Post Meal Energy Boluses Do Not Increase the Duration of Muscle Protein Synthesis Stimulation in Two Anabolic Resistant Situations

Background: When given in the long term, whey proteins alone do not appear to be an optimal nutritional strategy to prevent or slow down muscle wasting during aging or catabolic states. It has been hypothesized that the digestion of whey may be too rapid during a catabolic situation to sustain the anabolic postprandial amino acid requirement necessary to elicit an optimal anabolic response. Interestingly, it has been shown recently that the duration of the postprandial stimulation of muscle protein synthesis in healthy conditions can be prolonged by the supplementary ingestion of a desynchronized carbohydrate load after food intake. We verified this hypothesis in the present study in two different cases of muscle wasting associated with anabolic resistance, i.e., glucocorticoid treatment and aging. Methods: Multi-catheterized minipigs were treated or not with glucocorticoids for 8 days. Muscle protein synthesis was measured sequentially over time after the infusion of a 13C phenylalanine tracer using the arterio-venous method before and after whey protein meal ingestion. The energy bolus was given 150 min after the meal. For the aging study, aged rats were fed the whey meal and muscle protein synthesis was measured sequentially over time with the flooding dose method using 13C Valine. The energy bolus was given 210 min after the meal. Results: Glucocorticoid treatment resulted in a decrease in the duration of the stimulation of muscle protein synthesis. The energy bolus given after food intake was unable to prolong this stimulation despite a simultaneous increase of insulin and glucose following its absorption. In old rats, a similar observation was made with no effect of the energy bolus on the duration of the muscle anabolic response following whey protein meal intake. Conclusions. Despite very promising observations in healthy situations, the strategy aimed at increasing muscle protein synthesis stimulation by giving an energy bolus during the postprandial period remained inefficient in our two anabolic resistance models.

Key Roles of Glutamine Pathways in Reprogramming the Cancer Metabolism

Glutamine (GLN) is commonly known as an important metabolite used for the growth of cancer cells but the effects of its intake in cancer patients are still not clear. However, GLN is the main substrate for DNA and fatty acid synthesis. On the other hand, it reduces the oxidative stress by glutathione synthesis stimulation, stops the process of cancer cachexia, and nourishes the immunological system and the intestine epithelium, as well. The current paper deals with possible positive effects of GLN supplementation and conditions that should be fulfilled to obtain these effects. The analysis of GLN metabolism suggests that the separation of GLN and carbohydrates in the diet can minimize simultaneous supply of ATP (from glucose) and NADPH2(from glutamine) to cancer cells. It should support to a larger extent the organism to fight against the cancer rather than the cancer cells. GLN cannot be considered the effective source of ATP for cancers with the impaired oxidative phosphorylation and pyruvate dehydrogenase inhibition. GLN intake restores decreased levels of glutathione in the case of chemotherapy and radiotherapy; thus, it facilitates regeneration processes of the intestine epithelium and immunological system.

cfos and cjun antisense oligonucleotides block mitogenesis triggered by fibroblast growth factor-2 and ACTH in mouse Y1 adrenocortical cells

In G(0)/G(1) cell cycle-arrested mouse Y1 adrenocortical cells, short pulses (30 min to 2 h) of fibroblast growth factor-2 (FGF2) (5 pM to 1 nM) caused induction of cFos protein by 2 h and onset of DNA synthesis stimulation by 8-9 h. FGF2 dose-response curves for cFos induction (percent labeled nuclei with a specific anti-cFos antibody) and DNA synthesis stimulation (bromodeoxyuridine labeling index) were linearly correlated with a correlation coefficient of 0.969. Inhibition of cFos and cJun protein induction with antisense oligodeoxynucleotides (ODNs) to cfos and cjun mRNAs blocked DNA synthesis stimulation by FGF2. Pulses (up to 2 h) of synthetic ACTH(39) (1 pM to 1 nM) and natural porcine corticotropin A (10 pg/ml to 1 microg/ml) also induced cFos protein and DNA synthesis in G(0)/G(1)-arrested Y1 adrenal cells. ACTH dose-response curves for cFos induction and DNA synthesis stimulation were not correlated. But cfos and/or cjun antisense ODNs blocked DNA synthesis stimulation by ACTH. Thus, signals initiated in FGF2 and ACTH receptors appear to converge to the induction of cfos and cjun genes to trigger DNA synthesis stimulation.

Acylation-stimulating protein (ASP): structure–function determinants of cell surface binding and triacylglycerol synthetic activity

Acylation-stimulating protein (ASP or C3adesArg) is a potent lipogenic factor in human and murine adipocytes and fibroblasts. The arginated form of ASP, i.e. complement C3a (C3a), stimulates immunological responses in human granulocytes, mast cells, guinea pig platelets and guinea pig macrophages; however, ASP is inactive in stimulating these responses. Thus both ASP and C3a are bioactive across species but are not functionally interchangeable. Tertiary structure of both proteins by X-ray crystallography and NMR spectroscopy predicts a tightly linked core region consisting of three α-helices linked via three disulphide bonds, with one of the α-helices extending out from the core and terminating in a flexible conformationally irregular carboxy-tail region. The present studies were undertaken in order to define the functionally active domains of ASP, distinctive from those of C3a, using chemical modifications, enzymic cleavage and synthetic peptide fragments. The results indicate that: (i) the N-terminal region (< 10 amino acids) plays little role in ASP receptor binding and triacylglycerol synthesis stimulation; (ii) the native C-terminal region had no activity, but modifications which increased hydrophobicity increased receptor binding, and led to some activation of triacylglycerol synthesis stimulation; (iii) an intact disulphide-linked core region is essential for triacylglycerol synthesis stimulation activity but not for receptor interaction. Finally, basic charges in the carboxy region (His) are essential for ASP triacylglycerol synthesis stimulation but not for receptor binding, whereas both functions are eliminated by the modification of Lys in the disulphide-linked core region. The present results suggest that there are two functional domains in ASP, one that is responsible for the initial binding to the cell surface receptor, and a second domain that activates and increases triacylglycerol synthesis stimulation. This contrasts markedly with the structure-function studies of C3a where both binding competency and function were dependent on the C-terminal Arg. Thus ASP demonstrates distinct bioactivity.

Characterization of the in vitro hepatocyte model for toxicological evaluation: repeated growth stimulation and glutathione response

Hepatocytes in culture represent a useful model for investigating the effects of toxic agents on liver cells. However, further development of this model is hampered by the difficulty in promoting cell proliferation over prolonged periods and the lack of knowledge about the biochemical status of the cells relevant to the toxic response under proliferation conditions. In an effort to overcome these limitations, this work focused on the establishment of conditions to ameliorate the promotion of hepatocyte proliferation in vitro. It also examined the effects of growth stimuli on the levels of glutathione (GSH), a highly significant parameter influencing the resistance against toxic agents. In addition, albumin secretion was monitored as an indicator of liver-specific functions. Two modified L-15 media were developed: medium A for supporting cell differentiation, and medium B for promotion of proliferation. Collagen and Matrigel were used as substrata. In medium A, the time course of GSH levels was comparable for both substrata, with an initial increase followed by a plateau and then by a progressive decrease from the second to the fourth week. Hepatocytes cultured on collagen and sequentially exposed to medium B (containing epidermal growth factor ± norepinephrine) and medium A, showed repeated responsiveness to stimulation of DNA synthesis. Moreover, for cultures on collagen, a higher GSH content was observed in parallel with DNA synthesis stimulation, while albumin secretion was diminished. Although cells on Matrigel were refractory to DNA synthesis stimulation, GSH levels were still increased upon exposure to the growth factors, while under these conditions, albumin synthesis remained unaltered. These results show the possibility of expanding growth stimulus applications in hepatocyte cultures when it is of interest in cell pathology studies, such as those involving the effects of long-term exposure to xenobiotics, especially carcinogens. The results also suggest that hepatocytes subjected to a growth stimulus may have better protection against toxic injury.Key words: hepatocyte, glutathione, growth factors, long-term culture, collagen.