Maintenance of the branched-chain amino acid transporter LAT1 counteracts myotube atrophy following chemotherapy.

Prevention/management of cachexia remains a critical issue in muscle wasting conditions. The branched-chain amino acids (BCAA) have anabolic properties in skeletal muscle, but their use in treating cachexia has minimal benefits. This may be related to altered BCAA metabolism consequent to the use of chemotherapy, a main cancer treatment. Since this topic is minimally studied, we investigated the effect of chemotherapy on BCAA concentrations, transporter expression, and their metabolism. L6 myotubes were treated with vehicle (1.4 µL/mL DMSO) or a chemotherapy drug cocktail, FOLFIRI [CPT-11 (20 µg/mL), leucovorin (10 µg/mL), and 5-fluorouracil (50 µg/mL)] for 24-48 h. Chemotherapy reduced myotube diameter (-43%), myofibrillar protein content (-50%), and phosphorylation of the mechanistic target of rapamycin complex 1 (mTORC1) substrate S6K1thr389 (-80%). Drug-treated myotubes exhibited decreased BCAA concentrations (-52%) and expression of their transporter, L-type amino acid transporter 1 (LAT1; -67%). BCAA transaminase BCAT2 level was increased, but there was a reduction in PP2CM (-54%), along with increased inhibitory phosphorylation of BCKD-E1αser293 (+98%), corresponding with decreased BCKD enzyme activity (-23%) in chemotherapy-treated myotubes. Decreases in BCAA concentrations were a later response, preceded by decreases in LAT1 and BCKD activity. Although supplementation with the BCAA restored myotube BCAA levels, it had minimal effects on preventing the loss of myofibrillar proteins. However, RNAi-mediated depletion of neural precursor cell-expressed developmentally downregulated gene 4 (NEdd4), the protein ligase responsible for ubiquitin-dependent degradation of LAT1, attenuated the effects of chemotherapy on BCAA concentrations, anabolic signaling, protein synthesis, and myofibrillar protein abundance. Thus, if our findings are validated in preclinical models, interventions regulating muscle amino acid transporters might represent a promising strategy to treat cachexia.NEW & NOTEWORTHY This is the first study to attenuate chemotherapy-induced myotube atrophy by manipulating a BCAA transporter. Our findings suggest that positive regulation of amino acid transporters may be a promising strategy to treat cachexia.

Sex differences in cachexia and branched-chain amino acid metabolism following chemotherapy in mice.

Chemotherapy is a major contributor to cachexia, but studies often investigate male animals. Here, we investigated whether sex modifies the effects of chemotherapy on cachexia and BCAA metabolism. Ten-week-old CD2F1 male and female mice were treated with the chemotherapy drug cocktail folfiri (50 mg/kg 5-fluorouracil, 90 mg/kg leucovorin, and 24 mg/kg CPT11) (drug) or vehicle twice a week for 6 weeks. Insulin tolerance tests were conducted and BCAA levels and metabolism were measured in plasma and tissues. Drug treatment reduced body and skeletal muscle weights and anabolic signaling in both sexes, with females showing worsened outcomes (p < 0.05 for all). Drug treatment increased plasma BCAA only in males, but BCAA concentrations in the skeletal muscle of both sexes were decreased; this decrease was more profound in males (p = 0.0097). In addition, muscle expression of the BCAA transporter LAT1 was reduced; this reduction was more severe in females (p = 0.0264). In both sexes, the (inhibitory) phosphorylation of BCKD-E1αser293 was increased along with decreased BCKD activity. In the liver, drug treatment increased BCAA concentrations and LAT1 expression, but BCKD activity was suppressed in both sexes (p < 0.05 for all). Our results demonstrate that altered BCAA metabolism may contribute to chemotherapy-induced cachexia in a sex-dependent manner.

KIC (ketoisocaproic acid) and leucine have divergent effects on tissue insulin signaling but not on whole-body insulin sensitivity in rats.

Plasma levels of branched-chain amino acids and their metabolites, the branched-chain ketoacids are increased in insulin resistance. Our previous studies showed that leucine and its metabolite KIC suppress insulin-stimulated glucose uptake in L6 myotubes along with the activation of the S6K1-IRS-1 pathway. Because other tissue and fiber types can be differentially regulated by KIC, we analyzed the effect of KIC gavage on whole-body insulin sensitivity and insulin signaling in vivo. We hypothesized that KIC gavage would reduce whole-body insulin sensitivity and increase S6K1-IRS-1 phosphorylation in various tissues and muscle fibers. Five-week-old male Sprague-Dawley rats were starved for 24 hours and then gavaged with 0.75ml/100g of water, leucine (22.3g/L) or KIC (30g/L) twice, ten minutes apart. They were then euthanized at different time points post-gavage (0.5-3h), and muscle, liver, and heart tissues were dissected. Other sets of gavaged animals underwent an insulin tolerance test. Phosphorylation (ph) of S6K1 (Thr389), S6 (Ser235/6) and IRS-1 (Ser612) was increased at 30 minutes post leucine gavage in skeletal muscles irrespective of fiber type. Ph-S6 (Ser235/6) was also increased in liver and heart 30 minutes after leucine gavage. KIC gavage increased ph-S6 (Ser235/6) in the liver. Neither Leucine nor KIC influenced whole-body insulin tolerance, nor ph-Akt (Ser473) in skeletal muscle and heart. BCKD-E1 α abundance was highest in the heart and liver, while ph-BCKD-E1 α (Ser293) was higher in the gastrocnemius and EDL compared to the soleus. Our data suggests that only leucine activates the S6K1-IRS-1 signaling axis in skeletal muscle, liver and heart, while KIC only does so in the liver. The effect of leucine and KIC on the S6K1-IRS-1 signaling pathway is uncoupled from whole-body insulin sensitivity. These results suggest that KIC and leucine may not induce insulin resistance, and the contributions of other tissues may regulate whole-body insulin sensitivity in response to leucine/KIC gavage.

The silent killer: Previously undetected pulmonary emboli that result in death after discharge.

INTRODUCTION: Pulmonary embolism (PE) is a recognized cause of death in hospitalized trauma patients, yet less is known about PE after discharge. PATIENTS & METHODS: All post-discharge, autopsy-demonstrated, fatal PE resulting from trauma within a large US county over six years were analyzed. Counts, percentages, mean values, SD, and IQR were calculated for all variables. RESULTS: 1848 trauma deaths were reviewed, of which 85% had an autopsy. Eighty-five patients died from PE after discharge from their initial injury. 53% were initially treated at non-trauma centers, and 9% did not seek medical assistance. 75% were injured by falling, and most injuries occurred in the lower extremities. 86% had an ISS <16, but 87% needed assistance or were bed-bound after injury, despite 75% having no mobility limitations before the injury. 53% died within one month of injury, and 91% within the first year. Before death, only 11% were prescribed chemical thromboprophylaxis or an antiplatelet agent, and only 8% were diagnosed with venous thromboembolism before death. CONCLUSIONS: Fatal PE after discharge typically occurred following activity-limiting lower extremity injuries with an ISS<16.

The effect of a chemotherapy drug cocktail on myotube morphology, myofibrillar protein abundance, and substrate availability.

Cachexia, a condition prevalent in many chronically ill patients, is characterized by weight loss, fatigue, and decreases in muscle mass and function. Cachexia is associated with tumor burden and disease-related malnutrition, but other studies implicate chemotherapy as being causative. We investigated the effects of a chemotherapy drug cocktail on myofibrillar protein abundance and synthesis, anabolic signaling mechanisms, and substrate availability. On day 4 of differentiation, L6 myotubes were treated with vehicle (1.4 µl/ml DMSO) or a chemotherapy drug cocktail (a mixture of cisplatin [20 µg/ml], leucovorin [10 µg/ml], and 5-fluorouracil [5-FLU; 50 µg/ml]) for 24-72 h. Compared to myotubes treated with vehicle, those treated with the drug cocktail showed 50%-80% reductions in the abundance of myofibrillar proteins, including myosin heavy chain-1, troponin, and tropomyosin (p < 0.05). Cells treated with only a mixture of cisplatin and 5-FLU had identical reductions in myofibrillar protein abundance. Myotubes treated with the drug cocktail also showed >50% reductions in the phosphorylation of AKTSer473 and of mTORC1 substrates ribosomal protein S6Ser235/236 , its kinase S6K1Thr389 and eukaryotic translation initiation factor 4E-binding protein 1 (all p < 0.05). Drug treatment impaired peptide chain initiation in myofibrillar protein fractions and insulin-stimulated glucose uptake (p = 0.06) but increased the expression of autophagy markers beclin-1 and microtubule-associated proteins 1A/1B light chain 3B (p < 0.05), and of apoptotic marker, cleaved caspase 3 (p < 0.05). Drug treatment reduced the expression of mitochondrial markers cytochrome oxidase and succinate dehydrogenase (p < 0.05). The observed profound negative effects of this chemotherapy drug cocktail on myotubes underlie a need for approaches that can reduce the negative effects of these drugs on muscle metabolism.

Branched-chain Amino Acids: Catabolism in Skeletal Muscle and Implications for Muscle and Whole-body Metabolism.

Branched-chain amino acids (BCAAs) are critical for skeletal muscle and whole-body anabolism and energy homeostasis. They also serve as signaling molecules, for example, being able to activate mammalian/mechanistic target of rapamycin complex 1 (mTORC1). This has implication for macronutrient metabolism. However, elevated circulating levels of BCAAs and of their ketoacids as well as impaired catabolism of these amino acids (AAs) are implicated in the development of insulin resistance and its sequelae, including type 2 diabetes, cardiovascular disease, and of some cancers, although other studies indicate supplements of these AAs may help in the management of some chronic diseases. Here, we first reviewed the catabolism of these AAs especially in skeletal muscle as this tissue contributes the most to whole body disposal of the BCAA. We then reviewed emerging mechanisms of control of enzymes involved in regulating BCAA catabolism. Such mechanisms include regulation of their abundance by microRNA and by post translational modifications such as phosphorylation, acetylation, and ubiquitination. We also reviewed implications of impaired metabolism of BCAA for muscle and whole-body metabolism. We comment on outstanding questions in the regulation of catabolism of these AAs, including regulation of the abundance and post-transcriptional/post-translational modification of enzymes that regulate BCAA catabolism, as well the impact of circadian rhythm, age and mTORC1 on these enzymes. Answers to such questions may facilitate emergence of treatment/management options that can help patients suffering from chronic diseases linked to impaired metabolism of the BCAAs.

Fatal and Nonfatal Injury Patterns of Stranded Motorists.

ABSTRACT: Because nonfatal and fatal incidents for stranded motorists are not separated from vehicular accidents, little data are available on incident characteristics. To close this gap, data fields were inserted into databases at a medical examiner's office and two trauma centers to collect injury-related information. Forensic nurses and pathologists aided in forming a collaboration among the agencies involved and supported data collection efforts. Data collected over a 5-year period were examined for injury patterns to determine risk factors affecting these patterns. Of the total sample (N = 219), 24.7% had spinal injuries resulting in fatal injuries for 46 of 54 individuals. The odds were stranded motorists with spine-related injuries (C1-C7) had 9.13 times higher risk for a fatal outcome compared with those without spine-related injuries. Severe injuries (Abbreviated Injury Scale scores ≥ 4) noted for head/neck (29.7%) and chest (24.2%) were significantly associated with fatality. Of the 219 cases, 22.8% were inside of a stopped vehicle, and 77.2% were outside a vehicle at the time of injury. Outcomes illustrated the success of the interprofessional collaboration between trauma centers and a medicolegal death investigation agency that resulted in data useful for forensic nurses and pathologists documenting evidence, emergency and trauma responding personnel in patient priority stabilization, and injury prevention specialists for highway safety programs.

A survey of antifoulants in sediments from Ports and Marinas along the French Mediterranean coast.

Due to deleterious effects on non-target organisms, the use of organotin compounds on boat hulls of small vessels (<25 m) has been widely prohibited. The International Maritime Organisation (IMO) resolved that the complete prohibition on organotin compounds acting as biocides in antifouling systems should commence in 2008. As a result of restrictions on the use of organotin based paints, other antifouling formulations containing organic biocides have been utilised. This survey was conducted to assess the contamination of replacement biocides in the marine environment following the ban of TBT-based paints. Surface sediments samples were collected in the major ports and marinas along the France Mediterranean coastline (Cote d'Azur) and analysed for organotin compounds, Irgarol 1051, Sea-nine 211, Chlorothalonil, Dichlofluanid and Folpet. Every port and marina exhibited high levels of organotin compounds, with concentrations in sediments ranging from 37 ng Sn g(-1) dry wt in Menton Garavan to over 4000 ng Sn g(-1) dry wt close to the ship chandler within the port of Villefranche-sur-Mer. TBT degradation indexes suggested that fresh inputs are still made. Among the other antifoulants monitored, only Irgarol 1051 exhibited measurable concentrations in almost every port, with concentrations ranging from 40 ng g(-1) dry wt (Cannes) to almost 700 ng g(-1) dry wt (Villefranche-sur-Mer, ship chandler).

Arsenic in sediments from the southeastern Baltic Sea.

Arsenic occurs as a persistent constituent in many of the chemical weapons dumped into the Baltic Sea; it can be used as an indicator of leakage and dispersal of released munitions to the marine environment. Total arsenic was analysed in sediment samples taken from the Lithuanian economic zone in the Baltic Sea, which included samples from the chemical munitions dumpsite in the Gotland Basin and national monitoring stations in the southeastern Baltic Sea. Arsenic concentrations in sediments ranged from 1.1 to 19.0 mg kg(-1), with an average of 3.4 mg kg(-1). Although there was evidence of slightly elevated arsenic content in sediments near the weapons dumpsite, arsenic concentrations were nevertheless quite low relative to other investigations in the Baltic and North Seas.

Aliphatic and aromatic hydrocarbons in marine biota and coastal sediments from the Gulf and the Gulf of Oman.

The composition and spatial distribution of aliphatic and polycyclic aromatic hydrocarbons (PAHs) were investigated in biota and coastal sediments from four countries surrounding the Gulf (Bahrain, Qatar, United Arab Emirates and Oman). The levels of total petroleum hydrocarbons (TPH), aliphatic unresolved mixture and PAHs in sediments and biota were relatively low compared to world-wide locations reported to be chronically contaminated by oil. Only in the case of the sediments collected near the BAPCO oil refinery in Bahrain, having concentrations of 779 microg g(-1) total petroleum hydrocarbon equivalents and 6.6 microg g(-1) Sigma PAHs, can they be categorized as chronically contaminated. Some evidence of oil contamination was also apparent in sediments and bivalves around Akkah Head and Abu Dhabi in the UAE, and near Mirbat in Oman. Contaminant patterns in sediments and biota indicated that the PAHs were mainly from fossil sources, with the exception of the high PAH concentrations in sediments near the BAPCO refinery that contained substantial concentrations of carcinogenic PAH combustion products.

Chlorinated hydrocarbons in marine biota and coastal sediments from the Gulf and Gulf of Oman.

The spatial distribution of various organochlorinated compounds was investigated in the Gulf and Gulf of Oman based on marine biota (fish and various bivalves) and coastal sediment collected in Bahrain, Oman, Qatar and the United Arab Emirates (UAE) during 2000-2001. Several potential organic contaminants from agricultural (e.g., DDT and its breakdown products, lindane, endrin, dieldrin, endosulfan) and industrial (PCBs) sources were measured. Sediment burdens for all compounds, even for an apparent hot spot near a refinery in Bahrain, were amongst the lowest reported for surface sediments from other seas. Concentrations of DDTs were low in the muscle and liver of all fish (orange spotted grouper and the spangled emperor) analysed. Similarly, the levels of chlorinated hydrocarbons were low in the various bivalve species, notably rock oysters and pearl oysters. These results contribute to the sparse regional database for organochlorinated compounds in the marine environment. Moreover, they can be used as the most recent end point for elucidating temporal trends. Whereas the levels, albeit relatively low, SigmaDDTs in the rock oysters from the Gulf of Oman have remained uniform, there has been an irregular but generally decreasing trend in concentrations of summation SigmaPCBs during the last two decades.

Isolation of neutral and acidic lipid biomarker classes for compound-specific-carbon isotope analysis by means of solvent extraction and normal-phase high-performance liquid chromatography.

An analytical method for the quantitative determination of neutral and acidic lipid biomarkers in particulate and sediment samples has been developed. The method involves a first step with solvent extraction to isolate the neutral from the acidic compounds and a second step using normal-phase HPLC on a Nucleosil silica column to separate four different classes of neutral compounds: (1) aliphatic hydrocarbons, (2) aromatic hydrocarbons, (3) ketone compounds and (4) sterol and alcohol compounds. Recoveries of the individual spiked lipid biomarkers for the whole analytical procedure ranged from 88 to 106% for fatty acids, from 50 to 60% for aliphatic hydrocarbons (> or = n-C17), from 50 to 60% for polycyclic aromatic hydrocarbons (PAHs) (> or = 3 rings), 83% for friedelin and 60-80% for the sterol and alcohol compounds. The isolated compound classes were analysed by gas chromatography-combustion-isotope ratio mass spectrometry to measure the stable carbon isotope ratios in the individual compounds. The method enables the isolation of compound classes without fractionation for compound-specific carbon isotope analysis (delta13C). This analytical protocol has been applied, and proved suitable, for the determination of lipid biomarkers (sterols, fatty alcohols and fatty acids) in marine particulate material and for the determination of PAHs in sediment samples.

Assessment of organotin contamination in marine sediments and biota from the Gulf and adjacent region.

Butyltin species were measured in sediments from coastal locations in the Gulf and Gulf of Oman. Both butyltin and phenyltin species were measured in biota samples from four countries in this region. With tributyltin (TBT) concentrations up to 60 ngSng(-1), some sediments could be classified as contaminated (i.e. TBT>1.3 ngSng(-1)), namely Dukhan (Qatar), the BAPCO industrial complex and Askar (Bahrain), and Hilf and the Raysut Port Area (Oman). Higher concentrations of total butyltins were found in oysters relative to fish, but ranging from 6.5 to 488 ngSng(-1) dry weight they are nonetheless relatively low. Diphenyltin and triphenyltin were found in some fish and bivalves from the Gulf, but not in biota from the Gulf of Oman. The environmental levels of organotin species are comparatively low by global standards and pose no immediate public health problems.

Distribution of heavy metals in marine bivalves, fish and coastal sediments in the Gulf and Gulf of Oman.

An assessment of marine contamination due to heavy metals was made in the Gulf and Gulf of Oman based on marine biota (fish and various bivalves) and coastal sediment collected in Bahrain, Oman, Qatar, and the United Arab Emirates (UAE) during 2000-2001. Sediment metal loadings were generally not remarkable, although hot spots were noted in Bahrain (Cu, Hg, Pb, Zn) and on the east coast of the UAE (As, Co, Cr, Ni). Concentrations of As and Hg were typically low in sediments and the total Hg levels in top predator fish commonly consumed in the region were < 0.5 microg g(-1) and posed no threat to public health. Very high Cd concentrations (up to 195 microg g(-1)) in the liver of some fish from southern Oman may result from food-chain bioaccumulation of elevated Cd levels brought into the productive surface waters by upwelling in the region. Very high As concentrations (up to 156 microg g(-1)) were measured in certain bivalve species from the region. Although not certain, the As is probably derived from natural origins rather than anthropogenic contamination.

Organochlorinated compounds in Caspian Sea sediments.

Several organochlorinated contaminants, including numerous pesticides, were determined in coastal sediments from the Caspian Sea. The most important contaminants were p,p'-DDT (up to 7400 pg g(-1)) and its breakdown products, p,p'-DDD (up to 3400 pg g(-1)) and p,p'-DDE (up to 1300 pg g(-1)). Although the contamination was most severe in Azerbaijan, the sediment concentrations and percentage distribution of the three DDT-related compounds indicated that such contamination constitutes a contemporary and ubiquitous problem in the Caspian Sea. Lindane represented the second most significant contaminant, particularly in the Russian Federation, with concentrations up to 609 pg g(-1). The concentrations of HCB and other chlorinated pesticides (cis- and trans-chlordane, methoxychlor, heptachlor, heptachlor epoxide, aldrin, endrin, and endosulfans) were lower and not generally of concern, but the pesticides did demonstrate markedly different distributions reflecting differing agricultural usage in the region. The concentrations of Sigma PCBs were also quite low and ranged from 0.03 to 6.4 ng g(-1), with the highest amounts in the Russian Federation and Azerbaijan.

Aliphatic and aromatic hydrocarbons in coastal Caspian Sea sediments.

This investigation represents the first extensive study of the spatial distribution and sources of aliphatic (n-alkanes and unresolved complex mixture of fossil hydrocarbons) and polycyclic aromatic hydrocarbons (PAHs) in coastal sediments from the Caspian Sea. PAH concentrations, n-alkanes and biomarker profiles all suggested that there was limited petrogenic contamination in the shallow North Caspian Sea sediments, which are coarse with a low total organic carbon content. In contrast, moderate to high petrogenic contamination was found in the South Caspian Sea, in particular in the offshore oil fields near Baku, Azerbaijan. Contaminant patterns indicated that the PAHs were mainly from fossil sources, with higher contributions of pyrolytic only near industrialized and urban areas. A high contribution of perylene, a geochemically derived PAH, to the total PAHs was found in the west and south at sites influenced by the Kura, Safid Rud, Terek, Sulak and Samur Rivers.

An assessment of metal contamination in coastal sediments of the Caspian Sea.

An assessment of marine pollution due to metals was made in the Caspian Sea based on coastal sediment collected in Azerbaijan, Iran, Kazakhstan, Russia and Turkmenistan. Despite the high carbonate content, the distribution of most metals was largely controlled by terrigenous inputs. Several metals (As, Cr, Ni) exhibited concentrations that exceed sediment quality guidelines. Such metals have a high natural background but anthropogenic activities, notably mining, may further enhance concentrations. This would explain hot spots for Cu and Zn in Azerbaijan and Iran, and Cr at the mouth of the Ural River in Kazakhstan. Contamination by Hg was observed to the south of Baku Bay, Azerbaijan. Some anomalously high concentrations of Ba in the central Caspian are probably from offshore drilling operations, but the elevated U concentrations (up to 11.1 microg g(-1)) may be natural in origin. Several metals (Ag, Cd, Pb) have relatively low levels that pose no environmental concerns.