A transcriptional evaluation of the melanoma and squamous cell carcinoma TIL compartment reveals an unexpected spectrum of exhausted and functional T cells.

Introduction: Significant heterogeneity exists within the tumor-infiltrating CD8 T cell population, and exhausted T cells harbor a subpopulation that may be replicating and may retain signatures of activation, with potential functional consequences in tumor progression. Dysfunctional immunity in the tumor microenvironment is associated with poor cancer outcomes, making exploration of these exhausted T cell subpopulations critical to the improvement of therapeutic approaches. Methods: To investigate mechanisms associated with terminally exhausted T cells, we sorted and performed transcriptional profiling of CD8+ tumor-infiltrating lymphocytes (TILs) co-expressing the exhaustion markers PD-1 and TIM-3 from large-volume melanoma tumors. We additionally performed immunologic phenotyping and functional validation, including at the single-cell level, to identify potential mechanisms that underlie their dysfunctional phenotype. Results: We identified novel dysregulated pathways in CD8+PD-1+TIM-3+ cells that have not been well studied in TILs; these include bile acid and peroxisome pathway-related metabolism and mammalian target of rapamycin (mTOR) signaling pathways, which are highly correlated with immune checkpoint receptor expression. Discussion: Based on bioinformatic integration of immunophenotypic data and network analysis, we propose unexpected targets for therapies to rescue the immune response to tumors in melanoma.

Association of pancreatic fat on imaging with pediatric metabolic co-morbidities.

BACKGROUND: The relationship between pancreatic fat on imaging and metabolic co-morbidities has not been established in pediatrics. We sought to investigate the relationship between pancreatic fat measured by MRI and endocrine/exocrine dysfunctions along with the metabolic co-morbidities in a cohort of children. OBJECTIVE: To investigate relationships between pancreatic fat quantified by MRI and endocrine and exocrine conditions and metabolic co-morbidities in a cohort of children. MATERIALS AND METHODS: This was a retrospective review of pediatric patients (n = 187) who had a clinically indicated MRI examination between May 2018 and February 2020. After 51 patients without useable imaging data were excluded, the remaining 136 subjects comprised the study sample. Laboratory studies were assessed if collected within 6 months of MRI and patient charts were reviewed for demographic and clinical information. MRI proton density fat fraction (PDFF) sequence had been acquired according to manufacturer's specified parameters at a slice thickness of 3 mm. Two blinded radiologists independently collected PDFF data. RESULTS: The median age at MRI was 12.1 (IQR: 9.0-14.8) years and the majority of patients were Caucasian (79%), followed by African American and Hispanic at 12% and 11% respectively. There was a higher median pancreas fat fraction in patients with exocrine conditions (chronic pancreatitis or exocrine insufficiency) compared to those without (3.5% vs 2.2%, p = 0.03). There was also a higher median fat fraction in the head of pancreas in patients with endocrine insufficient conditions (insulin resistance, pre-diabetes, type 1 and type 2 diabetes) compared to those without endocrine insufficiency when excluding patients with active acute pancreatitis (3.5% vs 2.0%, p = 0.04). Patients with BMI > 85% had higher mean fat fraction compared to patients with BMI ≤ 85% (head: 3.8 vs 2.4%, p = 0.01; body: 3.8 vs 2.5%, p = 0.005; tail: 3.7 vs 2.7%, p = 0.049; overall pancreas fat fraction: 3.8 vs 2.6%, p = 0.002). CONCLUSION: Pancreas fat is elevated in patients with BMI > 85% and in those with exocrine and endocrine insufficiencies.

Image or scope: Magnetic resonance imaging and endoscopic testing for exocrine and endocrine pancreatic insufficiency in children.

OBJECTIVES: We sought to evaluate associations between Magnetic Resonance Imaging (MRI) findings, exocrine pancreatic insufficiency (EPI) and endocrine insufficiency (prediabetes or diabetes) in children. METHODS: This was a retrospective study that included patients<21 years of age who underwent MRI and endoscopic pancreatic function testing (ePFT; reference standard for pancreatic exocrine function) within 3 months. MRI variables included pancreas parenchymal volume, secreted fluid volume in response to secretin, and T1 relaxation time. Data were analyzed for the full sample as well as the subset without acute pancreatitis (AP) at the time of imaging. RESULTS: Of 72 patients, 56% (40/72) were female with median age 11.4 years. A 5 mL decrease in pancreas parenchymal volume was associated with increased odds of exocrine pancreatic dysfunction by both ePFT (OR = 1.16, p = 0.02 full sample; OR = 1.29, p = 0.01 no-AP subset), and fecal elastase (OR = 1.16, p = 0.04 full sample; OR = 1.23, p = 0.02 no-AP subset). Pancreas parenchymal volume had an AUC 0.71 (95% CI: 0.59, 0.83) for predicting exocrine pancreatic dysfunction by ePFT and when combined with sex and presence of AP had an AUC of 0.82 (95% CI: 0.72, 0.92). Regarding endocrine function, decreased pancreas parenchymal volume was associated with increased odds of diabetes (OR = 1.16, p = 0.03), and T1 relaxation time predicted glycemic outcomes with an AUC 0.78 (95% CI: 0.55-1), 91% specificity and 73% sensitivity. CONCLUSIONS: Pancreas parenchymal volume is an MRI marker of exocrine and endocrine pancreatic dysfunction in children. A model including sex, AP, and pancreas volume best predicted exocrine status. T1 relaxation time is also an MRI marker of endocrine insufficiency.

Quantification of Hepatic Steatosis by Ultrasound: Prospective Comparison With MRI Proton Density Fat Fraction as Reference Standard.

BACKGROUND. Multiple ultrasound platforms now provide quantitative measures of hepatic steatosis. One such measure is the ultrasound-derived fat fraction (UDFF), which combines attenuation and backscatter quantification. OBJECTIVE. The purpose of this study was to characterize agreement between UDFF and MRI proton-density fat fraction (PDFF) measurements. METHODS. This prospective cross-sectional study enrolled 56 overweight and obese adolescents and adults (age ≥ 16 years) who underwent investigational ultrasound (deep abdominal transducer) and MRI examinations of the liver during a single visit from August 2020 to October 2020. Ultrasound examinations included three UDFF acquisitions of five measurements each (15 measurements total), and an overall median of medians was computed (UDFFoverall). MRI examinations included three PDFF acquisitions with calculation of an overall median PDFF. Spearman rank-order correlation was computed between UDFF and MRI PDFF measurements. Intraclass correlation coefficients and Bland-Altman difference plots were used to assess agreement. ROC curves were used to assess diagnostic performance of UDFF for detecting MRI PDFF of 5.5% or more. RESULTS. Median participant age was 32.5 years (IQR, 24.0-39.0 years); 40 participants were female, and 16 were male. A total of 34 (60.7%) participants had an MRI PDFF of 5.5% or more. UDFFoverall was 10.5% (IQR, 5.0-20.0%); median MRI PDFF was 6.1% (IQR, 3.4-13.7%). UDFFoverall was positively associated with MRI PDFF (ρ, 0.82; p < .001; intraclass correlation coefficient, 0.84 [95% CI, 0.59-0.93]). Mean bias between UDFF and PDFF was 4.0% (95% limits of agreement, -7.9% to 15.9%), with similar bias if summarizing UDFF by the first five measurements (4.4%), first three measurements (4.4%), or first measurement (4.6%). UDFFoverall AUC was 0.90 (95% CI, 0.79-0.96) for MRI PDFF of 5.5% or more; AUC was not significantly different when it was based on the number of UDFF measurements (p = .11-.97 for all pairwise AUC comparisons). UDFFoverall cutoff of more than 5% had sensitivity of 94.1% and specificity of 63.6% for diagnosing MRI PDFF of 5.5% or more. CONCLUSION. Measurements of hepatic steatosis using UDFF show strong agreement with measurements by MRI PDFF. A UDFFoverall cutoff of more than 5% provides high AUC and sensitivity, albeit low specificity, for detection of MRI PDFF of 5.5% or more. CLINICAL IMPACT. UDFF may have a clinical role in detection of hepatic steatosis. A reduced number of individual measurements is likely sufficient for determining an overall UDFF value. TRIAL REGISTRATION. ClinicalTrials.gov: NCT04523584.

Agreement between serum estimates of glomerular filtration rate (GFR) and a reference standard of radioisotopic GFR in children with cancer.

BACKGROUND: Accurate assessment of renal function is important in the care of children with cancer because renal function has implications for anti-tumor medication dosing and eligibility for clinical trials. OBJECTIVE: To characterize agreement between serum estimates of glomerular filtration rate (GFR) and a reference standard of radioisotopic GFR in a large pediatric oncology cohort. MATERIALS AND METHODS: We conducted a retrospective cross-sectional study of children who had both radioisotopic GFR (99mTc-diethylenetriaminepentaacetic acid, or 99mTc-DTPA) and serum labs (creatinine, cystatin C) obtained <7 days apart between January 2017 and August 2019. We calculated estimated GFR from serum labs using published equations and calculated agreement using intraclass correlation coefficient (ICC) and Bland-Altman analysis with univariate regression to define predictors of agreement. RESULTS: We included 272 pairs of data. Mean patient age was (mean ± standard deviation) 7.8±5.7 years. Mean radioisotopic GFR was 112±33 mL/min/1.73 m2. Absolute agreement between radioisotopic GFR and serum estimates was only fair (ICC=0.46-0.58) with a mean difference of -26.6 to +0.12 mL/min/1.73 m2. For radioisotopic GFR measurements <60 mL/min/1.73 m2, mean differences were greater, with serum estimates overestimating GFR by a mean of 21.5-39.6 mL/min/1.73 m2. In multivariable modeling, significant predictors of agreement included age, height, acute kidney injury and tumor type. Sensitivity of serum estimates was 14-29% for a GFR <60 mL/min/1.73 m2. CONCLUSION: Agreement between radioisotopic GFR and serum estimates of GFR is only fair and serum estimates of GFR have poor sensitivity for clinically relevant GFR <60 mL/min/1.73 m2. Radioisotopic measurement of GFR likely remains necessary to assess renal function in pediatric oncology patients with decreased renal function.

Detection of urinary tract calculi on CT images reconstructed with deep learning algorithms.

BACKGROUND: Deep learning Computed Tomography (CT) reconstruction (DLR) algorithms promise to improve image quality but the impact on clinical diagnostic performance remains to be demonstrated. We aimed to compare DLR to standard iterative reconstruction for detection of urolithiasis by unenhanced CT in children and young adults. METHODS: This was an IRB approved retrospective study involving post-hoc reconstruction of clinically acquired unenhanced abdomen/pelvis CT scans. Images were reconstructed with six different manufacturer-standard DLR algorithms and reformatted in 3 planes (axial, sagittal, and coronal) at 3 mm intervals. De-identified reconstructions were loaded as independent examinations for review by 3 blinded radiologists (R1, R2, R3) tasked with identifying and measuring all stones. Results were compared to the clinical iterative reconstruction images as a reference standard. IntraClass correlation coefficients and kappa (k) statistics were used to quantify agreement. RESULTS: CT data for 14 patients (mean age: 17.3 ± 3.4 years, 5 males and 9 females, weight class: 31-70 kg (n = 6), 71-100 kg (n = 7), > 100 kg (n = 1)) were reconstructed into 84 total exams. 7 patients had urinary tract calculi. Interobserver agreement on the presence of any urinary tract calculus was substantial to almost perfect (k = 0.71-1) for all DLR algorithms. Agreement with the reference standard on number of calculi was excellent (ICC = 0.78-0.96) and agreement on the size of the largest calculus was fair to excellent (ICC = 0.51-0.97) depending on reviewer and DLR algorithm. CONCLUSION: Deep learning reconstruction of unenhanced CT images allows similar renal stone detectability compared to iterative reconstruction.

A high OXPHOS CD8 T cell subset is predictive of immunotherapy resistance in melanoma patients.

Immune checkpoint inhibitor (ICI) therapy continues to revolutionize melanoma treatment, but only a subset of patients respond. Major efforts are underway to develop minimally invasive predictive assays of ICI response. Using single-cell transcriptomics, we discovered a unique CD8 T cell blood/tumor-shared subpopulation in melanoma patients with high levels of oxidative phosphorylation (OXPHOS), the ectonucleotidases CD38 and CD39, and both exhaustion and cytotoxicity markers. We called this population with high levels of OXPHOS "CD8+ TOXPHOS cells." We validated that higher levels of OXPHOS in tumor- and peripheral blood-derived CD8+ TOXPHOS cells correlated with ICI resistance in melanoma patients. We then developed an ICI therapy response predictive model using a transcriptomic profile of CD8+ TOXPHOS cells. This model is capable of discerning responders from nonresponders using either tumor or peripheral blood CD8 T cells with high accuracy in multiple validation cohorts. In sum, CD8+ TOXPHOS cells represent a critical immune population to assess ICI response with the potential to be a new target to improve outcomes in melanoma patients.

Comparison of 0.3-mSv CT to Standard-Dose CT for Detection of Lung Nodules in Children and Young Adults With Cancer.

BACKGROUND. CT is the imaging modality of choice to identify lung metastasis. OBJECTIVE. The purpose of this study was to evaluate the performance of reduced-dose CT for the detection of lung nodules in children and young adults with cancer. METHODS. This prospective study enrolled patients 4-21 years old with known or suspected malignancy who were undergoing clinically indicated chest CT. Study participants underwent an additional investigational reduced-dose chest CT examination in the same imaging encounter. Separated deidentified CT examinations were reviewed in blinded fashion by three independent radiologists. One reviewer performed a subsequent secondary review to match nodules between the standard- and reduced-dose examinations. Diagnostic performance was computed for the reduced-dose examinations using the clinical examinations as the reference standard. Intraobserver agreement and interobserver agreement were calculated using Cohen kappa. RESULTS. A total of 78 patients (44 male patients and 34 female patients; mean age, 15.2 ± 3.8 [SD] years) were enrolled. The mean estimated effective dose was 1.8 ± 1.1 mSv for clinical CT and 0.3 ± 0.1 mSv for reduced-dose CT, which is an 83% dose reduction. Forty-five of the 78 (58%) patients had 162 total lung nodules (mean size, 3.4 ± 3.3 mm) detected on the clinical CT examinations. A total of 92% of nodules were visible on reduced-dose CT. The sensitivity and specificity of reduced-dose CT for nodules ranged from 63% to 77% and from 80% to 90%, respectively, across the three reviewers. Intraob-server agreement between clinical CT and reduced-dose CT was moderate to substantial for the presence of nodules (κ = 0.45-0.67) and was good to excellent for the number of nodules (κ = 0.68-0.84) and nodule size (κ = 0.69-0.86). Interobserver agreement for the presence of nodules was moderate for both reduced-dose (κ = 0.53) and clinical (κ = 0.54) CT. A median of one nodule was present on clinical CT in patients with a falsely negative reduced-dose CT examination. CONCLUSION. Reduced-dose CT depicts more than 90% of lung nodules in children and young adults with cancer. Reviewers identified the presence of nodules with moderate sensitivity and high specificity. CLINICAL IMPACT. CT performed at a 0.3-mSv mean effective dose has acceptable diagnostic performance for lung nodule detection in children and young adults and has the potential to reduce patient dose or expand CT utilization (e.g., to replace radiography in screening or monitoring protocols). TRIAL REGISTRATION. ClinicalTrials.gov NCT03681873.

Under-reporting of Hepatic Steatosis in Children: A Missed Opportunity for Early Detection.

OBJECTIVE: To determine the prevalence of underreporting of hepatic steatosis found incidentally on computed tomography (CT). STUDY DESIGN: Retrospective cross-sectional study including patients <18 years of age who had undergone unenhanced abdominal CT for evaluation of nephrolithiasis. Hepatic and splenic attenuation were measured independently by 2 reviewers. Hepatic steatosis was defined using various previously established criteria (4 original criteria designed to detect moderate/severe steatosis and 3 secondary criteria designed to identify mild steatosis). Radiology reports and clinical notes were reviewed for documentation of steatosis. Serum alanine aminotransferase levels were collected. Kappa statistics were used to assess agreement between reviewers. RESULTS: A total of 584 patients were included. Agreement between reviewers' measurements for categorical classification of presence of steatosis was excellent (kappa statistic agreement >87%). The prevalence of hepatic steatosis ranged from 3% to 35%, depending on the criterion. Using absolute liver attenuation <48 Hounsfield units (most likely reflective of the truth, given alanine aminotransferase distribution and body mass index data), the prevalence was 7% (n = 42). Steatosis was reported for only 12 of 42 (28%) of these patients and was documented in clinical notes in only 3 of those cases. CONCLUSIONS: Hepatic steatosis is underreported as an incidental finding of CT for nephrolithiasis. Given the prevalence and silent nature of nonalcoholic fatty liver disease, a high level of suspicion is needed, so as not to miss the opportunity to identify steatosis in childhood.

Activated Protein Phosphatase 2A Disrupts Nutrient Sensing Balance Between Mechanistic Target of Rapamycin Complex 1 and Adenosine Monophosphate-Activated Protein Kinase, Causing Sarcopenia in Alcohol-Associated Liver Disease.

BACKGROUND AND AIMS: Despite the high clinical significance of sarcopenia in alcohol-associated cirrhosis, there are currently no effective therapies because the underlying mechanisms are poorly understood. We determined the mechanisms of ethanol-induced impaired phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine monophosphate-activated protein kinase (AMPK) with consequent dysregulated skeletal muscle protein homeostasis (balance between protein synthesis and breakdown). APPROACH AND RESULTS: Differentiated murine myotubes, gastrocnemius muscle from mice with loss and gain of function of regulatory genes following ethanol treatment, and skeletal muscle from patients with alcohol-associated cirrhosis were used. Ethanol increases skeletal muscle autophagy by dephosphorylating mTORC1, circumventing the classical kinase regulation by protein kinase B (Akt). Concurrently and paradoxically, ethanol exposure results in dephosphorylation and inhibition of AMPK, an activator of autophagy and inhibitor of mTORC1 signaling. However, AMPK remains inactive with ethanol exposure despite lower cellular and tissue adenosine triphosphate, indicating a "pseudofed" state. We identified protein phosphatase (PP) 2A as a key mediator of ethanol-induced signaling and functional perturbations using loss and gain of function studies. Ethanol impairs binding of endogenous inhibitor of PP2A to PP2A, resulting in methylation and targeting of PP2A to cause dephosphorylation of mTORC1 and AMPK. Activity of phosphoinositide 3-kinase-γ (PI3Kγ), a negative regulator of PP2A, was decreased in response to ethanol. Ethanol-induced molecular and phenotypic perturbations in wild-type mice were observed in PI3Kγ-/- mice even at baseline. Importantly, overexpressing kinase-active PI3Kγ but not the kinase-dead mutant reversed ethanol-induced molecular perturbations. CONCLUSIONS: Our study describes the mechanistic underpinnings for ethanol-mediated dysregulation of protein homeostasis by PP2A that leads to sarcopenia with a potential for therapeutic approaches by targeting the PI3Kγ-PP2A axis.

Hyperammonaemia-induced skeletal muscle mitochondrial dysfunction results in cataplerosis and oxidative stress.

KEY POINTS: Hyperammonaemia occurs in hepatic, cardiac and pulmonary diseases with increased muscle concentration of ammonia. We found that ammonia results in reduced skeletal muscle mitochondrial respiration, electron transport chain complex I dysfunction, as well as lower NAD+ /NADH ratio and ATP content. During hyperammonaemia, leak of electrons from complex III results in oxidative modification of proteins and lipids. Tricarboxylic acid cycle intermediates are decreased during hyperammonaemia, and providing a cell-permeable ester of αKG reversed the lower TCA cycle intermediate concentrations and increased ATP content. Our observations have high clinical relevance given the potential for novel approaches to reverse skeletal muscle ammonia toxicity by targeting the TCA cycle intermediates and mitochondrial ROS. ABSTRACT: Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonaemia occurs in advanced hepatic, cardiac and pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non-hepatic ammonia disposal. Non-hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from α-ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonaemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonaemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonaemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced complex I/NADH oxidase activity and electron leak occurring at complex III of the electron transport chain. Consistently, lower NAD+ /NADH ratio was observed during hyperammonaemia with reduced TCA cycle intermediates compared to controls. Generation of reactive oxygen species resulted in increased content of skeletal muscle carbonylated proteins and thiobarbituric acid reactive substances during hyperammonaemia. A cell-permeable ester of α-ketoglutarate reversed the low TCA cycle intermediates and ATP content in myotubes during hyperammonaemia. However, the mitochondrial antioxidant MitoTEMPO did not reverse the lower ATP content during hyperammonaemia. We provide for the first time evidence that skeletal muscle hyperammonaemia results in mitochondrial dysfunction and oxidative stress. Use of anaplerotic substrates to reverse ammonia-induced mitochondrial dysfunction is a novel therapeutic approach.

Metabolic adaptation of skeletal muscle to hyperammonemia drives the beneficial effects of l-leucine in cirrhosis.

BACKGROUND & AIMS: Increased skeletal muscle ammonia uptake with loss of muscle mass adversely affects clinical outcomes in cirrhosis. Hyperammonemia causes reduced protein synthesis and sarcopenia but the cellular responses to impaired proteostasis and molecular mechanism of l-leucine induced adaptation to ammonia induced stress were determined. METHODS: Response to activation of amino acid deficiency sensor, GCN2, in the skeletal muscle from cirrhotic patients and the portacaval anastomosis (PCA) rat were quantified. During hyperammonemia and l-leucine supplementation, protein synthesis, phosphorylation of eIF2α, mTORC1 signaling, l-leucine transport and response to l-leucine supplementation were quantified. Adaptation to cellular stress via ATF4 and its target GADD34 were also determined. RESULTS: Activation of the eIF2α kinase GCN2 and impaired mTORC1 signaling were observed in skeletal muscle from cirrhotic patients and PCA rats. Ammonia activated GCN2 mediated eIF2α phosphorylation (eIF2α-P) and impaired mTORC1 signaling that inhibit protein synthesis in myotubes and MEFs. Adaptation to ammonia induced stress did not involve translational reprogramming by activation transcription factor 4 (ATF4) dependent induction of the eIF2α-P phosphatase subunit GADD34. Instead, ammonia increased expression of the leucine/glutamine exchanger SLC7A5, l-leucine uptake and intracellular l-leucine levels, the latter not being sufficient to rescue the inhibition of protein synthesis, due to potentially enhanced mitochondrial sequestration of l-leucine. l-leucine supplementation rescued protein synthesis inhibition caused by hyperammonemia. CONCLUSIONS: Response to hyperammonemia is reminiscent of the cellular response to amino acid starvation, but lacks the adaptive ATF4 dependent integrated stress response (ISR). Instead, hyperammonemia-induced l-leucine uptake was an adaptive response to the GCN2-mediated decreased protein synthesis. LAY SUMMARY: Sarcopenia or skeletal muscle loss is the most frequent complication in cirrhosis but there are no treatments because the cause(s) of muscle loss in liver disease are not known. Results from laboratory experiments in animals and muscle cells were validated in human patients with cirrhosis to show that ammonia plays a key role in causing muscle loss in patients with cirrhosis. We identified a novel stress response to ammonia in the muscle that decreases muscle protein content that can be reversed by supplementation with the amino acid l-leucine.

Metabolic and molecular responses to leucine-enriched branched chain amino acid supplementation in the skeletal muscle of alcoholic cirrhosis.

UNLABELLED: Skeletal muscle loss (sarcopenia) is a major clinical complication in alcoholic cirrhosis with no effective therapy. Skeletal muscle autophagic proteolysis and myostatin expression (inhibitor of protein synthesis) are increased in cirrhosis and believed to contribute to anabolic resistance. A prospective study was performed to determine the mechanisms of sarcopenia in alcoholic cirrhosis and potential reversal by leucine. In six well-compensated, stable, alcoholic patients with cirrhosis and eight controls, serial vastus lateralis muscle biopsies were obtained before and 7 hours after a single oral branched chain amino acid mixture enriched with leucine (BCAA/LEU). Primed-constant infusion of l-[ring-(2) H5 ]-phenylalanine was used to quantify whole-body protein breakdown and muscle protein fractional synthesis rate using liquid chromatography/mass spectrometry. Muscle expression of myostatin, mammalian target of rapamycin (mTOR) targets, autophagy markers, protein ubiquitination, and the intracellular amino acid deficiency sensor general control of nutrition 2 were quantified by immunoblots and the leucine exchanger (SLC7A5) and glutamine transporter (SLC38A2), by real-time polymerase chain reaction. Following oral administration, plasma BCAA concentrations showed a similar increase in patients with cirrhosis and controls. Skeletal muscle fractional synthesis rate was 9.63 ± 0.36%/hour in controls and 9.05 ± 0.68%/hour in patients with cirrhosis (P = 0.54). Elevated whole-body protein breakdown in patients with cirrhosis was reduced with BCAA/LEU (P = 0.01). Fasting skeletal muscle molecular markers showed increased myostatin expression, impaired mTOR signaling, and increased autophagy in patients with cirrhosis compared to controls (P < 0.01). The BCAA/LEU supplement did not alter myostatin expression, but mTOR signaling, autophagy measures, and general control of nutrition 2 activation were consistently reversed in cirrhotic muscle (P < 0.01). Expression of SLC7A5 was higher in the basal state in patients with cirrhosis than controls (P < 0.05) but increased with BCAA/LEU only in controls (P < 0.001). CONCLUSIONS: Impaired mTOR1 signaling and increased autophagy in skeletal muscle of patients with alcoholic cirrhosis is acutely reversed by BCAA/LEU.

Caspase 3 inactivation protects against hepatic cell death and ameliorates fibrogenesis in a diet-induced NASH model.

BACKGROUND/AIMS: Hepatocyte cell death is a key feature of nonalcoholic steatohepatitis (NASH). As the contribution of specific caspases remains unclear, our aim was to ascertain the effect of caspase 3 suppression on liver injury and fibrogenesis. METHODS: C57BL/6 wild-type (WT) and caspase 3 knock out (Casp3 (-/-)) mice were placed on a methionine- and choline-deficient (MCD) diet for 6 weeks to induce steatohepatitis and liver fibrosis. Thereafter, liver injury, liver fibrosis and hepatocellular apoptosis were quantified in liver sections. Additionally, expression of proteins associated with liver inflammation and fibrogenesis was analyzed. RESULTS: WT mice fed MCD diet showed marked activation of caspase 3 in hepatocytes, in conjunction with steatohepatitis and increased hepatic triglyceride levels, hepatocyte ballooning, inflammation and fibrosis. Casp3 (-/-) mice fed the MCD diet showed similar serum aminotransferase levels and NAFLD activity scores (NAS) compared with WT MCD-fed mice. However, Casp3 (-/-) mice on the MCD diet showed a marked reduction in expression of transcripts for profibrogenic genes, which translated into reduced hepatic collagen deposition. These changes were associated with decreased levels of apoptosis, and a significant reduction in the expression of cytokines involved in inflammatory signaling. Casp3 (-/-) mice on the MCD showed a reduction in expression of chemokine receptor 2 (CCR2) leading to ameliorated infiltration of inflammatory lymphocyte antigen 6 complex, locus C1 (Ly6c) positive monocytes. CONCLUSION: These findings support a prominent role for hepatocyte caspase 3 activation in NASH-related apoptosis, fibrogenesis and fibrosis which in part is mediated via CCR2-dependent infiltration of Ly6c positive monocytes.

Alcohol-induced autophagy contributes to loss in skeletal muscle mass.

Patients with alcoholic cirrhosis and hepatitis have severe muscle loss. Since ethanol impairs skeletal muscle protein synthesis but does not increase ubiquitin proteasome-mediated proteolysis, we investigated whether alcohol-induced autophagy contributes to muscle loss. Autophagy induction was studied in: A) Human skeletal muscle biopsies from alcoholic cirrhotics and controls, B) Gastrocnemius muscle from ethanol and pair-fed mice, and C) Ethanol-exposed murine C2C12 myotubes, by examining the expression of autophagy markers assessed by immunoblotting and real-time PCR. Expression of autophagy genes and markers were increased in skeletal muscle from humans and ethanol-fed mice, and in myotubes following ethanol exposure. Importantly, pulse-chase experiments showed suppression of myotube proteolysis upon ethanol-treatment with the autophagy inhibitor, 3-methyladenine (3MA) and not by MG132, a proteasome inhibitor. Correspondingly, ethanol-treated C2C12 myotubes stably expressing GFP-LC3B showed increased autophagy flux as measured by accumulation of GFP-LC3B vesicles with confocal microscopy. The ethanol-induced increase in LC3B lipidation was reversed upon knockdown of Atg7, a critical autophagy gene and was associated with reversal of the ethanol-induced decrease in myotube diameter. Consistently, CT image analysis of muscle area in alcoholic cirrhotics was significantly reduced compared with control subjects. In order to determine whether ethanol per se or its metabolic product, acetaldehyde, stimulates autophagy, C2C12 myotubes were treated with ethanol in the presence of the alcohol dehydrogenase inhibitor (4-methylpyrazole) or the acetaldehyde dehydrogenase inhibitor (cyanamide). LC3B lipidation increased with acetaldehyde treatment and increased further with the addition of cyanamide. We conclude that muscle autophagy is increased by ethanol exposure and contributes to sarcopenia.

Hyperammonemia in cirrhosis induces transcriptional regulation of myostatin by an NF-κB-mediated mechanism.

Loss of muscle mass, or sarcopenia, is nearly universal in cirrhosis and adversely affects patient outcome. The underlying cross-talk between the liver and skeletal muscle mediating sarcopenia is not well understood. Hyperammonemia is a consistent abnormality in cirrhosis due to impaired hepatic detoxification to urea. We observed elevated levels of ammonia in both plasma samples and skeletal muscle biopsies from cirrhotic patients compared with healthy controls. Furthermore, skeletal muscle from cirrhotics had increased expression of myostatin, a known inhibitor of skeletal muscle accretion and growth. In vivo studies in mice showed that hyperammonemia reduced muscle mass and strength and increased myostatin expression in wild-type compared with postdevelopmental myostatin knockout mice. We postulated that hyperammonemia is an underlying link between hepatic dysfunction in cirrhosis and skeletal muscle loss. Therefore, murine C2C12 myotubes were treated with ammonium acetate resulting in intracellular concentrations similar to those in cirrhotic muscle. In this system, we demonstrate that hyperammonemia stimulated myostatin expression in a NF-κB-dependent manner. This finding was also observed in primary murine muscle cell cultures. Hyperammonemia triggered activation of IκB kinase, NF-κB nuclear translocation, binding of the NF-κB p65 subunit to specific sites within the myostatin promoter, and stimulation of myostatin gene transcription. Pharmacologic inhibition or gene silencing of NF-κB abolished myostatin up-regulation under conditions of hyperammonemia. Our work provides unique insights into hyperammonemia-induced myostatin expression and suggests a mechanism by which sarcopenia develops in cirrhotic patients.

Adipocyte hypertrophy is associated with lysosomal permeability both in vivo and in vitro: role in adipose tissue inflammation.

Obesity in both humans and rodents is characterized by adipocyte hypertrophy and the presence of death adipocytes surrounded by macrophages forming "crown-like structures." However, the biochemical pathways involved in triggering adipocyte death as well as the role of death adipocytes in adipose tissue remodeling and macrophage infiltration remain poorly understood. We now show that induction of adipocyte hypertrophy by incubation of mature adipocytes with saturated fatty acids results in lysosomal destabilization and cathepsin B (ctsb), a key lysosomal cysteine protease, activation and redistribution into the cytosol. ctsb activation was required for the lysosomal permeabilization, and its inhibition protected cells against mitochondrial dysfunction. With the use of a dietary murine model of obesity, ctsb activation was detected in adipose tissue of these mice. This is an early event during weight gain that correlates with the presence of death adipocytes, and precedes macrophage infiltration of adipose tissue. Moreover, ctsb-deficient mice showed decreased lysosomal permeabilization in adipocytes and were protected against adipocyte cell death and macrophage infiltration to adipose tissue independent of body weight. These data strongly suggest that ctsb activation and lysosomal permeabilization in adipocytes are key initial events that contribute to the adipocyte cell death and macrophage infiltration into adipose tissue associated with obesity. Inhibition of ctsb activation may be a new therapeutic strategy for the treatment of obesity-associated metabolic complications.

Caspase-1-mediated regulation of fibrogenesis in diet-induced steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is typically associated with pro-apoptotic caspase activation. A potential role for pro-inflammatory caspases remains incompletely understood. Our aims were to examine a potential role of caspase-1 in the development of liver damage and fibrosis in NASH. C57BL/6 wild type (WT) developed marked steatohepatitis, activation, fibrosis and increased hepatic caspase-1 and interleukin-1ß expression when placed on the methionine- and choline-deficient (MCD) diet. Marked caspase-1 activation was detected in the liver of MCD-fed mice. Hepatocyte and non-parenchymal fractionation of the livers further demonstrated that caspase-1 activation after MCD feeding was mainly localized to non-parenchymal cells. Caspase-1-knockout (Casp1(-/-)) mice on the MCD diet showed marked reduction in mRNA expression of genes involved in inflammation and fibrogenesis (tumor necrosis factor-α was 7.6-fold greater in WT vs Casp1(-/-) MCD-fed mice; F4/80 was 1.5-fold greater in WT vs Casp1(-/-) MCD-fed mice; α-smooth muscle actin was 3.2-fold greater in WT vs Casp1(-/-) MCD-fed mice; collagen 1-α was 7.6-fold greater in WT vs Casp1(-/-) MCD-fed mice; transforming growth factor-ß was 2.4-fold greater in WT vs Casp1(-/-) MCD-fed mice; cysteine- and glycine-rich protein 2 was 3.2-fold greater in WT vs Casp1(-/-) MCD-fed mice). Furthermore, Sirius red staining for hepatic collagen deposition was significantly reduced in Casp1(-/-) MCD-fed mice compared with WT MCD-fed animals. However, serum alanine aminotransferase levels, caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells were similar in Casp1(-/-) and WT mice on the MCD diet. Selective Kupffer cell depletion by clodronate injection markedly suppressed MCD-induced caspase-1 activation and protected mice from fibrogenesis and fibrosis associated with this diet. The conclusion of this study is that it uncovers a novel role for caspase-1 in inflammation and fibrosis during NASH development.

Transcriptional profile of genes involved in oxidative stress and antioxidant defense in a dietary murine model of steatohepatitis.

Oxidative stress is a core abnormality responsible for disease progression in nonalcoholic steatohepatitis (NASH). However, the relevant pathways that contribute to oxidative damage in vivo remain poorly understood. Here we explore the gene-expression patterns related to oxidative stress, antioxidant defense, and reactive oxygen metabolism in an established dietary murine model of NASH. C57BL/6 mice were placed on either a methionine- and choline-deficient (MCD) or a control (CTL) diet for 6 weeks. Hepatic oxidative damage and the development of NASH were monitored by biochemical and histologic indices. Analysis of 84 oxidative stress-related genes was performed by real-time reverse transcription polymerase chain reaction (PCR) in the livers of the two groups of mice. Mice on the MCD diet showed increased ALT, histologic features of NASH, and oxidative liver damage with increases in 4-hydroxynonenal and 3-nitrotyrosine. Of the genes analyzed, the GPx family were most significantly upregulated, whereas SCD1 was most significantly downregulated. Other genes that were significantly upregulated included Fmo2 and peroxiredoxins, whereas genes downregulated included Catalase and Serpinb1b. Our data demonstrate that oxidative stress-related genes are differentially expressed in the livers of mice with diet-induced NASH. These findings have important implications for NASH pathogenesis and the development of novel therapeutic strategies for patients with this condition.

Adipocyte apoptosis, a link between obesity, insulin resistance, and hepatic steatosis.

Adipocyte death has been reported in both obese humans and rodents. However, its role in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity has not been studied. We now show using real-time reverse transcription-PCR arrays that adipose tissue of obese mice display a pro-apoptotic phenotype. Moreover, caspase activation and adipocyte apoptosis were markedly increased in adipose tissue from both mice with diet-induced obesity and obese humans. These changes were associated with activation of both the extrinsic, death receptor-mediated, and intrinsic, mitochondrial-mediated pathways of apoptosis. Genetic inactivation of Bid, a key pro-apoptotic molecule that serves as a link between these two cell death pathways, significantly reduced caspase activation, adipocyte apoptosis, prevented adipose tissue macrophage infiltration, and protected against the development of systemic insulin resistance and hepatic steatosis independent of body weight. These data strongly suggest that adipocyte apoptosis is a key initial event that contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis associated with obesity in both mice and humans. Inhibition of adipocyte apoptosis may be a new therapeutic strategy for the treatment of obesity-associated metabolic complications.