Carole Bennett

Sarcopenia associated with portosystemic shunting is reversed by follistatin

BACKGROUND & AIMS The distinct role of portosystemic shunting (PSS) in the pathogenesis of sarcopenia (skeletal muscle loss) that occurs commonly in cirrhosis is unclear. We have previously shown increased expression of myostatin (inhibitor of skeletal muscle mass) in the portacaval anastamosis (PCA) rat model of sarcopenia of PSS. The present study was performed to examine the mechanisms of sarcopenia following PCA. METHODS In PCA and sham operated pair fed control rats, the phenylalanine flooding dose method was used to quantify the fractional and absolute protein synthesis rates in the skeletal muscle over time and in response to follistatin, a myostatin antagonist. The expression of myostatin and markers of satellite cell (myocyte precursors) proliferation and differentiation were quantified by real-time PCR and Western blot analyses. RESULTS The absolute synthesis rate (ASR) was lower at 2, 4, and 6 weeks (p<0.05) and the fractional synthesis rate (FSR) of skeletal muscle protein was significantly lower (p<0.05) at week 2 in the PCA rats compared to control rats. Expression of myostatin was elevated while markers of satellite cell proliferation and differentiation were lower at 4 and 6 weeks after PCA. Follistatin increased skeletal muscle mass, muscle FSR and ASR, decreased expression of myostatin protein, and increased expression of markers of satellite cell function. CONCLUSIONS Sarcopenia associated with PSS is caused by impaired protein synthesis and reduced satellite cell function due to increased myostatin expression. Confirming these alterations in human patients with cirrhosis will provide novel therapeutic targets for sarcopenia of liver disease.

Elevated hepatic fatty acid oxidation, high plasma fibroblast growth factor 21, and fasting bile acids in nonalcoholic steatohepatitis

Background Data from studies in patients with nonalcoholic steatohepatitis (NASH) suggest an increased hepatic fatty acid oxidation. We have previously shown higher fasting plasma bile acid concentrations in patients with NASH. In-vivo and in-vitro studies suggest that bile acids by binding to peroxisome proliferator-activated receptor &agr; activate fibroblast growth factor 21 (FGF21) and increase hepatic fatty acid oxidation. Methods Plasma bile acid levels were quantified in healthy controls (n=38) and patients with biopsy-proven NASH (n=36). Plasma concentration of fatty acids, &bgr;-hydroxybutyrate, insulin, glucose, leptin, alanine aminotransferase, FGF21, and 8-hydroxydeoxyguanosine, a measure of oxidative stress, were measured in 16 healthy controls and 10 patients with NASH in the fasted state and in response to 3 h of infusion of intralipid. In a subgroup of these patients (n=6 each), plasma ceramide subspecies were quantified. Results Fasting plasma bile acids, FGF21, and leptin concentrations were significantly higher in patients with NASH. In response to intralipid infusion there was an increase in plasma &bgr;-hydroxybutyrate and free fatty acid levels in both controls and NASH; however, the ratio of &bgr;-hydroxybutyrate/free fatty acid was higher in NASH (P=0.02). Plasma FGF21 concentration increased in response to intralipid in patients with NASH only (P<0.01). Plasma leptin, insulin, glucose, and alanine transferase concentrations did not change in either group after infusion of intralipid. Increase in total ceramides in response to intralipid was greater in NASH. Conclusion Elevated bile acids and FGF21 may be responsible for the higher hepatic fatty acid oxidation in NASH.

Methionine and Protein Metabolism in Non Alcoholic Steatohepatitis: Evidence for Lower Rate of Transmethylation of Methionine

Hepatic metabolism of methionine is the source of cysteine, the precursor of glutathione, the major intracellular antioxidant in the body. Methionine also is the immediate precursor of SAM (S-adenosylmethionine) the key methyl donor for phosphatidylcholine synthesis required for the export of VLDL (very-low-density lipoprotein) triacylglycerols (triglycerides) from the liver. We have examined the kinetics of methionine, its transmethylation and trans-sulfuration with estimates of whole body rate of protein turnover and urea synthesis in clinically stable biopsy-confirmed subjects with NASH (non-alcoholic steatohepatitis). Subjects with NASH were more insulin-resistant and had significantly higher plasma concentrations of usCRP (ultrasensitive C-reactive protein), TNFα (tumour necrosis factor α) and other inflammatory cytokines. There was no significant effect of insulin resistance and NASH on whole body rate of protein turnover [phenylalanine Ra (rate of appearance)] and on the rate of urea synthesis. The rates of methylation of homocysteine and transmethylation of methionine were significantly lower in NASH compared with controls. There was no difference in the rate of trans-sulfuration of methionine between the two groups. Enteric mixed nutrient load resulted in a significant increase in all the measured parameters of methionine kinetics. Heterozygosity for MTHFR (5,10-methylene-tetrahydrofolate reductase) (677C→T) did not have an impact on methionine metabolism. We speculate that, as a result of oxidant stress possibly due to high fatty acid oxidation, the activity of methionine adenosyltransferase is attenuated resulting in a lower rate of transmethylation of methionine and of SAM synthesis. These results are the first evidence for perturbed metabolism of methionine in NASH in humans and provide a rationale for the development of targeted intervention strategies.

Metabolic and Genomic Response to Dietary Isocaloric Protein Restriction in the Rat

We have examined hepatic, genomic, and metabolic responses to dietary protein restriction in the non-pregnant Sprague-Dawley rat. Animals were pair-fed either a 6 or 24% casein-based diet for 7–10 days. At the end of the dietary period, a microarray analysis of the liver was performed, followed by validation of the genes of interest. The rates of appearance of phenylalanine, methionine, serine, and glucose and the contribution of pyruvate to serine and glucose were quantified using tracer methods. Plasma and tissue amino acid levels, enzyme activities, and metabolic intermediates were measured. Protein restriction resulted in significant differential expression of a number of genes involved in cell cycle, cell differentiation, transport, transcription, and metabolic processes. RT-PCR showed that the expression of genes involved in serine biosynthesis and fatty acid oxidation was higher, and those involved in fatty acid synthesis and urea synthesis were lower in the liver of protein-restricted animals. Free serine and glycine levels were higher and taurine levels lower in all tissues examined. Tracer isotope studies showed an ∼50% increase in serine de novo synthesis. Pyruvate was the primary (∼90%) source of serine in both groups. Transmethylation of methionine was significantly higher in the protein-restricted group. This was associated with a higher S-adenosylmethionine/S-adenosylhomocysteine ratio and lower cystathione β-synthase and cystathionine γ-lyase activity. Dietary isocaloric protein restriction results in profound changes in hepatic one-carbon metabolism within a short period. These may be related to high methylation demands placed on the organism and caused by possible changes in cellular osmolarity as a result of the efflux of the intracellular taurine.

Metabolomic Endotype of Asthma

Metabolomics, the quantification of small biochemicals in plasma and tissues, can provide insight into complex biochemical processes and enable the identification of biomarkers that may serve as therapeutic targets. We hypothesized that the plasma metabolome of asthma would reveal metabolic consequences of the specific immune and inflammatory responses unique to endotypes of asthma. The plasma metabolomic profiles of 20 asthmatic subjects and 10 healthy controls were examined using an untargeted global and focused metabolomic analysis. Individuals were classified based on clinical definitions of asthma severity or by levels of fraction of exhaled NO (FENO), a biomarker of airway inflammation. Of the 293 biochemicals identified in the plasma, 25 were significantly different among asthma and healthy controls (p < 0.05). Plasma levels of taurine, lathosterol, bile acids (taurocholate and glycodeoxycholate), nicotinamide, and adenosine-5-phosphate were significantly higher in asthmatics compared with healthy controls. Severe asthmatics had biochemical changes related to steroid and amino acid/protein metabolism. Asthmatics with high FENO, compared with those with low FENO, had higher levels of plasma branched-chain amino acids and bile acids. Asthmatics have a unique plasma metabolome that distinguishes them from healthy controls and points to activation of inflammatory and immune pathways. The severe asthmatic and high FENO asthmatic have unique endotypes that suggest changes in NO-associated taurine transport and bile acid metabolism.

Plasma levels of asymmetric dimethylarginine in patients with biopsy-proven nonalcoholic fatty liver disease

Asymmetric (ADMA) and symmetric dimethylarginine (SDMA) are produced by breakdown of proteins that have been methylated posttranslationally at an arginine residue. Plasma levels of ADMA are elevated in insulin resistance states. Nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance and varying degrees of hepatic dysfunction. Because ADMA is metabolized in the liver, we hypothesized that ADMA levels will be high in patients with NAFLD as a consequence of hepatic dysfunction and insulin resistance. Plasma levels of ADMA, SDMA, total homocysteine, glucose, and insulin were measured in nondiabetic patients with biopsy-proven NAFLD (11 steatosis and 24 nonalcoholic steatohepatitis) and 25 healthy subjects. Plasma ADMA levels were significantly higher (P = .029) in patients with biopsy-proven NAFLD (0.43 ± 0.21 μmol/L) compared with controls (0.34 ± 0.10 μmol/L). However, when adjusted for insulin resistance (homeostasis model assessment), the difference between 2 groups was not evident. Plasma SDMA levels were similar in all 3 groups. Plasma levels of ADMA were positively correlated with plasma total homocysteine levels (P = .003). Plasma levels of SDMA were negatively correlated with estimated glomerular filtration rate (P = .016) and positively correlated with plasma total homocysteine levels (P = .003). The ratio of ADMA/SDMA was positively correlated with body mass index (P = .027). Elevated plasma concentrations of ADMA in biopsy-proven NAFLD were primarily related to insulin resistance. Hepatic dysfunction in NAFLD does not appear to make significant contribution to changes in plasma methylarginine levels.

All-trans Retinoic Acid Induces Arginase-1 and Inducible Nitric Oxide Synthase–Producing Dendritic Cells with T Cell Inhibitory Function

Hepatic stellate cells (HSC) are a major source of the immunoregulatory metabolite all-trans retinoic acid (ATRA), which may contribute to the generation of tolerogenic dendritic cells (DCs) in the liver. The present study seeks to clarify the mechanism(s) through which ATRA promotes the development of tolerogenic DCs. Although bone marrow–derived ATRA-treated DCs (RA-DCs) and conventional DCs had comparable surface phenotype, RA-DCs had diminished stimulatory capacity and could directly inhibit the expansion of DC/OVA-stimulated OT-II T cells. Arginase-1 (Arg-1) was found promote suppression because 1) ATRA was a potent inducer of Arg-1 protein and activity, 2) the Arg-1 inhibitor Nw-hydroxy nor-l-arginine partially reversed suppression, and 3) the suppressive function of RA-DCs was partially compromised using OT-II T cells from GCN2−/− mice, which are insensitive to Arg-1. Inducible NO synthase (iNOS), however, was found to be a more significant contributor to RA-DC function because 1) ATRA potentiated the expression of IFN-γ–induced iNOS, 2) suppressive function in RA-DCs was blocked by the iNOS inhibitor NG-monomethyl-l-arginine, monoacetate salt, and 3) RA-DCs derived from iNOS−/− mice exhibited near complete loss of tolerogenic function, despite sustained Arg-1 activity. The expression of iNOS and the suppressive function of RA-DCs were dependent on both IFN-γ and ATRA. Furthermore, the in vivo behavior of RA-DCs proved to be consistent with their in vitro behavior. Thus, we conclude that ATRA enhances both Arg-1 and iNOS expression in IFN-γ–treated DCs, resulting in a tolerogenic phenotype. These findings elucidate mechanisms through which ATRA may contribute to liver immune tolerance.

Glycine and urea kinetics in nonalcoholic steatohepatitis in human: effect of intralipid infusion

The rates of oxidation of glycine and ureagenesis were quantified in the basal state and in response to an intravenous infusion of intralipid with heparin (IL) in healthy subjects (n = 8) and in subjects with nonalcoholic steatohepatitis (NASH) (n = 6). During fasting, no significant difference in weight-specific rate of appearance (R(a)) of glycine, glycine oxidation, and urea synthesis was observed. Intralipid infusion resulted in a significant increase in plasma beta-hydroxybutyrate in both groups. The correlation between free fatty acids and beta-hydroxybutyrate concentration in plasma was 0.94 in NASH compared with 0.4 in controls, indicating greater hepatic fatty acid oxidation in NASH. Intralipid infusion resulted in a significant decrease in urea synthesis and glycine R(a) in both groups and did not impact glycine oxidation. The fractional contribution of glycine carbon to serine was lower in subjects with NASH before and after IL infusion. In contrast, the fractional contribution of serine carbon to cystathionine was higher in NASH before and following IL infusion. These results suggest that hepatic fatty acid oxidation is higher in NASH compared with controls and that glycine oxidation and urea synthesis are not altered. An increase in oxidative stress, induced by a higher rate of fatty acid oxidation in NASH, may have caused an increase in the contribution of serine to cystathionine to meet the higher demands for glutathione.

Increased mitochondrial arginine metabolism supports bioenergetics in asthma

High levels of arginine metabolizing enzymes, including inducible nitric oxide synthase (iNOS) and arginase (ARG), are typical in asthmatic airway epithelium; however, little is known about the metabolic effects of enhanced arginine flux in asthma. Here, we demonstrated that increased metabolism sustains arginine availability in asthmatic airway epithelium with consequences for bioenergetics and inflammation. Expression of iNOS, ARG2, arginine synthetic enzymes, and mitochondrial respiratory complexes III and IV was elevated in asthmatic lung samples compared with healthy controls. ARG2 overexpression in a human bronchial epithelial cell line accelerated oxidative bioenergetic pathways and suppressed hypoxia-inducible factors (HIFs) and phosphorylation of the signal transducer for atopic Th2 inflammation STAT6 (pSTAT6), both of which are implicated in asthma etiology. Arg2-deficient mice had lower mitochondrial membrane potential and greater HIF-2α than WT animals. In an allergen-induced asthma model, mice lacking Arg2 had greater Th2 inflammation than WT mice, as indicated by higher levels of pSTAT6, IL-13, IL-17, eotaxin, and eosinophils and more mucus metaplasia. Bone marrow transplants from Arg2-deficient mice did not affect airway inflammation in recipient mice, supporting resident lung cells as the drivers of elevated Th2 inflammation. These data demonstrate that arginine flux preserves cellular respiration and suppresses pathological signaling events that promote inflammation in asthma.

Arginine metabolic endotypes related to asthma severity

Aims Arginine metabolism via inducible nitric oxide synthase (iNOS) and arginase 2 (ARG2) is higher in asthmatics than in healthy individuals. We hypothesized that a sub-phenotype of asthma might be defined by the magnitude of arginine metabolism categorized on the basis of high and low fraction of exhaled nitric oxide (FENO). Methods To test this hypothesis, asthmatics (n = 52) were compared to healthy controls (n = 51) for levels of FENO, serum arginase activity, and airway epithelial expression of iNOS and ARG2 proteins, in relation to clinical parameters of asthma inflammation and airway reactivity. In parallel, bronchial epithelial cells were evaluated for metabolic effects of iNOS and ARG2 expression in vitro. Results Asthmatics with high FENO (≥ 35 ppb; 44% of asthmatics) had higher expression of iNOS (P = 0.04) and ARG2 (P = 0.05) in the airway, indicating FENO is a marker of the high arginine metabolic endotype. High FENO asthmatics had the lowest FEV1% (P < 0.001), FEV1/FVC (P = 0.0002) and PC20 (P < 0.001) as compared to low FENO asthmatics or healthy controls. Low FENO asthmatics had near normal iNOS and ARG2 expression (both P > 0.05), and significantly higher PC20 (P < 0.001) as compared to high FENO asthmatics. In vitro studies to evaluate metabolic effects showed that iNOS overexpression and iNOS+ARG2 co-expression in a human bronchial epithelial cell line led to greater reliance on glycolysis with higher rate of pyruvate going to lactate. Conclusions The high FENO phenotype represents a large portion of the asthma population, and is typified by greater arginine metabolism and more severe and reactive asthma.