Acute acidosis attenuates leucine stimulated signal transduction and protein synthesis in rat skeletal muscle.

BACKGROUND: Critical illnesses are often complicated by acute metabolic acidosis, which if persistent, adversely affects outcome. Among the harmful effects that it might cause are impaired utilization of nutrients, increased proteolysis and depressed protein synthesis, leading to muscle wasting. As the amino acid leucine stimulates protein synthesis by activating mTOR signaling, we explored whether in acidosis, impaired leucine-stimulated signaling might be a contributor to the depressed protein synthesis. METHODS: Male pair-fed rats were gavaged with NH4Cl (acidosis) or NaCl (control) for 2 days and then gavaged once with leucine and sacrificed 45 min later. Extensor digitorum longus muscles were isolated, incubated with or without leucine and protein synthesis measured. The anterior tibial muscle signaling was analysed by Western immunobloting. RESULTS: Despite pair-feeding, acidotic rats lost body and muscle weight vs. controls. Moreover, leucine-induced protein synthesis in isolated muscle from acidotic rats was impaired. In-vivo, 45 min after an oral leucine load, anterior tibial muscle mTOR and 4E-BP1 phosphorylation increased significantly and comparably in control and acidotic rats. In contrast, leucine-stimulated phosphorylation of S6K1, a regulator of translation initiation and protein synthesis, was attenuated to approximately 56% of the control value (p < 0.05). CONCLUSION: This study reveals that an acute metabolic acidosis impairs leucine-stimulated protein synthesis and activation of signaling downstream of mTOR at the level of S6K1. We propose that this S6K1 abnormality may account in part, for the resistance to leucine-stimulated muscle protein synthesis, and may thereby contribute to the impaired nutrient utilization and ultimately the muscle wasting that develops in acidosis.

Acute uremia suppresses leucine-induced signal transduction in skeletal muscle.

Adequate nutrient intake in acute uremia is a key part of patient management especially as food utilization is usually impaired. Leucine is important as it comprises about one-fifth of essential amino acid needs and, apart from serving as a substrate, it directly activates the mTOR signaling pathway stimulating protein synthesis and inhibiting autophagy. Here we tested whether leucine activation of the mTOR signaling pathway in muscle is severely disrupted in acute uremia. Several abnormalities were identified in bilateral ureteral ligated (model of acute uremia) compared to sham-operated pair-fed control rats. Levels of several signaling proteins increased significantly while leucine-induced phosphorylation of mTOR and downstream proteins, 4e-BP1 and S6K1, was completely suppressed. Levels of LC3B-II, a specific autophagosomal membrane-associated protein used as a marker of autophagy, increased threefold in uremia. Furthermore, while leucine suppressed LC3B-II levels in controls, it failed to do so in uremic rats. Muscle IL-6 mRNA levels increased, while IGF-1 mRNA levels decreased in uremia. These findings establish that, in acute uremia, severe resistance to leucine-induced activation of the mTOR anabolic signaling pathway develops. Thus, leucine resistance, together with the reduction in IGF-1 and increase in IL-6 expression, may explain why the anabolic effect of nutritional therapy is diminished in acute uremic patients.

A marked deficiency in circulating and renal IGF-I peptide does not inhibit compensatory renal enlargement in uninephrectomized mice.

OBJECTIVE: Increase in kidney IGF-I levels due to its increased trapping from the circulation was hypothesized to be a key mediator of compensatory renal enlargement. We tested this hypothesis using genetically engineered mice with extremely low circulating IGF-I levels. DESIGN: Both IGF-I deficient (ID) and normal (N) mice underwent a uninephrectomy (UNx) and sacrificed 2 or 9days later. RESULTS: Initial body weight (BW) and kidney weight (KW) were significantly reduced in ID vs. N mice, while KW/BW ratios were similar. KW increased post-UNx to a comparable extent in ID and N mice (125±4 and 118±6% of pre-UNx KW, p<0.05 vs. C). Kidney IGF-I mRNA levels were similar in the ID and N mice and did not change post-UNx. Kidney IGF-I peptide levels pre-UNx were significantly lower in ID vs. N mice (25±5 vs. 305±39ng/g) and increased in both groups after UNx, remaining low in ID mice (45±4 in ID vs 561±64ng/g in N). IGF type 1 receptor phosphorylation was unchanged. CONCLUSION: While a severe deficiency of circulating IGF-I impairs body growth, UNx induces a significant and proportional increase in renal mass in ID mice despite markedly decreased kidney IGF-I levels (>90% reduction) and no significant change in receptor phosphorylation. This all suggests that factors other than circulating and locally produced IGF-I are responsible for compensatory renal enlargement.

Leucine-stimulated mTOR signaling is partly attenuated in skeletal muscle of chronically uremic rats.

The branched-chain amino acid leucine stimulates muscle protein synthesis in part by directly activating the mTOR signaling pathway. Furthermore, leucine, if given in conjunction with resistance exercise, enhances the exercise-induced mTOR signaling and protein synthesis. Here we tested whether leucine can activate the mTOR anabolic signaling pathway in uremia and whether it can enhance work overload (WO)-induced signaling through this pathway. Chronic kidney disease (CKD) and control rats were studied after 7 days of surgically induced unilateral plantaris muscle WO and a single leucine or saline load. In the basal state, 4E-BP1 phosphorylation was modestly depressed in non-WO muscle of CKD rats, whereas rpS6 phosphorylation was nearly completely suppressed. After oral leucine mTOR, S6K1 and rpS6 phosphorylation increased similarly in both groups, whereas the phospho-4E-BP1 response was modestly attenuated in CKD. WO alone activated the mTOR signaling pathway in control and CKD rats. In WO CKD, muscle leucine augmented mTOR and 4E-BP1 phosphorylation, but its effect on S6K1 phosphorylation was attenuated. Taken together, this study has established that the chronic uremic state impairs basal signaling through the mTOR anabolic pathway, an abnormality that may contribute to muscle wasting. However, despite this abnormality, leucine can stimulate this signaling pathway in CKD, although its effectiveness is partially attenuated, including in skeletal muscle undergoing sustained WO. Thus, although there is some resistance to leucine in CKD, the data suggest a potential role for leucine-rich supplements in the management of uremic muscle wasting.

Uremia attenuates growth hormone-stimulated insulin-like growth factor-1 expression, a process worsened by inflammation.

Growth hormone (GH) resistance is common in uremia and together with resistance to insulin-like growth factor-1 (IGF-1) contributes to uremic growth retardation and muscle wasting. Previously, we found decreased GH-stimulated janus-kinase 2-signal transducers and activators of transcription 5 (STAT5) phosphorylation and nuclear translocation in uremia; however, it is unclear whether there are more distal defects. Therefore, we tested whether the binding of phosphorylated STAT5b to DNA is intact in uremia. Using uremic rats we found that in addition to impaired hepatic STAT5b phosphorylation, the binding of available phospho-STAT5b to DNA is decreased thus contributing to impaired IGF-1 gene expression. As sepsis-induced inflammation causes a loss of body protein and as Gram-negative infections are relatively common in uremia, we also characterized mechanisms in which acute inflammation might contribute to GH resistance in uremia. Endotoxin-induced inflammation markedly increased the resistance to GH-mediated STAT5b signaling, and further decreased STAT5b binding to DNA and IGF-1 gene expression. These perturbations appear to be related to increased cytokine expression. Thus, our findings indicate that hepatic resistance to GH-induced IGF-1 expression in uremia arises due to defects in STAT5b phosphorylation and its impaired binding to DNA, processes further aggravated by inflammation.

Endotoxin-induced growth hormone resistance in skeletal muscle.

Inflammation-induced skeletal muscle wasting is a serious clinical problem and arises in part because of resistance to GH-stimulated IGF-I expression. Although it is established that in the liver, resistance develops because of impaired signaling through the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) transduction pathway, together with a more distal defect in STAT5 DNA-binding activity, the situation in skeletal muscle is unclear. Accordingly, we set out to characterize the mechanisms behind the skeletal muscle resistance to GH in rats with acute inflammation induced by endotoxin. Endotoxin caused significant declines in GH-stimulated STAT5a/b phosphorylation and IGF-I gene expression, and this occurred despite a lack of change in signaling protein levels or phosphorylation of JAK2. In whole muscle, GH-stimulated phospho-STAT5a/b levels were reduced by half, and in the nucleus, phospho-STAT5b levels were similarly reduced. Furthermore, the binding of phosphorylated STAT5b to DNA was reduced and to a similar extent to the reduction in nuclear phosphorylated STAT5b. Interestingly, GH-induced androgen receptor gene expression was also suppressed. Thus, it appears that skeletal muscle resistance to GH-stimulated IGF-I expression in acute endotoxemia arises from a defect in STAT5b signaling, with a proportionate reduction in STAT5b DNA binding. Finally, it appears that resistance to GH-induced androgen receptor expression also develops and, together with the attenuated GH-induced IGF-I expression, likely plays an important role in the muscle wasting that arises in endotoxin-induced inflammation.

Low-dose growth hormone is cardioprotective in uremia.

Growth hormone (GH) is required to maintain normal cardiac structure and function and has a positive effect on cardiac remodeling in experimental and possibly human disease. Cardiac resistance to GH develops in the uremic state, perhaps predisposing to the characteristic cardiomyopathy associated with uremia. It was hypothesized that administration of low-dosage GH may have a salutary effect on the cardiac remodeling process in uremia, but because high levels of GH have adverse cardiac effects, administration of high-dosage GH may worsen uremic cardiomyopathy. In rats with chronic renal failure, quantitative cardiac morphology revealed a decrease in total capillary length and capillary length density and an increase in mean intercapillary distance and fibroblast volume density evident. Low-dosage GH prevented these changes. Collagen and TGF-beta immunostaining, increased in chronic renal failure, were also reduced by GH, suggesting a mechanism for its salutary action. Low-dosage GH also prevented thickening of the carotid artery but did not affect aortic pathology. In contrast, high-dosage GH worsened several of these variables. These results suggest that low-dosage GH may benefit the heart and possibly the carotid arteries in chronic renal failure.

Gene expression changes associated with Barrett's esophagus and Barrett's-associated adenocarcinoma cell lines after acid or bile salt exposure.

BACKGROUND: Esophageal reflux and Barrett's esophagus represent two major risk factors for the development of esophageal adenocarcinoma. Previous studies have shown that brief exposure of the Barrett's-associated adenocarcinoma cell line, SEG-1, or primary cultures of Barrett's esophageal tissues to acid or bile results in changes consistent with cell proliferation. In this study, we determined whether similar exposure to acid or bile salts results in gene expression changes that provide insights into malignant transformation. METHODS: Using previously published methods, Barrett's-associated esophageal adenocarcinoma cell lines and primary cultures of Barrett's esophageal tissue were exposed to short pulses of acid or bile salts followed by incubation in culture media at pH 7.4. A genome-wide assessment of gene expression was then determined for the samples using cDNA microarrays. Subsequent analysis evaluated for statistical differences in gene expression with and without treatment. RESULTS: The SEG-1 cell line showed changes in gene expression that was dependent on the length of exposure to pH 3.5. Further analysis using the Gene Ontology, however, showed that representation by genes associated with cell proliferation is not enhanced by acid exposure. The changes in gene expression also did not involve genes known to be differentially expressed in esophageal adenocarcinoma. Similar experiments using short-term primary cultures of Barrett's esophagus also did not result in detectable changes in gene expression with either acid or bile salt exposure. CONCLUSION: Short-term exposure of esophageal adenocarcinoma SEG-1 cells or primary cultures of Barrett's esophagus does not result in gene expression changes that are consistent with enhanced cell proliferation. Thus other model systems are needed that may reflect the impact of acid and bile salt exposure on the esophagus in vivo.

Seroprevalence of HIV infection among the foreign students at Agra during a fifteen year period (1988-2002).

The present study reports a retrospective analysis of data of HIV testing of foreign students from Sub-Saharan Africa, South-East Asia and Europe, studying as well as staying at Agra, over a period of 15 yr (1988 to 2002). Of the 2653 [2092 (78.85%) were from the Sub-Saharan African countries, 377 (14.21%) from the South-East Asian countries, and 184 (6.93%) from the European countries], foreign students tested for HIV, only 26 were found to be positive for HIV-1/2 antibodies by the ELISA, rapid and Western Blot assays. Out of 26 HIV-positive, 17 males and 7 females were from Sub-Saharan Africa and 2 males were from the European countries. The range of HIV-positivity over a period of 15 yr varied greatly. When the five-year (1988-1992, 1993-1997 and 1998-2002) results were compared, the HIV-seropositivity showed a decline from 1.85, 0.50 to 0.36 per cent in the first, second and third 5 yr slots, respectively. While the data were not representative of all foreign students in India, this reflected the population tested in this centre was not a growing focus of HIV infection in this part of the country.

A simple and rapid spectrophotometric method for determination of thiophanate-methyl in commercial formulations and its residues in foodstuffs.

A new, simple, rapid, and sensitive spectrophotometric method for the determination of thiophanate-methyl, based on its reaction with cobalt(II) in the presence of triethylamine, has been developed. The yellowish green color that develops instantaneously on mixing the fungicide with the reagents in dimethylformamide is stable for at least 2 h and has maximum absorbance at 360 nm. The method has been successfully applied to the determination of thiophanate-methyl in its commercial formulations and residues on grains and apples. A photometric titration procedure for formulation analysis of the fungicide has also been developed.