Insulin resistance is a significant determinant of sarcopenia in advanced kidney disease.

Maintenance hemodialysis (MHD) patients display significant nutritional abnormalities. Insulin is an anabolic hormone with direct effects on skeletal muscle (SM). We examined the anabolic actions of insulin, whole-body (WB), and SM protein turnover in 33 MHD patients and 17 participants without kidney disease using hyperinsulinemic-euglycemic-euaminoacidemic (dual) clamp. Gluteal muscle biopsies were obtained before and after the dual clamp. At baseline, WB protein synthesis and breakdown rates were similar in MHD patients. During dual clamp, controls had a higher increase in WB protein synthesis and a higher suppression of WB protein breakdown compared with MHD patients, resulting in statistically significantly more positive WB protein net balance [2.02 (interquartile range [IQR]: 1.79 and 2.36) vs. 1.68 (IQR: 1.46 and 1.91) mg·kg fat-free mass-1·min-1 for controls vs. for MHD patients, respectively, P < 0.001]. At baseline, SM protein synthesis and breakdown rates were higher in MHD patients versus controls, but SM net protein balance was similar between groups. During dual clamp, SM protein synthesis increased statistically significantly more in controls compared with MHD patients ( P = 0.03), whereas SM protein breakdown decreased comparably between groups. SM net protein balance was statistically significantly more positive in controls compared with MHD patients [67.3 (IQR: 46.4 and 97.1) vs. 15.4 (IQR: -83.7 and 64.7) µg·100 ml-1·min-1 for controls and MHD patients, respectively, P = 0.03]. Human SM biopsy showed a positive correlation between glucose and leucine disposal rates, phosphorylated AKT to AKT ratio, and muscle mitochondrial markers in controls but not in MHD patients. Diminished response to anabolic actions of insulin in the stimulated setting could lead to muscle wasting in MHD patients.

Omega-3 fatty acids inhibit the up-regulation of endothelial chemokines in maintenance hemodialysis patients.

BACKGROUND: Chronic systemic inflammation is common in patients with chronic kidney disease on dialysis (CKD5D) and has been considered a key mediator of the increased cardiovascular risk in this patient population. In this study, we tested the hypothesis that supplementation of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) will attenuate the systemic inflammatory process in CKD5D patients. METHODS: The design was a randomized, double-blinded, placebo controlled pilot trial (NCT00655525). Thirty-eight patients were randomly assigned in a 1 : 1 fashion to receive 2.9 g of eicosapentaenoic acid (C20:5, n-3) plus docosahexaenoic acid (C22:6, n-3) versus placebo for 12 weeks. The primary outcome was change in pro-inflammatory chemokines measured by lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs). Secondary outcomes were changes in systemic inflammatory markers. Analysis of covariance was used to compare percent change from baseline to 12 weeks. RESULTS: Thirty-one patients completed 12 weeks and three patients completed 6 weeks of the study. Median age was 52 (interquartile range 45, 60) years, 74% were African-American and 79% were male. Supplementation of ω-3 PUFAs effectively decreased the LPS-induced PBMC expression of RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) and MCP-1 (Monocyte Chemotactic Protein-1; unadjusted P = 0.04 and 0.06; adjusted for demographics P = 0.02 and 0.05, respectively). There was no significant effect of the intervention on serum inflammatory markers (C-reactive protein, interleukin-6 and procalcitonin). CONCLUSIONS: The results of this pilot study suggest that supplementation of ω-3 PUFAs is beneficial in decreasing the levels of endothelial chemokines, RANTES and MCP-1. Studies of larger sample size and longer duration are required to further evaluate effects of ω-3 PUFAs on systemic markers of inflammation, other metabolic parameters and clinical outcomes, particularly cardiovascular outcomes in CKD5D patients.

Association of plasma F2-isoprostanes and isofurans concentrations with erythropoiesis-stimulating agent resistance in maintenance hemodialysis patients.

BACKGROUND: In patients undergoing maintenance hemodialysis (HD), hyporesponsiveness to erythropoiesis stimulating agents (ESAs) is associated with adverse clinical outcomes. Systemic inflammation is highly prevalent in HD patients and is associated with ESA hyporesponsiveness. Oxidative stress is also highly prevalent in HD patients, but no previous study has determined its association with ESA response. This study assessed the association of plasma markers of oxidative stress and inflammation with ESA resistance in patients undergoing maintenance HD. METHODS: We analyzed data from 165 patients enrolled in the Provision of Antioxidant Therapy in Hemodialysis study, a randomized controlled trial evaluating antioxidant therapy in prevalent HD patients. Linear and mixed-effects regression were used to assess the association of baseline and time-averaged high sensitivity F2-isoprostanes, isofurans, C-reactive protein (hsCRP), and interleukin-6 (IL-6) with ESA resistance index (ERI), defined as the weekly weight-adjusted ESA dose divided by blood hemoglobin level. Unadjusted models as well as models adjusted for potential confounders were examined. Predicted changes in ERI per month over study follow-up among baseline biomarker quartiles were also assessed. RESULTS: Patients with time-averaged isofurans in the highest quartile had higher adjusted mean ERI compared with patients in the lowest quartile (ß = 14.9 ng/ml; 95% CI 7.70, 22.2; reference group <0.26 ng/ml). The highest quartiles of hsCRP and IL-6 were also associated with higher adjusted mean ERI (ß = 10.8 mg/l; 95% CI 3.52, 18.1 for hsCRP; ß = 10.2 pg/ml; 95% CI 2.98, 17.5 for IL-6). No significant association of F2-isoprostanes concentrations with ERI was observed. Analyses restricted to baseline exposures and ERI showed similar results. Baseline hsCRP, IL-6, and isofurans concentrations in the highest quartiles were associated with greater predicted change in ERI over study follow-up compared to the lowest quartiles (P = 0.008, P = 0.004, and P = 0.04, respectively). There was no association between baseline F2-isoprostanes quartile and change in ERI. CONCLUSIONS: In conclusion, higher concentrations of isofurans, hsCRP and IL-6, but not F2-isoprostanes, were associated with greater resistance to ESAs in prevalent HD patients. Further research is needed to test whether interventions that successfully decrease oxidative stress and inflammation in patients undergoing maintenance HD improve ESA responsiveness.

Obesity, diabetes and survival in maintenance hemodialysis patients.

Increased body mass index (BMI) confers a survival advantage in maintenance hemodialysis (MHD) patients. Diabetic (diabetes mellitus (DM)) patients undergoing MHD have worse survival. There are limited studies examining the effect of obesity on the risk of death among MHD patients with diabetes. Ninety-eight MHD patients were studied for median follow-up time of 33 months. Patients were classified according to the presence of obesity (BMI ≥ 30 kg/m(2)) or DM. Primary outcome was all-cause mortality. Cox regression was used to evaluate the effect of obesity on time to death. Effect modification and mediation analysis were also performed. Mean age was 49 ± 13 years, 66% were male, 48% were obese and 34% were diabetic. Mortality rates (per 100 person-years) were: 3.4 for non-diabetic obese, 8.6 for non-diabetic non-obese, 14.3 for diabetic non-obese and 18.1 for diabetic obese patients. Log-rank comparing diabetic obese versus non-diabetic obese was significant (p=0.007). Diabetes was associated with an increased risk of mortality after adjustment for potential mediators. Effect modification of obesity in the mortality risk was different between patients with and without diabetes. With adjustment for adipokines, a greater effect modification by diabetes was observed; whereas, adjustment for inflammatory marker did not influence the effect modification. Diabetic obese MHD patients have increased mortality risk compared to non-diabetic obese. Obesity does not offer survival benefits in diabetic obese MHD patients and potentially may have detrimental effects. Larger studies evaluating the effect of adipokines and obesity in outcomes in the diabetic MHD population need to be undertaken.

A pilot study of active vitamin D administration and insulin resistance in African American patients undergoing chronic hemodialysis.

OBJECTIVE: Insulin resistance (IR) is associated with increased cardiovascular risk in multiple patient populations, including those undergoing chronic hemodialysis (CHD). Active vitamin D deficiency has been proposed to play a role in the extent of IR observed in patients with CHD. We postulated that administration of paracalcitol, an active vitamin D medication, influences IR in patients with CHD. DESIGN AND METHODS: This was a pilot randomized controlled trial. Ten prevalent CHD patients receiving a stable dose of paracalcitol were recruited. Paracalcitol was withheld for 8 weeks in all patients (phase I). Parathyroid hormone levels were managed with the calcium-sensing receptor agonist cinacalcet. At week 8, patients were randomized to continue cinacalcet or to restart paracalcitol for 8 weeks (phase II). The primary outcome was the change in IR measured by the glucose disposal rate (GDR) using hyperinsulinemic euglycemic clamp (HEGC) method. Secondary outcomes included changes in IR between groups in indirect indices of IR, biomarkers of inflammation, and adipokine levels. RESULTS: The mean age was 49 years (range, 46-57 years) and 40% of patients were women. There was no detectable change in the GDR at the end of phase I (P = .7) when compared with baseline values. There was also no statistically significant difference in GDR between groups at the end of phase II (P = .9). No changes were observed in indirect indices of IR, adipokine levels, or biomarkers of inflammation in either phase. CONCLUSION: The results of this pilot study suggest that withdrawal of paracalcitol over 8 to 16 weeks and replacement for 8 weeks after withdrawal does not influence IR measured by HEGC in patients receiving CHD.

Insulin resistance and protein metabolism in chronic hemodialysis patients.

OBJECTIVE: Loss of lean body mass (sarcopenia) is associated with increased morbidity and mortality in patients receiving chronic hemodialysis (CHD). Insulin resistance (IR), which is highly prevalent in patients receiving CHD, has been proposed to play a critical role in the development of sarcopenia. The aim of this study was to examine the effect of IR on amino acid metabolism in patients receiving CHD. DESIGN: This was a cross-sectional study. SUBJECTS: The study included 12 prevalent (i.e., patients that have been on dialysis for more than 90 days) African American patients receiving CHD. METHODS: IR was measured as glucose disposal rate (GDR) determined from hyperinsulinemic euglycemic clamp (HGEC) studies performed 3 consecutive times. Plasma amino acid (AA) concentrations were measured by real-time high-performance liquid chromatography (HPLC) throughout the clamp study. The primary outcome was percentage change in leucine concentrations during the clamp study. The main predictor was the GDR measured simultaneously during the HGEC studies. Mixed model analysis was used to account for repeated measures. RESULTS: All individual AA concentrations declined significantly in response to high-dose insulin administration (P < .001). There was a significant direct association between GDR by HECG studies and the percentage change in leucine concentration (P = .02). Although positive correlations were observed between GDR values and concentration changes from baseline for other AAs, these associations did not reach statistical significance. CONCLUSIONS: Our results suggest that the severity of IR of carbohydrate metabolism is associated with a lesser decline in plasma leucine concentrations, suggesting a similar resistance to protein anabolism. Insulin resistance represents a potential mechanism for sarcopenia commonly observed in patients receiving CHD.

IL-1ß receptor antagonist reduces inflammation in hemodialysis patients.

Chronic inflammation is highly prevalent in maintenance hemodialysis (MHD) patients and associates with increased mortality. IL-1ß, a pro-inflammatory cytokine, is elevated in MHD patients. A balance between IL-1ß and its naturally occurring antagonist may determine the inflammatory response and its consequences in this population. We performed a pilot randomized placebo-controlled trial to evaluate the efficacy of the administration of recombinant human IL-1 receptor antagonist (IL-1ra) on biomarkers of inflammation and nutrition in MHD patients with three consecutive high sensitivity C-reactive protein (hsCRP) measurements >5 mg/L. We randomly assigned 22 patients to placebo or IL-1ra (1:1) for 4 weeks; 14 completed the trial. Patients in the IL-1ra arm had a 53% reduction in mean hsCRP compared with 1% in the placebo arm (P = 0.008), a 40% reduction in mean IL-6 levels compared with a 20% increase in the placebo arm (P = 0.03), and a 23% increase in mean prealbumin compared with 6% in the placebo arm (P = NS). In conclusion, the administration of IL-1ra in MHD patients can lower biomarkers of inflammation. Whether IL-1ra administration improves survival in this population requires additional long-term studies.

The NOTCH pathway contributes to cell fate decision in myelopoiesis.

BACKGROUND: Controversy persists regarding the role of Notch signaling in myelopoiesis. We have used genetic approaches, employing two Notch zebrafish mutants deadly seven (DES) and beamter (BEA) with disrupted function of notch1a and deltaC, respectively, and Notch1a morphants to analyze the development of leukocyte populations in embryonic and mature fish. DESIGN AND METHODS: Myelomonocytes were quantified in early embryos by in situ hybridization using a myeloper-oxidase (mpx) probe. Morpholinos were used to knock down expression of Notch1a or DeltaC. Wound healing assays and/or flow cytometry were used to quantify myelomonocytes in 5-day post-fertilization (dpf) Notch mutants (BEA and DES), morphants or pu.1:GFP, mpx:GFP and fms:RFP transgenic embryos. Flow cytometry was performed on 2-3 month old mutant fish. RESULTS: The number of mpx(+) cells in embryos was reduced at 48 hpf (but not at 26 hpf) in DES compared to WT. At 5 dpf this was reflected by a reduction in the number of myelomonocytic cells found at the wound site in mutants and in Notch1a morphants. This was due to a reduced number of myelomonocytes developing rather than a deficit in the migratory ability since transient inhibition of Notch signaling using DAPT had no effect. The early deficit in myelopoiesis was maintained into later life, 2-3 month old BEA and DES fish having a decreased proportion of myelomonocytes in both the hematopoietic organ (kidney marrow) and the periphery (coelomic cavity). CONCLUSIONS: Our results indicate that defects in Notch signaling affect definitive hematopoiesis, altering myelopoiesis from the early stages of development into the adult.

Lysophospholipid micelles sustain the stability and catalytic activity of diacylglycerol kinase in the absence of lipids.

There has been a renewal of interest in interactions of membrane proteins with detergents and lipids, sparked both by recent results that illuminate the structural details of these interactions and also by the realization that some experimental membrane protein structures are distorted by detergent-protein interactions. The integral membrane enzyme diacylglycerol kinase (DAGK) has long been thought to require the presence of lipid as an obligate "cofactor" in order to be catalytically viable in micelles. Here, we report that near-optimal catalytic properties are observed for DAGK in micelles composed of lysomyristoylphosphatidylcholine (LMPC), with significant activity also being observed in micelles composed of lysomyristoylphosphatidylglycerol and tetradecylphosphocholine. All three of these detergents were also sustained high stability of the enzyme. NMR measurements revealed significant differences in DAGK-detergent interactions involving LMPC micelles versus micelles composed of dodecylphosphocholine. These results highlight the fact that some integral membrane proteins can maintain native-like properties in lipid-free detergent micelles and also suggest that C(14)-based detergents may be worthy of more widespread use in studies of membrane proteins.

Translational bypass of nonsense mutations in zebrafish rep1, pax2.1 and lamb1 highlights a viable therapeutic option for untreatable genetic eye disease.

The extensive molecular genetic heterogeneity seen with inherited eye disease is a major barrier to the development of gene-based therapeutics. The underlying molecular pathology in a considerable proportion of these diseases however are nonsense mutations leading to premature termination codons. A therapeutic intervention targeted at this abnormality would therefore potentially be relevant to a wide range of inherited eye diseases. We have taken advantage of the ability of aminoglycoside drugs to suppress such nonsense mutations and partially restore full-length, functional protein in a zebrafish model of choroideraemia (chm(ru848); juvenile chorio-retinal degeneration) and in two models of ocular coloboma (noi(tu29a) and gup(m189); congenital optic fissure closure defects). In vitro cell-based assays showed significant readthrough with two drugs, gentamicin and paromomycin, which was confirmed by western blot and in vitro prenylation assays. The presence of either aminoglycoside during zebrafish development in vivo showed remarkable prevention of mutant ocular phenotypes in each model and a reduction in multisystemic defects leading to a 1.5-1.7-fold increase in survival. We also identified a significant reduction in abnormal cell death shown by TUNEL assay. To test the hypothesis that optic fissure closure was apoptosis-dependent, the anti-apoptotic agents, curcumin and zVAD-fmk, were tested in gup(m189) embryos. Both drugs were found to reduce the size of the coloboma, providing molecular evidence that cell death is required for optic fissure remodelling. These findings draw attention to the value of zebrafish models of eye disease as useful preclinical drug screening tools in studies to identify molecular mechanisms amenable to therapeutic intervention.

Linear electrode arrays for stimulation and recording within cardiac tissue space constants.

In this paper, we document a fabrication process that yields linear arrays of rectangular platinum black electrodes spaced 25 mum apart with edge-to-edge separation of 20 microm. The spatial arrangement is therefore sufficiently fine to insure stimulation and recording within cardiac tissue space constants, as six electrodes with dimensions of either 5 x 100 microm2, 5 x 250 microm2, or 5 x 500 microm2 were positioned in a 130-microm2 span in the arrays. Despite the small electrode sizes and available surface areas, favorable impedance characteristics were identifed. Averages ranged from 111 kOmega to 146 kOmega at 0.5 Hz and from 14 kOmega 39 kOmega at 500 Hz. Differences in impedances between the electrode sizes tested were small. Potential differences (deltaphis) recorded using the two central electrodes during stimulation with combinations at separations of only 75 microm, 100 microm, and 125 microm had low signal noise. As a preliminary test of the use of these arrays for possible application to impedance measurements in cardiac tissue, the deltaphis recorded during stimulation were compared to deltaphis obtained from finite-difference simulations using an isotropic volume conductor model. Anticipated decays in deltaphi with widening electrode separation identified in those simulations matched the decays in the recorded deltaphis closely. These findings are significant because they suggest intracellular and interstitial microimpedance mesurements in heart experiments will be straightforward.

Tissue sodium accumulation and peripheral insulin sensitivity in maintenance hemodialysis patients.

BACKGROUND: Recent data suggest that sodium (Na+ ) is stored in the muscle and skin without commensurate water retention in maintenance hemodialysis (MHD) patients. In this study, we hypothesized that excessive Na+ accumulation would be associated with abnormalities in peripheral insulin action. METHODS: Eleven MHD patients and eight controls underwent hyperinsulinemic-euglycemic-euaminoacidemic clamp studies to measure glucose (GDR) and leucine disposal rates (LDR), as well as lower left leg 23 Na magnetic resonance imaging to measure Na+ concentration in the muscle and skin tissue. RESULTS: The median GDR and LDR levels were lower, and the median muscle Na+ concentration was higher in MHD patients compared with controls. No significant difference was found regarding skin Na+ concentration between group comparisons. Linear regression revealed inverse relationships between muscle Na+ concentration and GDR and LDR in MHD patients, whereas no relationship was observed in controls. There was no association between skin Na+ content and GDR or LDR in either MHD patients or controls. CONCLUSIONS: These data suggest that excessive muscle Na+ content might be a determinant of IR in MHD patients, although the causality and mechanisms remain to be proven.

Systemic inflammation is associated with exaggerated skeletal muscle protein catabolism in maintenance hemodialysis patients.

BACKGROUND: Systemic inflammation and muscle wasting are highly prevalent and coexist in patients on maintenance hemodialysis (MHD). We aimed to determine the effects of systemic inflammation on skeletal muscle protein metabolism in MHD patients. METHODS: Whole body and skeletal muscle protein turnover were assessed by stable isotope kinetic studies. We incorporated expressions of E1, E214K, E3αI, E3αII, MuRF-1, and atrogin-1 in skeletal muscle tissue from integrin ß1 gene KO CKD mice models. RESULTS: Among 129 patients with mean (± SD) age 47 ± 12 years, 74% were African American, 73% were male, and 22% had diabetes mellitus. Median high-sensitivity C-reactive protein (hs-CRP) concentration was 13 (interquartile range 0.8, 33) mg/l. There were statistically significant associations between hs-CRP and forearm skeletal muscle protein synthesis, degradation, and net forearm skeletal muscle protein balance (P < 0.001 for all). The associations remained statistically significant after adjustment for clinical and demographic confounders, as well as in sensitivity analysis, excluding patients with diabetes mellitus. In attempting to identify potential mechanisms involved in this correlation, we show increased expressions of E1, E214K, E3αI, E3αII, MuRF-1, and atrogin-1 in skeletal muscle tissue obtained from an animal model of chronic kidney disease. CONCLUSION: These data suggest that systemic inflammation is a strong and independent determinant of skeletal muscle protein homeostasis in MHD patients, providing rationale for further studies using anticytokine therapies in patients with underlying systemic inflammation. FUNDING: This study was in part supported by NIH grants R01 DK45604 and 1K24 DK62849, the Clinical Translational Science Award UL1-TR000445 from the National Center for Advancing Translational Sciences, the Veterans Administration Merit Award I01 CX000414, the SatelliteHealth Normon Coplon Extramural Grant Program, and the FDA grant 000943.

Leucine disposal rate for assessment of amino acid metabolism in maintenance hemodialysis patients.

BACKGROUND: Protein energy wasting (PEW) is common in patients undergoing maintenance hemodialysis (MHD) and closely associated with poor outcomes. Insulin resistance and associated alterations in amino acid metabolism are potential pathways leading to PEW. We hypothesized that the measurement of leucine disposal during a hyperinsulinemic- euglycemic-euaminoacidemic clamp (HEAC) procedure would accurately measure the sensitivity to insulin for its actions on concomitant carbohydrate and protein metabolism in MHD patients. METHODS: We examined 35 MHD patients and 17 control subjects with normal kidney function by hyperinsulinemic-euglycemic clamp (HEGC) followed by HEAC clamp procedure to obtain leucine disposal rate (LDR) along with isotope tracer methodology to assess whole body protein turnover. RESULTS: The glucose disposal rate (GDR) by HEGC was 5.1 ± 2.1 mg/kg/min for the MHD patients compared to 6.3 ± 3.9 mg/kg/min for the controls (p = 0.38). The LDR during HEAC was 0.09 ± 0.03 mg/kg/min for the MHD patients compared to 0.11 ± 0.05 mg/kg/min for the controls (p = 0.009). The LDR level was correlated with whole body protein synthesis (r = 0.25; p = 0.08), with whole body protein breakdown (r = -0.38 p = 0.01) and net protein balance (r = 0.85; p < 0.001) in the overall study population. Correlations remained significant in subgroup analysis. The GDR derived by HEGC and LDR correlated well in the controls (r = 0.79, p < 0.001), but less so in the MHD patients (r = 0.58, p < 0.001). CONCLUSIONS: Leucine disposal rate reliably measures amino acid utilization in MHD patients and controls in response to high dose insulin.

High Dose Omega-3 Fatty Acid Administration and Skeletal Muscle Protein Turnover in Maintenance Hemodialysis Patients.

BACKGROUND AND OBJECTIVES: Protein energy wasting and systemic inflammation are prevalent in maintenance hemodialysis (MHD) patients. Omega-3 (ω-3) fatty acids have anti-inflammatory properties and have been shown to improve protein homeostasis. We hypothesized that administration of high-dose (2.9 g/d) ω-3 would be associated with decreased muscle protein breakdown in MHD patients with systemic inflammation. DESIGN, SETTING, PARTICIPANTS & MEASUREMENTS: This is a substudy from a randomized, placebo-controlled study (NCT00655525). Patients were recruited between September 2008 and June 2011. Primary inclusion criteria included signs of chronic inflammation (average C-reactive protein of ≥5 mg/L over three consecutive measurements), lack of active infectious or inflammatory disease, no hospitalization within 1 month prior to the study, and not receiving steroids (>5 mg/d) and/or immunosuppressive agents. The primary outcomes were forearm muscle and whole body protein breakdown and synthesis before and after the intervention. The patients received ω-3 (n=11) versus placebo (n=9) for 12 weeks. Analysis of covariance was used to compare outcome variables at 12 weeks. Models were adjusted for a propensity score that was derived from age, sex, race, baseline high sensitivity C-reactive protein, diabetes mellitus, and fat mass because the groups were not balanced for several characteristics. RESULTS: Compared with placebo, ω-3 supplementation was significantly associated with decreased muscle protein breakdown at 12 weeks (-31, [interquartile range, -98--13] versus 26 [interquartile range, 13-87] µg/100 ml per min; P=0.01), which remained significant after multivariate adjustment (-46, [95% confidence interval, -102 to -1] µg/100 ml per min). ω-3 Supplementation resulted in decreased forearm muscle protein synthesis while the rate in the placebo group increased; however, there is no longer a statistically significant difference in skeletal muscle protein synthesis or in net protein balance after multivariate adjustment. There was no statistically significant effect of ω-3 supplementation on whole body protein synthesis or breakdown. CONCLUSIONS: High-dose ω-3 supplementation over 12 weeks in MHD patients with systemic inflammation was associated with attenuation of forearm muscle protein breakdown but did not influence skeletal muscle protein synthesis, skeletal muscle net protein balance or any component of the whole-body protein balance. These results should be interpreted cautiously given the imbalance in the two groups and the short duration of the intervention.

Measurement and Correlation of Indices of Insulin Resistance in Patients on Peritoneal Dialysis.

UNLABELLED: ♦ BACKGROUND: Insulin resistance (IR) is common in maintenance dialysis patients and is associated with excess mortality. Hyperinsulinemic euglycemic glucose clamp (HEGC) is the gold standard for measuring IR. There are limited studies using HEGC for comparison to other indirect indices of IR in peritoneal dialysis (PD) patients, nor have there been direct comparisons between patients receiving PD and those on maintenance hemodialysis (MHD) with regard to severity of IR, methods of measurement, or factors associated with the development of IR. ♦ METHODS: This is a cross-sectional, single-center study performed in 10 prevalent PD patients of median age 48 years (range 41 - 54); 50% were female and 60% were African American. Insulin resistance was assessed by HEGC (glucose disposal rate [GDR]), homeostatic model assessment of IR (HOMA-IR), HOMA-IR corrected by adiponectin (HOMA-AD), leptin adiponectin ratio (LAR), quantitative insulin sensitivity check index (QUICKI), McAuley's index, and oral glucose tolerance test (OGTT) at each time point for a total of 18 studies. Retrospective analysis compared this cohort to 12 hemodialysis patients who had previously undergone similar testing. ♦ RESULTS: The median GDR was 6.4 mg/kg/min (interquartile range [IQR] 6.0, 7.8) in the PD cohort compared with the MHD group, which was 5.7 mg/kg/min (IQR 4.3, 6.6). For both the PD and MHD cohorts, the best predictors of GDR by HEGC after adjusting for age, gender, and body mass index (BMI), were HOMA-AD (PD: r = -0.69, p = 0.01; MHD: r = -0.78, p = 0.03) and LAR (PD: r = -0.68, p < 0.001; MHD: r = -0.65, p = 0.04). In both groups, HOMA-IR and QUICKI failed to have strong predictive value. Eight of 10 PD patients had at least 1 abnormal OGTT, demonstrating impaired glucose tolerance. ♦ CONCLUSIONS: Insulin resistance is highly prevalent in PD patients. The adipokine based formulas, HOMA-AD and LAR, correlated well in both the PD and MHD populations in predicting GDR by HEGC, outperforming HOMA-IR. The use of these novel markers could be considered for large-scale, epidemiological outcome studies.

Membrane protein preparation for TROSY NMR screening.

The first steps toward undertaking an NMR structural study of a new protein is very often to purify the protein and then to acquire an HSQC or TROSY NMR spectrum, the quality of which is used to assess the feasibility of an NMR-based structural determination. Relatively few integral membrane proteins (IMPs) have been subjected even to this very preliminary stage of NMR analysis. Here, NMR feasibility testing methods are outlined that are tailored for hexahistidine-tagged IMPs that have been expressed in Escherichia coli. Generally applicable protocols are presented for expression testing, purification, and NMR sample preparation. A 2D TROSY pulse sequence that has been optimized for use with IMPs is also presented.

Administration of IL-1ra improves adiponectin levels in chronic hemodialysis patients.

BACKGROUND: Adiponectin, an adipose tissue derived hormone, is known to have insulin-sensitizing, anti-inflammatory, and anti-atherogenic properties in the general population. Adiponectin secretion is suppressed by systemic inflammation, a highly prevalent condition in maintenance hemodialysis (MHD) patients. We evaluated whether short-term administration of interleukin 1 receptor antagonist (IL-1ra) improves adiponectin levels and insulin sensitivity in MHD patients. METHODS: Ad hoc analysis was performed on a pilot randomized placebo-controlled trial of the administration of IL-1ra in chronically inflamed MHD patients. Twenty-two patients were randomly assigned to receive 100 mg of IL-1ra or placebo (1:1) for 4 weeks, and 14 completed the trial. ANCOVA was used to compare percent change from baseline to 4 weeks. The primary outcome was percent change in adiponectin and the secondary outcomes were changes in leptin, homeostatic model assessment of insulin resistance (HOMA-IR) and the leptin-to-adiponectin ratio (LAR). RESULTS: Patients' mean age was 49 ± 13 years, and 71 % were males. At baseline, the median values for adiponectin, leptin, LAR and HOMA-IR were 11.5 µg/ml [interquartile range (IQR) 9, 28.5], 17.8 ng/ml (3.9, 50.0), 2.20 (0.13, 3.98), and 2.8 (2.0, 3.6), respectively. IL-1ra administration resulted in a mean percent increase in serum adiponectin of 22 % vs. 14 % decrease in the placebo arm (p = 0.003). Leptin, LAR or HOMA-IR levels did not change in either arm. CONCLUSIONS: Short-term administration of IL-1ra significantly increased adiponectin levels among prevalent MHD patients. The intervention did not impact insulin sensitivity parameters. Studies of longer duration and larger sample size are needed to further evaluate the potential effect of anti-inflammatory interventions on metabolic markers and insulin sensitivity in MHD patients.

A comparison of novel and commonly-used indices of insulin sensitivity in African American chronic hemodialysis patients.

BACKGROUND: Insulin resistance (IR) is highly prevalent in chronic hemodialysis (CHD) patients and is associated with poor cardiovascular outcomes. Hyperinsulinemic euglycemic glucose clamp (HEGC) is the gold standard for measuring IR. The comparison of commonly-used indirect indices of IR to HEGC has not been adequately performed in this population. Furthermore, the validity of newly proposed adipokine-based IR indices has not been explored. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This is an observational study performed in a single center, involving 12 prevalent CHD patients (50 ± 9 years old, 100% African American, 33% women, body mass index of 34.4 ± 7.6 kg/m(2)) who were studied three consecutive times. IR was assessed by HEGC (glucose-disposal rate [GDR]), homeostatic model assessment of IR (HOMA-IR), HOMA-IR corrected by adiponectin (HOMA-AD), leptin adiponectin ratio (LAR), QUICKI, and the McAuley's index at each time point. RESULTS: Eighty-three percent of the subjects displayed either glucose intolerance or overt insulin resistance by HEGC (GDR median, 5.71; interquartile range [IQR], 4.16, 6.81). LAR and HOMA-AD were the best correlates of IR measured by HEGC (r=-0.72, P<0.001, and -0.67, P<0.001), respectively. Fat percentage, interleukin-6, and adipokines (leptin, adiponectin, and resistin) were strongly associated with GDR. HEGC, LAR, and HOMA-AD had the best intraclass correlation coefficients. CONCLUSION: IR is common in CHD patients. Adipokine-based indices are the best correlates of IR measurements by HEGC. HOMA-IR and QUICKI are reasonable alternatives. Use of these indices may allow better detection of alterations in insulin sensitivity in CHD patients.

Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase.

Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK's three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK's lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis.