Adaptation of renal ammonia production in the diabetic ketoacidotic rat

Adaptation of renal ammonia production in the diabetic ketoacidotic rat. Renal ammonia production was measured in diabetic, ketoacidotic rats. Rats were made diabetic by the i.v. injection of streptozotocin (150mg/kg of body wt). They were maintained on insulin for 1 week. Ketoacidosis was induced by withdrawing insulin for 3 days. At that time, blood and kidney ketone body (D-β-hydroxybutyrate and acetoacetate) levels were markedly elevated (approx. 6mM), and plasma total carbon dioxide concentration was strongly depressed (approx. 11mM). Renal ammonia production (ammonia released into renal vein plus that excreted in the urine) was stimulated sevenfold by the diabetic ketoacidosis. In a separate study, we examined the effects of ketone bodies on renal ammonia production in ammonium-chloride-induced acidotic, nondiabetic rats. Infusion of β-hydroxybutyrate had no significant effect on either urinary ammonia excretion (at relatively constant urinary pH), total renal ammonia production, or renal glutamine extraction. In vitro studies showed that β-hydroxybutyrate (4.0mM) markedly inhibited (61%) conversion ofL-glutamine (0.6mM) to ammonia by renal cortical slices prepared from normal rats. Inhibition was greatly reduced with slices prepared from kidneys of acidotic (ammonium-chloride-induced or diabetic ketoacidosis) rats. These results indicate that (1) renal ammonia production is markedly stimulated in diabetic ketoacidosis, and (2) in contrast to findings previously obtained by others in the acidotic dog, ketone bodies do not appear to inhibit renal ammonia production in vivo and only weakly in vitro in the acidotic rat.Adaptation de la production rénale d'ammoniac chez le rat diabétique en acidocétose. La production rénale d'ammoniaque a été mesurée chez le rat diabétique en acidocétose. Les rats ont été rendus diabétiques par une injection i.v. de streptozotocine (150 mg/kg body wt) et traités par l'insuline pendant une semaine. L'acidocétose a été déclenchée par la suppresion de l'insuline pendant trois jours. A ce moment, les concentrations sanguines et rénales de corps cétoniques (D-β-hydroxybutyrate et acéto-acétate) étaient très augmentées (approximativement 6mM) et le CO2 total du plasma très abaissé (approximativement 11mM). La production rénale d'ammoniac (ammoniac de la veine rénale plus ammoniaque de l'urine) était multipliée par 7. Dans un protocole différent, nous avons étudié les effets des corps cétoniques sur la production rénale d'ammoniac chez des rats non diabétiques, en acidose parle chlorure d'ammonium. La perfusion de β-hydroxybutyrate n'a eu d'effet significatif ni sur l'excétion urinaire d'ammoniaque (à pH relativement constant), ni sur la production rénale d'ammoniac ou l'extraction rénale de la glutamine. Des études in vitro ont montré que le β-hydroxybutyrate (4,0mM) inhibe de façon importante (61%) la conversion de laL-glutamine (0,6mM) en ammoniac par les tranches de cortex de rein préparées à partir de rats normaux. L'inhibition est moindre avec des tranches préparées à partir de rats en acidose (chlorure d'ammonium ou acidocétose diabétique). Ces résultats indiquent que (1) la production rénale d'ammoniac est fortement stimulée au cours de l'acidocétose diabétique et, (2) contrairement aux résultats obtenus par d'autres chez le chien en acidose, les corps cétoniques ne paraissent pas inhiber la production rénale d'ammoniac in vivo, et seulement faiblement in vitro, chez le rat en acidose

Rapamycin Response in Tumorigenic and Non-Tumorigenic Hepatic Cell Lines

The mTOR inhibitor rapamycin has anti-tumor activity across a variety of human cancers, including hepatocellular carcinoma. However, resistance to its growth inhibitory effects is common. We hypothesized that hepatic cell lines with varying rapamycin responsiveness would show common characteristics accounting for resistance to the drug.We profiled a total of 13 cell lines for rapamycin-induced growth inhibition. The non-tumorigenic rat liver epithelial cell line WB-F344 was highly sensitive while the tumorigenic WB311 cell line, originally derived from the WB-F344 line, was highly resistant. The other 11 cell lines showed a wide range of sensitivities. Rapamycin induced inhibition of cyclin E-dependent kinase activity in some cell lines, but the ability to do so did not correlate with sensitivity. Inhibition of cyclin E-dependent kinase activity was related to incorporation of p27(Kip1) into cyclin E-containing complexes in some but not all cell lines. Similarly, sensitivity of global protein synthesis to rapamycin did not correlate with its anti-proliferative effect. However, rapamycin potently inhibited phosphorylation of two key substrates, ribosomal protein S6 and 4E-BP1, in all cases, indicating that the locus of rapamycin resistance was downstream from inhibition of mTOR Complex 1. Microarray analysis did not disclose a unifying mechanism for rapamycin resistance, although the glycolytic pathway was downregulated in all four cell lines studied.We conclude that the mechanisms of rapamycin resistance in hepatic cells involve alterations of signaling downstream from mTOR and that the mechanisms are highly heterogeneous, thus predicting that maintaining or promoting sensitivity will be highly challenging

Resveratrol Inhibits Protein Translation in Hepatic Cells

Resveratrol is a plant-derived polyphenol that extends lifespan and healthspan in model organism. Despite extensive investigation, the biological processes mediating resveratrol's effects have yet to be elucidated. Because repression of translation shares many of resveratrol's beneficial effects, we hypothesized that resveratrol was a modulator of protein synthesis. We studied the effect of the drug on the H4-II-E rat hepatoma cell line. Initial studies showed that resveratrol inhibited global protein synthesis. Given the role of the mammalian Target of Rapamycin (mTOR) in regulating protein synthesis, we examined the effect of resveratrol on mTOR signaling. Resveratrol inhibited mTOR self-phosphorylation and the phosphorylation of mTOR targets S6K1 and eIF4E-BP1. It attenuated the formation of the translation initiation complex eIF4F and increased the phosphorylation of eIF2α. The latter event, also a mechanism for translation inhibition, was not recapitulated by mTOR inhibitors. The effects on mTOR signaling were independent of effects on AMP-activated kinase or AKT. We conclude that resveratrol is an inhibitor of global protein synthesis, and that this effect is mediated through modulation of mTOR-dependent and independent signaling

Phosphoproteomic Profiling of In Vivo Signaling in Liver by the Mammalian Target of Rapamycin Complex 1 (mTORC1)

Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood.We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO(2) affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates.In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo

Taking it personally: the effect of ethnic attachment on preferences for regionalism

This article presents three related findings on regional decentralization. We use an original dataset collected in Uganda to establish, for the first time in a developing country context, that individuals have meaningful preferences over the degree of regional decentralization they desire, ranging from centralism to secessionism. Second, multilevel models suggest that a small share of this variation is explained at the district and ethnic group levels. The preference for regional decentralization monotonically increases with an ethnic group or a district’s average ethnic attachment. However, the relationship with an ethnic group or district’s income is U-shaped: both the richest and the poorest groups desire more regionalism, reconciling interest-based and identity-based explanations for regionalism. Finally, we show that higher individual ethnic attachment increases preferences for regionalism using fixed effects and a new matching method

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Hepatic translation control in the late-gestation fetal rat

We have investigated the regulation of translation during the period of rapid liver growth that occurs at the end of gestation in the rat. This work was based on our prior observation that fetal hepatocyte proliferation is resistant to the inhibitory effects of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a nutrient-sensing kinase that controls ribosome biogenesis and protein translation. We hypothesized that translation control in late-gestation fetal liver differs from that in adult liver. We first examined the ability of rapamycin to inhibit the translation of mRNAs encoding ribosomal proteins. Consistent with the effect of rapamycin on proliferation, the activation of adult liver 5′-terminal oligopyrimidine tracts (5′-TOP) translation that occurred during refeeding after food deprivation was sensitive to rapamycin. Fetal liver 5′-TOP translation was insensitive. We went on to examine the eukaryotic initiation factor (eIF) 4F cap-binding complex that controls global protein synthesis. The molecular weights of the multiple eIF4G1 isoforms present in fetal and adult liver eIF4F complexes differed. In addition, fetal liver expressed the eIF4A1 form of the eIF4A helicase, whereas adult liver contained eIF4A1 and eIF4A2. Rapamycin administration before refeeding in adult rats inhibited formation of the preinitiation complex to a much greater degree than rapamycin administration to fetal rats in situ. We conclude that there are major structural and functional differences in translation control between late-gestation fetal and adult liver. These differences may confer differential sensitivity to the growth inhibitory effects of rapamycin