Protein synthesis in the liver and small intestine of the rat

This thesis, presented in four sections, is concerned with the regulation of protein metabolism in the liver and small intestine of the rat. Section 1. A method was developed which allows for the measurement of rates of protein synthesis in tissues such as liver and intestine which synthesise and degrade protein very rapidly. The method employed a flooding amount of (14C)leucine (100umoles per 100g body weight) injected intravenously with measurement of the rate of incorporation of labelled leucine into protein over the following 10 minutes. Several experiments were performed to show that protein synthesis, per se, was unaltered by the flooding amount of leucine. Section 2. Employing the method described in Section 1, protein synthesis was measured in the liver and the individual components of the gastro-intestinal tract and in the whole animal. These measurements demonstrated the importance of these rapidly turning over tissues to overall protein metabolism in the whole animal. Section 3. Nutritional and hormonal perturbations leading to the loss of body protein were investigated. Rates of protein synthesis in the liver and small intestine have been examined under three conditions; namely, starvation, dietary protein-deprivation and diabetes. Each of these conditions caused a loss of protein from the liver and a lowered rate of synthesis but by different Intracellular mechanisms. In the intestine the synthesis of protein was depressed by starvation and protein-deprivation but maintained in diabetes. Section 4. The role of leucine as a regulator of protein synthesis was examined. Protein synthesis was measured using a flooding amount of (-H)phenylalanine in the presence and absence of l00umoles of unlabelled leucine. The impact of leucine on protein synthesis in liver, intestine and muscle of fed, starved and protein-deprived animals appeared to be negligible in vivo, inspite of many reports that leucine stimulated protein synthesis in vitro

A Role for Drosophila dFoxO and dFoxO 5′UTR Internal Ribosomal Entry Sites during Fasting

One way animals may cope with nutrient deprivation is to broadly repress translation by inhibiting 5′-cap initiation. However, under these conditions specific proteins remain essential to survival during fasting. Such peptides may be translated through initiation at 5′UTR Internal Ribosome Entry Sites (IRES). Here we show that the Drosophila melanogaster Forkhead box type O (dFoxO) transcription factor is required for adult survival during fasting, and that the 5′UTR of dfoxO has the ability to initiate IRES-mediated translation in cell culture. Previous work has shown that insulin negatively regulates dFoxO through AKT-mediated phosphorylation while dFoxO itself induces transcription of the insulin receptor dInR, which also harbors IRES. Here we report that IRES-mediated translation of both dFoxO and dInR is activated in fasted Drosophila S2 cells at a time when cap-dependent translation is reduced. IRES mediated translation of dFoxO and dInR may be essential to ensure function and sensitivity of the insulin signaling pathway during fasting

Effects of leucine supplemented diet on intestinal absorption in tumor bearing pregnant rats

BACKGROUND: It is known that amino acid oxidation is increased in tumor-bearing rat muscles and that leucine is an important ketogenic amino acid that provides energy to the skeletal muscle. METHODS: To evaluate the effects of a leucine supplemented diet on the intestinal absorption alterations produced by Walker 256, growing pregnant rats were distributed into six groups. Three pregnant groups received a normal protein diet (18% protein): pregnant (N), tumor-bearing (WN), pair-fed rats (Np). Three other pregnant groups were fed a diet supplemented with 3% leucine (15% protein plus 3% leucine): leucine (L), tumor-bearing (WL) and pair-fed with leucine (Lp). Non pregnant rats (C), which received a normal protein diet, were used as a control group. After 20 days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption. RESULTS: Tumor-bearing pregnant rats showed impairment in food intake, body weight gain and muscle protein content, which were less accentuated in WL than in WN rats. These metabolic changes led to reduction in both fetal and tumor development. Leucine absorption slightly increased in WN group. In spite of having a significant decrease in leucine and methionine absorption compared to L, the WL group has shown a higher absorption rate of methionine than WN group, probably due to the ingestion of the leucine supplemented diet inducing this amino acid uptake. Glucose absorption was reduced in both tumor-bearing groups. CONCLUSIONS: Leucine supplementation during pregnancy in tumor-bearing rats promoted high leucine absorption, increasing the availability of the amino acid for neoplasic cells and, mainly, for fetus and host utilization. This may have contributed to the better preservation of body weight gain, food intake and muscle protein observed in the supplemented rats in relation to the non-supplemented ones

Dysregulation of Mitochondrial Dynamics and the Muscle Transcriptome in ICU Patients Suffering from Sepsis Induced Multiple Organ Failure

BACKGROUND: Septic patients treated in the intensive care unit (ICU) often develop multiple organ failure including persistent skeletal muscle dysfunction which results in the patient's protracted recovery process. We have demonstrated that muscle mitochondrial enzyme activities are impaired in septic ICU patients impairing cellular energy balance, which will interfere with muscle function and metabolism. Here we use detailed phenotyping and genomics to elucidate mechanisms leading to these impairments and the molecular consequences. METHODOLOGY/PRINCIPAL FINDINGS: Utilising biopsy material from seventeen patients and ten age-matched controls we demonstrate that neither mitochondrial in vivo protein synthesis nor expression of mitochondrial genes are compromised. Indeed, there was partial activation of the mitochondrial biogenesis pathway involving NRF2alpha/GABP and its target genes TFAM, TFB1M and TFB2M yet clearly this failed to maintain mitochondrial function. We therefore utilised transcript profiling and pathway analysis of ICU patient skeletal muscle to generate insight into the molecular defects driving loss of muscle function and metabolic homeostasis. Gene ontology analysis of Affymetrix analysis demonstrated substantial loss of muscle specific genes, a global oxidative stress response related to most probably cytokine signalling, altered insulin related signalling and a substantial overlap between patients and muscle wasting/inflammatory animal models. MicroRNA 21 processing appeared defective suggesting that post-transcriptional protein synthesis regulation is altered by disruption of tissue microRNA expression. Finally, we were able to demonstrate that the phenotype of skeletal muscle in ICU patients is not merely one of inactivity, it appears to be an actively remodelling tissue, influenced by several mediators, all of which may be open to manipulation with the aim to improve clinical outcome. CONCLUSIONS/SIGNIFICANCE: This first combined protein and transcriptome based analysis of human skeletal muscle obtained from septic patients demonstrated that losses of mitochondria and muscle mass are accompanied by sustained protein synthesis (anabolic process) while dysregulation of transcription programmes appears to fail to compensate for increased damage and proteolysis. Our analysis identified both validated and novel clinically tractable targets to manipulate these failing processes and pursuit of these could lead to new potential treatments

Protein metabolism in the pectoralis muscle and liver of hibernating bats, Eptesicus fuscus

Seasonal variations in protein metabolism of the pectoralis muscle and liver of the big brown bat, Eptesicus fuscus , are examined in relation to seasonal changes in physiological status. A technique is described for the determination of protein synthetic rates in vivo in animals too small for conventional methods. The results indicate no detectable rates of protein synthesis in hibernating bats during torpor bouts (Table 2). Rates of synthesis in hibernating bats during periods of arousal are comparable to those of active summer bats (Table 2), despite the fact that the hibernating bats had not eaten in over 2 months. Rates of protein degradation were calculated from the rate of urea formation in torpid bats (Figs. 4, 5), the overall loss of pectoralis muscle and liver protein mass during hibernation (Table 3), the proportion of the total time of hibernation spent in torpor and arousal (Table 1), and the observed rates of protein synthesis (Table 2). These estimates (Table 4) indicate negligible rates of protein degradation in torpid bats. However, protein degradation during periodic arousals is comparable to that of summer bats after an overnight fast. These findings are consistent with earlier observations suggesting that significant gluconeogenesis from tissue protein occurs during spontaneous arousals from hibernation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47129/1/360_2004_Article_BF00689738.pd