Arginase 2 deficiency results in spontaneous steatohepatitis: a novel link between innate immune activation and hepatic de novo lipogenesis.

BACKGROUND & AIMS: Innate immune activation has been postulated as a central mechanism for disease progression from hepatic steatosis to steatohepatitis in obesity-related fatty liver disease. Arginase 2 competes with inducible nitric oxide synthase (iNOS) for its substrate and the balance between these two enzymes plays a crucial role in regulating immune responses and macrophage activation. Our aim was to test the hypothesis that arginase 2 deficiency in mice favours progression from isolated hepatic steatosis, induced by high fat feeding, to steatohepatitis. METHODS: Arginase 2-knockout (Arg2(-/-)) mice were studied for changes in liver histology and metabolic phenotype at baseline and after a short term course (7 week) feeding with a high fat (HFAT) diet. In additional experiments, Arg2(-/-) mice received tail vein injections of liposome-encapsulated clodronate (CLOD) over a three-week period to selectively deplete liver macrophages. RESULTS: Unexpectedly, Arg2(-/-) mice showed profound changes in their livers at baseline, characterized by significant steatosis as demonstrated with histological and biochemical analysis. These changes were independent of systemic metabolic parameters and associated with marked mRNA level increases of genes involved in hepatic de novo lipogenesis. Liver injury and inflammation were present with elevated serum ALT, marked infiltration of F4/80 positive cells, and increased mRNA levels of inflammatory genes. HFAT feeding exacerbated these changes. Macrophage depletion after CLOD injection significantly attenuated lipid deposition and normalized lipogenic mRNA profile of livers from Arg2(-/-) mice. CONCLUSIONS: This study identifies arginase 2 as a novel link between innate immune responses, hepatic lipid deposition, and liver injury.

Differential regulation of inflammation and apoptosis in Fas-resistant hepatocyte-specific Bid-deficient mice.

BACKGROUND & AIMS: Activation of Fas death receptor results in apoptosis in multiple organs, particularly liver, in a process dependent on Bid cleavage. Mice injected with an anti-Fas antibody die within hours of acute liver failure associated with massive apoptosis and hemorrhage. Our aim was to investigate the crosstalk of apoptotic and inflammatory pathways and the contribution of selective hepatocellular apoptosis during in vivo Fas activation. METHODS: We generated hepatocyte-specific Bid deficient mice (hBid(-/-)). Acute liver injury was induced by Fas-activating antibody (Jo2) in a time-course study. RESULTS: In contrast to controls, nearly all Jo2 injected hBid(-/-) survived. Their livers showed complete protection against hepatocellular apoptosis with minimal focal hemorrhagic changes and mainly non-parenchymal cell apoptosis. In agreement, the hepatocytes had no mitochondrial cytochrome c release in cytosol, or caspase 3 activation. hBid(-/-) livers showed marked increase in acute inflammatory foci composed of neutrophils and monocytes associated with the increased expression of proinflammatory chemokines and cytokines, in the manner dependent on non-canonical interleukin-1ß activation and amplified in the absence of caspase-3 activation. In addition, hBid(-/-) mice were completely protected from hepatotoxicity and the infiltrated cells were cleared 2 weeks post single Jo2 injection. CONCLUSIONS: Hepatocyte Bid suppression is critical for the resistance to the lethal effects of Fas activation in vivo. Fas signaling induces differential activation of non-canonical interleukin-1ß maturation, amplified in the absence of apoptotic Bid-mitochondrial loop, in hepatocytes. These findings may have important pathophysiological and therapeutic implications in a variety of liver disorders associated with Fas activation.

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.

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.

An apoptosis panel for nonalcoholic steatohepatitis diagnosis.

BACKGROUND & AIMS: The extrinsic death receptor-mediated pathway of apoptosis is involved in nonalcoholic steatohepatitis (NASH) development. Our aims were to create and validate a noninvasive prediction model for NASH diagnosis based on specific circulating markers of apoptosis. METHODS: Our initial cohort consisted of 95 consecutive patients undergoing a liver biopsy for clinically suspected NASH. Blood was obtained from each patient at the time of liver biopsy. Plasma caspase 3 generated cytokeratin-18 fragments (CK-18), soluble Fas (sFas), and soluble Fas ligand (sFasL) were measured. Histology was assessed by an experienced hepatopathologist. The validation cohort consisted of 82 consecutive patients that underwent liver biopsy at the time of bariatric surgery. RESULTS: Patients with NASH had significantly higher levels of CK-18 and sFas than patients in the "not NASH" group [median (25th, 75th percentile): 508 (280, 846) U/L versus 176 (131, 224) U/L (p<0.001), and 11.8 (7.8, 12.5) ng/ml versus 5.9 (4.8, 8.3) ng/ml (p<0.001), respectively]. A significant positive correlation was revealed between the apoptosis markers and liver histopathology independent of other metabolic factors. A prediction model was generated including CK-18 fragments and sFas levels that showed an AUC of 0.93 and 0.79 in the initial and validation cohorts, respectively. A cutoff value using this model predicted NASH with a sensitivity and specificity of 88% and 89%, respectively. CONCLUSIONS: Quantification of circulating levels of two apoptotic markers accurately predicts the presence of NASH, supporting the potential usefulness of these markers in clinical practice for noninvasive diagnosis of NASH.

HSD3B1(1245A>C) variant regulates dueling abiraterone metabolite effects in prostate cancer.

BACKGROUND: A common germline variant in HSD3B1(1245A>C) encodes for a hyperactive 3ß-hydroxysteroid dehydrogenase 1 (3ßHSD1) missense that increases metabolic flux from extragonadal precursor steroids to DHT synthesis in prostate cancer. Enabling of extragonadal DHT synthesis by HSD3B1(1245C) predicts for more rapid clinical resistance to castration and sensitivity to extragonadal androgen synthesis inhibition. HSD3B1(1245C) thus appears to define a subgroup of patients who benefit from blocking extragonadal androgens. However, abiraterone, which is administered to block extragonadal androgens, is a steroidal drug that is metabolized by 3ßHSD1 to multiple steroidal metabolites, including 3-keto-5α-abiraterone, which stimulates the androgen receptor. Our objective was to determine if HSD3B1(1245C) inheritance is associated with increased 3-keto-5α-abiraterone synthesis in patients. METHODS: First, we characterized the pharmacokinetics of 7 steroidal abiraterone metabolites in 15 healthy volunteers. Second, we determined the association between serum 3-keto-5α-abiraterone levels and HSD3B1 genotype in 30 patients treated with abiraterone acetate (AA) after correcting for the determined pharmacokinetics. RESULTS: Patients who inherit 0, 1, and 2 copies of HSD3B1(1245C) have a stepwise increase in normalized 3-keto-5α-abiraterone (0.04 ng/ml, 2.60 ng/ml, and 2.70 ng/ml, respectively; P = 0.002). CONCLUSION: Increased generation of 3-keto-5α-abiraterone in patients with HSD3B1(1245C) might partially negate abiraterone benefits in these patients who are otherwise more likely to benefit from CYP17A1 inhibition. FUNDING: Prostate Cancer Foundation Challenge Award, National Cancer Institute.

Microparticles release by adipocytes act as "find-me" signals to promote macrophage migration.

Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical "find-me" signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel "find me" signals that contributes to macrophage infiltration associated with obesity.

Lipidomic profiling of serum and pancreatic fluid in chronic pancreatitis.

OBJECTIVES: Oxidative stress is intimately involved in the pathogenesis of chronic pancreatitis (CP), and its quantification may represent a useful biomarker. The aim was to determine whether oxidized fatty acid (OxFA) levels in serum and/or pancreatic fluid are elevated in CP. METHODS: Patients evaluated for abdominal pain were classified into 3 groups (controls, mild CP, and severe CP). Serum and secretin-stimulated pancreatic fluid samples were stored under conditions to minimize artificial oxidation. Ten arachidonic and linoleic acid oxidation products were simultaneously measured using liquid chromatography-electrospray ionization-tandem mass spectrometry. RESULTS: Most OxFAs were significantly elevated in the serum of patients with mild and severe CP compared to controls and correlated with structural features on endoscopic ultrasound. For example, the mean (SD) ratio of serum 13-HODE to its precursor linoleic acid was 0.03 (0.004) in controls, 0.06 (0.02) in mild CP, and 0.10 (0.04) in severe CP (analysis of variance, P = 0.004) and had a strong correlation with endoscopic ultrasound features (Spearman r = 0.84, P < 0.001). In the pancreatic fluid, statistically significant increases of OxFA products were observed in mild CP compared to controls. CONCLUSIONS: Oxidized fatty acid products are increased in the serum and pancreatic fluid of patients with CP, suggesting a potential role as biomarkers. ABBREVIATIONS: AA - arachidonic acidANOVA - analysis of varianceAP - acute pancreatitisCP - chronic pancreatitisePFT - endoscopic pancreatic function testEUS - endoscopic ultrasoundHPLC - high-pressure liquid chromatographyHETE - hydroxy-eicosatetranoic acidHODE - hydroxy-octadecadienoic acidLA - linoleic acidLC-ESI-MS/MS - liquid chromatography-electrospray ionization-tandem mass spectrometryM - molarOxFA - oxidized fatty acidsPFT - pancreatic function test.