p = 0.05). LEU had no effect and CIT + LEU supplementation had few effects, limited to adipose mass and fatigue force. The results of this study highlight the ability of CIT alone to preserve muscle function during dietary restriction. Surprisingly, LEU antagonized some effects of CIT. The mechanisms involved in this antagonistic effect warrant further study.
The Dd PK2 gene codes for a putative protein of 648 amino acids with a C-terminal half sharing high homology with protein kinase A catalytic subunits from other organisms. In order to find out more about the physiological role of the Dd PK2 kinase, its gene, and a version having a frame shift mutation in the middle of the catalytic region, were overexpressed in developing Dictyostelium cells. Both the intact gene (K-) and the frame shift mutant (Kdel-) caused rapid development with spores formed in 16–18 hours compared to the 24 hours required by their parent. This result was confirmed by the pattern of expression of some developmentally regulated genes. Other rapid developing strains (rde) are activated in the cAMP second messenger system. Both K- and Kdel-containing strains have lower cAMP levels than the parental strain during late development, thus resembling rdeC mutants. K-cells (but not Kdel-cells) produced bizarre fruiting bodies with many prostrate forms. The parallel with rde mutants was confirmed by demonstrating that K-cells are able to form spores in submerged monolayer culture. Furthermore, K-cells have about four times more protein kinase A (cAPK) activity than wild-type cells. These results indicate that the N-terminal domain of Dd PK2 is sufficient to influence cAMP levels and to provoke rapid development, whereas kinase activity seems to be required for the sporogenous phenotype. The association between elevated cAPK and Dd PK2 overexpression phenotype further indicates a role for cAPK in the formation of spores.
Age related muscle wasting leads to overall reductions of lean body mass, reduced muscle strength, and muscle function resulting in compromised quality of life. Utilizing novel nutritional strategies to attenuate such losses is of great importance in elderly individuals. We aimed to test if a complete dietary supplement containing 25 g of milk proteins and ingested in the evening before bed would improve protein metabolism in terms of whole body protein balance over a 10 h overnight period following ingestion of the test drink in healthy middle-aged male subjects. In addition we also assessed the rates of muscle protein synthesis during the second half of the night in order to see if previously reported extended amino acidemia during sleep results in increased rates of muscle protein synthesis. Seventeen healthy middle-aged male subjects (59.4 ± 3.2 year) consumed a dietary supplement drink at 21:00 containing either 25 g milk protein concentrate, 25 g maltodextrin, 7.75 g canola oil (treatment group), or an isocaloric protein void drink (placebo group). Muscle protein synthesis was assessed from a muscle biopsy following the continuous intravenous infusion of 13C-phenylalanine for 5 h (from 03:00 to 08:00). Whole body protein balance was greater in the treatment group (−0.13 ± 11.30 g prot/10 h) compared to placebo (−12.22 ± 6.91 g prot/10 h) (P ≤ 0.01). In contrast, no changes were observed on rates of muscle protein synthesis during the second half of the night. Ingestion of a dietary supplement containing 25 g of milk proteins significantly reduced the negative protein balance observed during the night. Therefore, pre-bedtime protein ingestion may attenuate overnight losses of lean tissue in healthy elderly men. Despite increases in aminoacidemia during the second part of the night, no changes were observed in the rates of muscle protein synthesis during this time.Clinical Trial Registration:
www.ClinicalTrials.gov, identifier: NCT02041143.
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