Loss of mitochondrial pyruvate transport initiates cardiac glycogen accumulation and heart failure.

Background: Heart failure involves metabolic alterations including increased glycolysis despite unchanged or decreased glucose oxidation. The mitochondrial pyruvate carrier (MPC) regulates pyruvate entry into the mitochondrial matrix, and cardiac deletion of the MPC in mice causes heart failure. How MPC deletion results in heart failure is unknown. Methods: We performed targeted metabolomics and isotope tracing in wildtype (fl/fl) and cardiac-specific Mpc2-/- (CS-Mpc2-/-) hearts after in vivo injection of U-13C-glucose. Cardiac glycogen was assessed biochemically and by transmission electron microscopy. Cardiac uptake of 2-deoxyglucose was measured and western blotting performed to analyze insulin signaling and enzymatic regulators of glycogen synthesis and degradation. Isotope tracing and glycogen analysis was also performed in hearts from mice fed either low-fat diet or a ketogenic diet previously shown to reverse the CS-Mpc2-/- heart failure. Cardiac glycogen was also assessed in mice infused with angiotensin-II that were fed low-fat or ketogenic diet. Results: Failing CS-Mpc2-/- hearts contained normal levels of ATP and phosphocreatine, yet these hearts displayed increased enrichment from U-13C-glucose and increased glycolytic metabolite pool sizes. 13C enrichment and pool size was also increased for the glycogen intermediate UDP-glucose, as well as increased enrichment of the glycogen pool. Glycogen levels were increased ~6-fold in the failing CS-Mpc2-/- hearts, and glycogen granules were easily detected by electron microscopy. This increased glycogen synthesis occurred despite enhanced inhibitory phosphorylation of glycogen synthase and reduced expression of glycogenin-1. In young, non-failing CS-Mpc2-/- hearts, increased glycolytic 13C enrichment occurred, but glycogen levels remained low and unchanged compared to fl/fl hearts. Feeding a ketogenic diet to CS-Mpc2-/- mice reversed the heart failure and normalized the cardiac glycogen and glycolytic metabolite accumulation. Cardiac glycogen levels were also elevated in mice infused with angiotensin-II, and both the cardiac hypertrophy and glycogen levels were improved by ketogenic diet. Conclusions: Our results indicate that loss of MPC in the heart causes glycogen accumulation and heart failure, while a ketogenic diet can reverse both the glycogen accumulation and heart failure. We conclude that maintaining mitochondrial pyruvate import and metabolism is critical for the heart, unless cardiac pyruvate metabolism is reduced by consumption of a ketogenic diet.

Prior Treatment with AICAR Causes the Selective Phosphorylation of mTOR Substrates in C2C12 Cells.

Metabolic stress in skeletal muscle cells causes sustained metabolic changes, but the mechanisms of the prolonged effects are not fully known. In this study, we tested C2C12 cells with the AMP-activated protein kinase (AMPK) stimulator AICAR and measured the changes in the metabolic pathways and signaling kinases. AICAR caused an acute increase in the phosphorylation of the AMPK target ULK1, the mTORC1 substrate S6K, and the mTORC2 target Akt. Intriguingly, prior exposure to AICAR only decreased glucose-6 phosphate dehydrogenase activity when it underwent three-hour recovery after exposure to AICAR in a bicarbonate buffer containing glucose (KHB) instead of Dulbecco's Minimum Essential Medium (DMEM). The phosphorylation of the mTORC1 target S6K was increased after recovery in DMEM but not KHB, although this appeared to be specific to S6K, as the phosphorylation of the mTORC1 target site on ULK1 was not altered when the cells recovered in DMEM. The phosphorylation of mTORC2 target sites was also heterogenous under these conditions, with Akt increasing at serine 473 while other targets (SGK1 and PKCα) were unaffected. The exposure of cells to rapamycin (an mTORC1 inhibitor) and PP242 (an inhibitor of both mTOR complexes) revealed the differential phosphorylation of mTORC2 substrates. Taken together, the data suggest that prior exposure to AICAR causes the selective phosphorylation of mTOR substrates, even after prolonged recovery in a nutrient-replete medium.

Photoglobin, a distinct family of non-heme binding globins, defines a potential photosensor in prokaryotic signal transduction systems.

Globins constitute an ancient superfamily of proteins, exhibiting enormous structural and functional diversity, as demonstrated by many heme-binding families and two non-heme binding families that were discovered in bacterial stressosome component RsbR and in light-harvesting phycobiliproteins (phycocyanin) in cyanobacteria and red algae. By comprehensively exploring the globin repertoire using sensitive computational analyses of sequences, structures, and genomes, we present the identification of the third family of non-heme binding globins-the photoglobin. By conducting profile-based comparisons, clustering analyses, and structural modeling, we demonstrate that photoglobin is related to, but distinct from, the phycocyanin family. Photoglobin preserves a potential ligand-binding pocket, whose residue configuration closely resembles that of phycocyanin, indicating that photoglobin potentially binds to a comparable linear tetrapyrrole. By exploring the contextual information provided by the photoglobin's domain architectures and gene-neighborhoods, we found that photoglobin is frequently associated with the B12-binding light sensor domain and many domains typical of prokaryotic signal transduction systems. Structural modeling using AlphaFold2 demonstrated that photoglobin and B12-binding domains form a structurally conserved hub among different domain architecture contexts. Based on these strong associations, we predict that the coupled photoglobin and B12-binding domains act as a light-sensing regulatory bundle, with each domain sensing different wavelengths of light resulting in switch-like regulation of downstream signaling effectors. Thus, based on the above lines of evidence, we present a distinct non-heme binding globin family and propose that it may define a new type of light sensor, by means of a linear tetrapyrrole, in complex prokaryotic signal transduction systems.

Stimulation of Akt Phosphorylation and Glucose Transport by Metalloporphyrins with Peroxynitrite Decomposition Catalytic Activity.

Iron porphyrin molecules such as hemin and iron(III) 4,4',4″,4‴-(porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) (FeTBAP) have previously been shown to influence insulin signaling and glucose metabolism. We undertook this study to determine whether a catalytic action of iron porphyrin compounds would be related to their stimulation of insulin signaling and glucose uptake in C2C12 myotubes. FeTBAP did not display nitrite reductase activity or alter protein S-nitrosylation in myotubes, eliminating this as a candidate mode by which FeTBAP could act. FeTBAP displayed peroxynitrite decomposition catalytic activity in vitro. Additionally, in myotubes FeTBAP decreased protein nitration. The peroxynitrite decomposition catalyst Fe(III)5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato chloride (FeTPPS) also decreased protein nitration in myotubes, but the iron porphyrin Fe(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachlorideporphyrin pentachloride (FeTMPyP) did not. FeTBAP and FeTPPS, but not FeTMPyP, showed in vitro peroxidase activity. Further, FeTBAP and FeTPPs, but not FeTMPyP, increased Akt phosphorylation and stimulated glucose uptake in myotubes. These findings suggest that iron porphyrin compounds with both peroxynitrite decomposition activity and peroxidase activity can stimulate insulin signaling and glucose transport in skeletal muscle cells.

Mitigation of radiation exposure during surgical hepatectomy after yttrium-90 radioembolization.

Yttrium-90 (Y-90) radioembolization for the treatment of hepatocellular carcinoma can present safety challenges when transplanting recently treated Y-90 patients. To reduce surgeons' contact with radioactive tissue and remain within occupational dose limits, current guidelines recommend delaying transplants at least 14 days, if possible. We wanted to determine the level of radiation exposure to the transplant surgeon when explanting an irradiated liver before the recommended decay period. Anex-vivoradiation exposure analysis was conducted on the explanted liver of a patient who received Y-90 therapy 46 h prior to orthotopic liver transplant. To estimate exposure to the surgeon's hands, radiation dosimeter rings were placed inside three different surgical glove configurations and exposed to the explanted liver. Estimated radiation doses corrected for Y-90 decay were calculated. Radiation safety gloves performed best, with an average radiation exposure rate of 5.36 mSV h-1in the static hand position, an 83% reduction in exposure over controls with no glove (31.31 mSv h-1). Interestingly, non-radiation safety gloves also demonstrated reduced exposure rates, well below occupational regulation limits. Handling of Y-90 radiated organs within the immediate post-treatment period can be done safely and does not exceed federal occupational dose limits if appropriate gloves and necessary precautions are exercised.

Safety and Efficacy of a Steroid Avoidance Immunosuppression Regimen in Renal Transplant Patients With De Novo or Preformed Donor-Specific Antibodies: A Single-Center Study.

Although interest in the role of donor-specific antibodies (DSAs) in kidney transplant rejection, graft survival, and histopathological outcomes is increasing, their impact on steroid avoidance or minimization in renal transplant populations is poorly understood. Primary outcomes of graft survival, rejection, and histopathological findings were assessed in 188 patients who received transplants between 2012 and 2015 at the Scripps Center for Organ Transplantation, which follows a steroid avoidance protocol. Analyses were performed using data from the United Network for Organ Sharing. Cohorts included kidney transplant recipients with de novo DSAs (dnDSAs; n = 27), preformed DSAs (pfDSAs; n = 15), and no DSAs (nDSAs; n = 146). Median time to dnDSA development (classes I and II) was shorter (102 days) than in previous studies. Rejection of any type was associated with DSAs to class I HLA (P < .05) and class II HLA (P < .01) but not with graft loss. Although mean fluorescence intensity (MFI) independently showed no association with rejection, an MFI >5000 showed a trend toward more antibody-mediated rejection (P < .06), though graft loss was not independently associated. Banff chronic allograft nephropathy scores and a modified chronic injury score were increased in the dnDSA cohort at 6 months, but not at 2 years (P < .001 and P < .08, respectively). Our data suggest that dnDSAs and pfDSAs impact short-term rejection rates but do not negatively impact graft survival or histopathological outcomes at 2 years. Periodic protocol post-transplant DSA monitoring may preemptively identify patients who develop dnDSAs who are at a higher risk for rejection.

Reversible Oxidative Modifications in Myoglobin and Functional Implications.

Myoglobin (Mb), an oxygen-binding heme protein highly expressed in heart and skeletal muscle, has been shown to undergo oxidative modifications on both an inter- and intramolecular level when exposed to hydrogen peroxide (H2O2) in vitro. Here, we show that exposure to H2O2 increases the peroxidase activity of Mb. Reaction of Mb with H2O2 causes covalent binding of heme to the Mb protein (Mb-X), corresponding to an increase in peroxidase activity when ascorbic acid is the reducing co-substrate. Treatment of H2O2-reacted Mb with ascorbic acid reverses the Mb-X crosslink. Reaction with H2O2 causes Mb to form dimers, trimers, and larger molecular weight Mb aggregates, and treatment with ascorbic acid regenerates Mb monomers. Reaction of Mb with H2O2 causes formation of dityrosine crosslinks, though the labile nature of the crosslinks broken by treatment with ascorbic acid suggests that the reversible aggregation of Mb is mediated by crosslinks other than dityrosine. Disappearance of a peptide containing a tryptophan residue when Mb is treated with H2O2 and the peptide's reappearance after subsequent treatment with ascorbic acid suggest that tryptophan side chains might participate in the labile crosslinking. Taken together, these data suggest that while exposure to H2O2 causes Mb-X formation, increases Mb peroxidase activity, and causes Mb aggregation, these oxidative modifications are reversible by treatment with ascorbic acid. A caveat is that future studies should demonstrate that these and other in vitro findings regarding properties of Mb have relevance in the intracellular milieu, especially in regard to actual concentrations of metMb, H2O2, and ascorbate that would be found in vivo.

A peroxidase mimetic protects skeletal muscle cells from peroxide challenge and stimulates insulin signaling.

Reactive oxygen species such as hydrogen peroxide have been implicated in causing metabolic dysfunction such as insulin resistance. Heme groups, either by themselves or when incorporated into proteins, have been shown to scavenge peroxide and demonstrate protective effects in various cell types. Thus, we hypothesized that a metalloporphyrin similar in structure to heme, Fe(III)tetrakis(4-benzoic acid)porphyrin (FeTBAP), would be a peroxidase mimetic that could defend cells against oxidative stress. After demonstrating that FeTBAP has peroxidase activity with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and NADH as reducing substrates, we determined that FeTBAP partially rescued C2C12 myotubes from peroxide-induced insulin resistance as measured by phosphorylation of AKT (S473) and insulin receptor substrate 1 (IRS-1, Y612). Furthermore, we found that FeTBAP stimulates insulin signaling in myotubes and mouse soleus skeletal muscle to about the same level as insulin for phosphorylation of AKT, IRS-1, and glycogen synthase kinase 3ß (S9). We found that FeTBAP lowers intracellular peroxide levels and protects against carbonyl formation in myotubes exposed to peroxide. Additionally, we found that FeTBAP stimulates glucose transport in myotubes and skeletal muscle to about the same level as insulin. We conclude that a peroxidase mimetic can blunt peroxide-induced insulin resistance and also stimulate insulin signaling and glucose transport, suggesting a possible role of peroxidase activity in regulation of insulin signaling.

Regulation of Myogenic Activity by Substrate and Electrical Stimulation In Vitro.

Skeletal muscle has a remarkable regenerative capacity in response to mild injury. However, when muscle is severely injured, muscle regeneration is impaired due to the loss of muscle-resident stem cells, known as satellite cells. Fibrotic tissue, primarily comprising collagen I (COL), is deposited with this critical loss of muscle. In recent studies, supplementation of laminin (LM)-111 has been shown to improve skeletal muscle regeneration in several models of disease and injury. Additionally, electrical stimulation (E-stim) has been investigated as a possible rehabilitation therapy to improve muscle's functional recovery. This study investigated the role of E-stim and substrate in regulating myogenic response. C2C12 myoblasts were allowed to differentiate into myotubes on COL- and LM-coated polydimethylsiloxane molds. The myotubes were subjected to E-stim and compared with nonstimulated controls. While E-stim resulted in increased myogenic activity, irrespective of substrate, LM supported increased proliferation and uniform distribution of C2C12 myoblasts. In addition, C2C12 myoblasts cultured on LM showed higher Sirtuin 1, mammalian target of rapamycin, desmin, nitric oxide, and vascular endothelial growth factor expression. Taken together, these results suggest that an LM substrate is more conducive to myoblast growth and differentiation in response to E-stim in vitro.

Myoglobin as a versatile peroxidase: Implications for a more important role for vertebrate striated muscle in antioxidant defense.

Myoglobins (Mb) are ubiquitous proteins found in striated muscle of nearly all vertebrate taxa. Although their function is most commonly associated with facilitating oxygen storage and diffusion, Mb has also been implicated in cellular antioxidant defense. The oxidized (Fe3+) form of Mb (metMB) can react with hydrogen peroxide (H2O2) to produce ferrylMb. FerrylMb can be reduced back to metMb for another round of reaction with H2O2. In the present study, we have shown that horse skeletal muscle Mb displays peroxidase activity using 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) as reducing substrates, as well as the biologically-relevant substrates NADH/NADPH, ascorbate, caffeic acid, and resveratrol. We have also shown that ferrylMb can be reduced by both ethanol and acetaldehyde, which are known to accumulate in some vertebrate tissues under anaerobic conditions, such as anoxic goldfish and crucian carp, implying a potential mechanism for ethanol detoxification in striated muscle. We found that metMb peroxidase activity is pH-dependent, increasing as pH decreases from 7.4 to 6.1, which is biologically relevant to anaerobic vertebrate muscle when incurring intracellular lactic acidosis. Finally, we found that metMb reacts with hypochlorite in a heme-dependent fashion, indicating that Mb could play a role in hypochlorite detoxification. Taken together, these data suggest that Mb peroxidase activity might be an important antioxidant mechanism in vertebrate cardiac and skeletal muscle under a variety of physiological conditions, such as those that might occur in contracting skeletal muscle or during hypoxia.

C-peptide levels do not correlate with pancreas allograft failure: Multicenter retrospective analysis and discussion of the new OPT definition of pancreas allograft failure.

The OPTN Pancreas Transplantation Committee performed a multicenter retrospective study to determine if undetectable serum C-peptide levels correspond to center-reported pancreas graft failures. C-peptide data from seven participating centers (n = 415 graft failures for transplants performed from 2002 to 2012) were analyzed pretransplant, at graft failure, and at return to insulin. One hundred forty-nine C-peptide values were submitted at pretransplant, 94 at return to insulin, and 233 at graft failure. There were 77 transplants with two available values (at pretransplant and at graft failure). For recipients in the study with pretransplant C-peptide <0.75 ng/mL who had a posttransplant C-peptide value available (n = 61), graft failure was declared at varying levels of C-peptide. High C-peptide values at graft failure were not explained by nonfasting testing or by individual center bias. Transplant centers declare pancreas graft failure at varying levels of C-peptide and do not consistently report C-peptide data. Until February 28, 2018, OPTN did not require reporting of posttransplant C-peptide levels and it appears that C-peptide levels are not consistently used for evaluating graft function. C-peptide levels should not be used as the sole criterion for the definition of pancreas graft failure.

Glucose-dependent trans-plasma membrane electron transport and p70S6k phosphorylation in skeletal muscle cells.

The reduction of extracellular oxidants by intracellular electrons is known as trans-plasma membrane electron transport (tPMET). The goal of this study was to characterize a role of tPMET in the sensing of glucose as a physiological signal. tPMET from C2C12 myotubes was monitored using a cell-impermeable extracellular electron acceptor, water-soluble tetrazolium salt-1 (WST-1). Superoxide dismutase in the incubation medium or exposure to an NADPH oxidase (NOX) isoform 1/4 inhibitor suppressed WST-1 reduction by 70%, suggesting a role of NOXs in tPMET. There was a positive correlation between medium glucose concentration and WST-1 reduction, suggesting that tPMET is a glucose-sensing process. WST-1 reduction was also decreased by an inhibitor of the pentose phosphate pathway, dehydroepiandrosterone. In contrast, glycolytic inhibitors, 3PO and sodium fluoride, did not affect WST-1 reduction. Thus, it appears that glucose uptake and processing in the pentose phosphate pathway drives NOX-dependent tPMET. Western blot analysis demonstrated that p70S6k phosphorylation is glucose-dependent, while the phosphorylation of AKT and MAPK did not differ in the presence or absence of glucose. Further, phosphorylation of p70S6k was dependent upon NOX enzymes. Finally, glucose was required for full stimulation of p70S6k by insulin, again in a fashion prevented by NOX inhibition. Taken together, the data suggest that muscle cells have a novel glucose-sensing mechanism dependent on NADPH production and NOX activity, culminating in increased p70S6k phosphorylation.

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

Trans-Plasma Membrane Electron Transport and Ascorbate Efflux by Skeletal Muscle.

Trans-plasma membrane electron transport (tPMET) and the antioxidant roles of ascorbate reportedly play a role in protection of cells from damage by reactive oxygen species, which have been implicated in causing metabolic dysfunction such as insulin resistance. Skeletal muscle comprises the largest whole-body organ fraction suggesting a potential role of tPMET and ascorbate export as a major source of extracellular antioxidant. We hypothesized that skeletal muscle is capable of tPMET and ascorbate efflux. To measure these processes, we assayed the ability of cultured muscle cells, satellite cells, and isolated extensor digitorum longus (EDL) and soleus (SOL) to reduce two extracellular electron acceptors, water soluble tetrazolium salt 1 (WST-1), and dichlorophenolindophenol (DPIP). Ascorbate oxidase (AO) was utilized to determine which portion of WST-1 reduction was dependent on ascorbate efflux. We found that muscle cells can reduce extracellular electron acceptors. In C2C12 myotubes and satellite cells, a substantial portion of this reduction was dependent on ascorbate. In myotubes, glucose transporter 1 (GLUT1) inhibitors along with a pan-GLUT inhibitor suppressed tPMET and ascorbate efflux, while a GLUT4 inhibitor had no effect. The adenosine 5'-monophosphate (AMP)-activated protein kinase activator 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) suppressed both tPMET and ascorbate efflux by myotubes, while insulin had no effect. Taken together, our data suggest that muscle cells are capable of tPMET and ascorbate efflux supported by GLUT1, thus illustrating a model in which resting muscle exports electrons and antioxidant to the extracellular environment.

A role for ataxia telangiectasia mutated in insulin-independent stimulation of glucose transport.

Literature reports suggest that ataxia telangiectasia mutated (ATM) can activate the AMP-activated protein kinase (AMPK), a protein that can stimulate glucose transport in skeletal muscle. We hypothesized that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, would increase glucose transport in mouse extensor digitorum longus (EDL) muscles in an ATM-dependent manner. AICAR-stimulated glucose transport was prevented by the ATM inhibitor KU-55933 despite normal stimulation of AMPK phosphorylation. Consistent with this, AICAR caused AMPK phosphorylation but not an increase of glucose transport in ATM-deficient (ATM-/-) muscles. S231 of TBC1D1 matches the sequence motif of ATM substrates, and phosphorylation of this site is known to inhibit TBC1D1 and lead to increased glucose transport. Accordingly, we assessed TBC1D1 phosphorylation and found that AICAR-stimulated phosphorylation of TBC1D1 at S231 did not occur in ATM-/- muscles. However, activation of ATM without activation of AMPK was insufficient to increase TBC1D1 phosphorylation. The data suggest that ATM plays a role in AICAR-stimulated glucose transport downstream of AMPK.

Chloroquine increases phosphorylation of AMPK and Akt in myotubes.

AIMS: There are reports that ataxia telangiectasia mutated (ATM) can activate the AMP-activated protein kinase (AMPK) and also Akt, two kinases that play integral parts in cardioprotection and metabolic function. We hypothesized that chloroquine and resveratrol, both known ATM activators, would also activate AMPK and Akt. MAIN METHODS: Phosphorylation of AMPK and Akt was assessed after C2C12 myotubes were exposed to chloroquine or resveratrol. Additional experiments were done in cells expressing shRNA against ATM or in the presence of the ATM inhibitor KU55933. The effects of chloroquine on intracellular calcium were assessed with the fluorescent probe Calcium Green-1 AM. KEY FINDINGS: 0.5 mM chloroquine increased AMPK phosphorylation by nearly 4-fold (P<0.05), and 0.25 mM chloroquine roughly doubled Akt phosphorylation (P<0.05). Chloroquine also increased autophosphorylation of ATM by ~50% (P<0.05). Resveratrol (0.15 mM) increased AMPK phosphorylation about three-fold (P<0.05) but in contrast to chloroquine sharply decreased Akt phosphorylation. Chloroquine increased AMPK and Akt phosphorylation in myotubes expressing shRNA against ATM that reduced ATM protein levels by about 90%. Likewise, chloroquine-stimulated phosphorylation of AMPK and Akt and resveratrol-stimulated phosphorylation of AMPK were not altered by inhibition of ATM. Chloroquine decreased intracellular calcium by >50% concomitant with a decrease in glucose transport. SIGNIFICANCE: These ATM-independent effects of chloroquine on AMPK and Akt and the additional effect to decrease intracellular calcium are likely to partially underlie the positive metabolic effects of chloroquine that have been reported in the literature.

Views of US Voters on Compensating Living Kidney Donors.

IMPORTANCE: Patients in the United States waiting for kidney transplantation die in increasing numbers owing to the severe kidney shortage, which might be alleviated by compensating living kidney donors. OBJECTIVE: To determine the willingness of voting US citizens to become living kidney donors and to ascertain the potential influence of compensation for donation. DESIGN, SETTING, AND PARTICIPANTS: A professionally designed quantitative survey was administered by an international polling firm in June 2014. Information was collected on willingness to donate a kidney and the potential influence of compensation ($50 000); survey data included respondent age, income, education level, sex, US region, race/ethnicity, marital status, political affiliation, likelihood to vote, and employment status. The survey was performed via a random-digit dialing process that selected respondents via both landlines and mobile telephones to improve population representation. The survey included 1011 registered US voters likely to vote. MAIN OUTCOMES AND MEASURES: The degree to which the US voting public is willing to donate a kidney and the perceptions of current voters toward paying living kidney donors. RESULTS: Of the 1011 respondents, 427 were male and 584 were female, with 43% of participants between ages 45 and 64 years. With respondents grouped by willingness to donate, we found that 689 (68%) would donate a kidney to anyone and 235 (23%) only to certain persons; 87 (9%) would not donate. Most (59%) indicated that payment of $50 000 would make them even more likely to donate a kidney, 32% were unmoved by compensation, and 9% were negatively influenced by payment. CONCLUSIONS AND RELEVANCE: Most US voters view living kidney donation positively, and most would be motivated toward donor nephrectomy if offered a payment of $50 000. Because most registered voters favor such payments, and because thousands of lives might be saved should compensation increase the number of transplantable kidneys, laws and regulations prohibiting donor compensation should be modified to allow pilot studies of financial incentives for living kidney donors. Outcomes of such trials could then result in evidence-based policies, which would incorporate fair and just compensation to those persons willing to undergo donor nephrectomy.

Role of GLUT1 in regulation of reactive oxygen species.

In skeletal muscle cells, GLUT1 is responsible for a large portion of basal uptake of glucose and dehydroascorbic acid, both of which play roles in antioxidant defense. We hypothesized that conditions that would decrease GLUT1-mediated transport would cause increased reactive oxygen species (ROS) levels in L6 myoblasts, while conditions that would increase GLUT1-mediated transport would result in decreased ROS levels. We found that the GLUT1 inhibitors fasentin and phloretin increased the ROS levels induced by antimycin A and the superoxide generator pyrogallol. However, indinavir, which inhibits GLUT4 but not GLUT1, had no effect on ROS levels. Ataxia telangiectasia mutated (ATM) inhibitors and activators, previously shown to inhibit and augment GLUT1-mediated transport, increased and decreased ROS levels, respectively. Mutation of an ATM target site on GLUT1 (GLUT1-S490A) increased ROS levels and prevented the ROS-lowering effect of the ATM activator doxorubicin. In contrast, expression of GLUT1-S490D lowered ROS levels during challenge with pyrogallol, prevented an increase in ROS when ATM was inhibited, and prevented the pyrogallol-induced decrease in insulin signaling and insulin-stimulated glucose transport. Taken together, the data suggest that GLUT1 plays a role in regulation of ROS and could contribute to maintenance of insulin action in the presence of ROS.

Postprandial plasma incretin hormones in exercise-trained versus untrained subjects.

INTRODUCTION: After food ingestion, the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are secreted by the intestines into circulation where they act on the pancreas to promote insulin secretion. We evaluated the hypothesis that low postprandial plasma insulin levels in lean exercise-trained individuals are associated with low concentrations of incretin hormones. METHODS: A cross-sectional study was performed to compare postprandial incretin hormone levels in lean endurance exercise-trained individuals (EX; n = 14, ≥40 yr) and age- and sex-matched, nonobese, sedentary control subjects (CON, n = 14). The main outcome measures were GLP-1, GIP, insulin, and glucose incremental areas under the curve (AUC) as measured in plasma samples collected during a 2-h,75-g oral glucose tolerance test (OGTT). RESULTS: The EX group had lower body fat percentage (14.6% ± 1.1% vs 23.3% ± 1.7%, P = 0.0002) and higher maximal oxygen uptake (53 ± 2 vs 34 ± 2, P < 0.0001) than CON. Glucose AUC did not differ between groups (P = 0.20). Insulin AUC was lower in EX (2.5 ± 0.5 vs 4.2 ± 1.2 µU·mL·1000 min, P = 0.02). No differences were observed between groups (EX and CON, respectively) for GLP-1 AUC (3.5 ± 0.7 vs 4.1 ± 1.1 pmol·min·100 L, P = 0.61) or GIP AUC (19.2 ± 1.4 vs 18.0 ± 1.4 pg·min·1000 mL; P = 0.56). In CON, insulin AUC was correlated with AUC for GLP-1 (r = 0.53, P = 0.05) and GIP (r = 0.71, P = 0.004), but no such correlations were observed in EX (both P ≥ 0.67). CONCLUSIONS: Low postprandial insulin levels in lean exercise-trained individuals are not attributable to lower incretin hormone concentrations. However, exercise may decrease the dependency of postprandial insulin levels on incretin hormones.