Nutritional stress exacerbates hepatic steatosis induced by deletion of the histidine nucleotide-binding (Hint2) mitochondrial protein.

The histidine nucleotide-binding protein, Hint2, is a mitochondrial phosphoramidase expressed in liver, brown fat, pancreas, and muscle. The livers of Hint2 knockout (Hint2(-/-)) mice accumulate triglycerides and show a pattern of mitochondrial protein lysine hyperacetylation. The extent and nature of the lysine acetylation changes and the response of Hint2(-/-) mice to nutritional challenges that elicit a modification of protein acetylation have not been investigated. To compare the adaptation of Hint2(-/-) and control (Hint2(+/+)) mice with episodes of fasting and high-fat diet (HFD), we subjected animals to either feeding ad libitum or fasting for 24 h, and to either a HFD or control diet for 8 wk. Triglyceride content was higher in Hint2(-/-) than in Hint2(+/+) livers, whereas plasma triglycerides were fourfold lower. Malonyl-CoA levels were increased twofold in Hint2(-/-) livers. After 24 h fasting, Hint2(-/-) displayed a decrease in body temperature, commensurate with a decrease in mass of brown fat and downregulation of uncoupling protein 1. HFD-treated Hint2(-/-) livers showed more steatosis, and plasma insulin and cholesterol were higher than in Hint(+/+) mice. Several proteins identified as substrates of sirtuin 3 and 5 and active in intermediary and ketone metabolism were hyperacetylated in liver and brown fat mitochondria after both HFD and fasting regimens. Glutamate dehydrogenase activity was downregulated in fed and fasted livers, and this was attributed to an increase in acetylation and ADP-ribosylation. The absence of Hint2 deregulates the posttranslational modification of several mitochondrial proteins, which impedes the adaptation to episodes of nutritional stress.

Disruption of tumor suppressor gene Hint1 leads to remodeling of the lipid metabolic phenotype of mouse liver.

A lipidomic and metabolomic investigation of serum and liver from mice was performed to gain insight into the tumor suppressor gene Hint1. A major reprogramming of lipid homeostasis was found in both serum and liver of Hint1-null (Hint(-/-)) mice, with significant changes in the levels of many lipid molecules, as compared with gender-, age-, and strain-matched WT mice. In the Hint1(-/-) mice, serum total and esterified cholesterol were reduced 2.5-fold, and lysophosphatidylcholines (LPCs) and lysophosphatidic acids were 10-fold elevated in serum, with a corresponding fall in phosphatidylcholines (PCs). In the liver, MUFAs and PUFAs, including arachidonic acid (AA) and its metabolic precursors, were also raised, as was mRNA encoding enzymes involved in AA de novo synthesis. There was also a significant 50% increase in hepatic macrophages in the Hint1(-/-) mice. Several hepatic ceramides and acylcarnitines were decreased in the livers of Hint1(-/-) mice. The changes in serum LPCs and PCs were neither related to hepatic phospholipase A2 activity nor to mRNAs encoding lysophosphatidylcholine acetyltransferases 1-4. The lipidomic phenotype of the Hint1(-/-) mouse revealed decreased inflammatory eicosanoids with elevated proliferative mediators that, combined with decreased ceramide apoptosis signaling molecules, may contribute to the tumor suppressor activity of Hint1.

Tissue metabolomics of hepatocellular carcinoma: tumor energy metabolism and the role of transcriptomic classification.

UNLABELLED: Hepatocellular carcinoma (HCC) is one of the commonest causes of death from cancer. A plethora of metabolomic investigations of HCC have yielded molecules in biofluids that are both up- and down-regulated but no real consensus has emerged regarding exploitable biomarkers for early detection of HCC. We report here a different approach, a combined transcriptomics and metabolomics study of energy metabolism in HCC. A panel of 31 pairs of HCC tumors and corresponding nontumor liver tissues from the same patients was investigated by gas chromatography-mass spectrometry (GCMS)-based metabolomics. HCC was characterized by ∼2-fold depletion of glucose, glycerol 3- and 2-phosphate, malate, alanine, myo-inositol, and linoleic acid. Data are consistent with a metabolic remodeling involving a 4-fold increase in glycolysis over mitochondrial oxidative phosphorylation. A second panel of 59 HCC that had been typed by transcriptomics and classified in G1 to G6 subgroups was also subjected to GCMS tissue metabolomics. No differences in glucose, lactate, alanine, glycerol 3-phosphate, malate, myo-inositol, or stearic acid tissue concentrations were found, suggesting that the Wnt/ß-catenin pathway activated by CTNNB1 mutation in subgroups G5 and G6 did not exhibit specific metabolic remodeling. However, subgroup G1 had markedly reduced tissue concentrations of 1-stearoylglycerol, 1-palmitoylglycerol, and palmitic acid, suggesting that the high serum α-fetoprotein phenotype of G1, associated with the known overexpression of lipid catabolic enzymes, could be detected through metabolomics as increased lipid catabolism. CONCLUSION: Tissue metabolomics yielded precise biochemical information regarding HCC tumor metabolic remodeling from mitochondrial oxidation to aerobic glycolysis and the impact of molecular subtypes on this process.

Disruption of the histidine triad nucleotide-binding hint2 gene in mice affects glycemic control and mitochondrial function.

UNLABELLED: The histidine triad nucleotide-binding (HINT2) protein is a mitochondrial adenosine phosphoramidase expressed in the liver and pancreas. Its physiological function is unknown. To elucidate the role of HINT2 in liver physiology, the mouse Hint2 gene was deleted. Hint2(-/-) and Hint2(+/+) mice were generated in a mixed C57Bl6/J × 129Sv background. At 20 weeks, the phenotypic changes in Hint2(-/-) relative to Hint2(+/+) mice were an accumulation of hepatic triglycerides, decreased tolerance to glucose, a defective counter-regulatory response to insulin-provoked hypoglycemia, and an increase in plasma interprandial insulin but a decrease in glucose-stimulated insulin secretion and defective thermoregulation upon fasting. Leptin messenger RNA (mRNA) in adipose tissue and plasma leptin were elevated. In mitochondria from Hint2(-/-) hepatocytes, state 3 respiration was decreased, a finding confirmed in HepG2 cells where HINT2 mRNA was silenced. The linked complex II-III electron transfer was decreased in Hint2(-/-) mitochondria, which was accompanied by a lower content of coenzyme Q. Hypoxia-inducible factor-2α expression and the generation of reactive oxygen species were increased. Electron microscopy of mitochondria in Hint2(-/-) mice aged 12 months revealed clustered, fused organelles. The hepatic activities of 3-hydroxyacyl-coenzyme A dehydrogenase short chain and glutamate dehydrogenase (GDH) were decreased by 68% and 60%, respectively, without a change in protein expression. GDH activity was similarly decreased in HINT2-silenced HepG2 cells. When measured in the presence of purified sirtuin 3, latent GDH activity was recovered (126% in Hint2(-/-) versus 83% in Hint2(+/+) ). This suggests a greater extent of acetylation in Hint2(-/-) than in Hint2(+/+) . CONCLUSION: Hint2/HINT2 positively regulates mitochondrial lipid metabolism and respiration and glucose homeostasis. The absence of Hint2 provokes mitochondrial deformities and a change in the pattern of acetylation of selected proteins.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury.

UNLABELLED: The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.

Continuous administration of sorafenib in combination with transarterial chemoembolization in patients with hepatocellular carcinoma: results of a phase I study.

BACKGROUND AND AIM: It is unknown whether sorafenib can be combined with transarterial chemoembolization (TACE) in patients with hepatocellular carcinoma. This study assesses the safety and tolerability of a continuous regimen of sorafenib combined with TACE. METHODS: This was an open-label phase I study testing a continuous administration of sorafenib (dose escalation from 200 mg twice daily [bid] to 400 mg bid) starting 7 days prior to TACE with doxorubicin (50 mg). RESULTS: Twenty-one patients were screened and 14 received sorafenib combined with TACE. Because there were no dose-limiting toxicities in the first three patients who received sorafenib at a dose of 200 mg bid, subsequent patients received 400 mg bid. Twenty-seven procedures were performed (median, two per patient) and two local therapy-related severe adverse events occurred. The median duration of sorafenib therapy was 246 days (range, 14-547 days). Sorafenib-related adverse events of grade ≥3 were hand-foot skin reaction (n = 3), weight loss (n = 2), diarrhea (n = 1), abdominal pain (n = 1), and thrombocytopenia (n = 3). After treatment with sorafenib and TACE, there was a significant decrease in the concentration of plasma vascular endothelial growth factor (VEGF) from 93 ng/l to 67 ng/l. CONCLUSIONS: Continuous administration of sorafenib at a dose of 400 mg bid combined with TACE was tolerable. The adverse event profile of this regimen was comparable with that of sorafenib monotherapy with the exception of thrombocytopenia, which may be more frequent. There were no increases in the circulating VEGF levels after TACE with this combined regimen. (Swiss Association for the Study of the Liver study number 25; ClinicalTrials.gov trial identifier, NCT00478374).

Aberrant lipid metabolism in hepatocellular carcinoma revealed by plasma metabolomics and lipid profiling.

There has been limited analysis of the effects of hepatocellular carcinoma (HCC) on liver metabolism and circulating endogenous metabolites. Here, we report the findings of a plasma metabolomic investigation of HCC patients by ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS), random forests machine learning algorithm, and multivariate data analysis. Control subjects included healthy individuals as well as patients with liver cirrhosis or acute myeloid leukemia. We found that HCC was associated with increased plasma levels of glycodeoxycholate, deoxycholate 3-sulfate, and bilirubin. Accurate mass measurement also indicated upregulation of biliverdin and the fetal bile acids 7α-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochol-4,6-dien-24-oic acid in HCC patients. A quantitative lipid profiling of patient plasma was also conducted by ultraperformance liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (UPLC-ESI-TQMS). By this method, we found that HCC was also associated with reduced levels of lysophosphocholines and in 4 of 20 patients with increased levels of lysophosphatidic acid [LPA(16:0)], where it correlated with plasma α-fetoprotein levels. Interestingly, when fatty acids were quantitatively profiled by gas chromatography-mass spectrometry (GC-MS), we found that lignoceric acid (24:0) and nervonic acid (24:1) were virtually absent from HCC plasma. Overall, this investigation illustrates the power of the new discovery technologies represented in the UPLC-ESI-QTOFMS platform combined with the targeted, quantitative platforms of UPLC-ESI-TQMS and GC-MS for conducting metabolomic investigations that can engender new insights into cancer pathobiology.

The bile acid nuclear receptor FXR and the bile acid binding protein IBABP are differently expressed in colon cancer.

Bile acids have been implicated in the development of colorectal cancers. We investigated the expression of the transcription factor regulated by bile acids, farnesoid X receptor (FXR), as well as other components of this pathway in human colorectal tumors and cell lines. The most significant changes were a decrease in FXR mRNA levels in adenomas (5-fold average) and carcinomas (10 fold average) and an increase in peroxisome proliferator activated receptor-gamma (2-fold average). FXR was not expressed in undifferentiated colon adenocarcinoma SW480 cells and metastasis derived SW620 cells. In Caco-2 and HT-29 cells, the level of FXR expression increased with the degree of differentiation. Intestinal bile acid binding protein was activated by chenodeoxycholic acid and the synthetic FXR agonist GW4064 in Caco-2 and HT-29 but not in SW cells unless FXR was transfected. The down-regulation of the nuclear receptor FXR in colon cancer might be of clinical and pharmacological importance.