New modes of data partitioning based on PARS peak alignment for improved multivariate biomarker/biopattern detection in 1H-NMR spectroscopic metabolic profiling of urine

This paper addresses the possibility of mathematically partition and process urine ¹H-NMR spectra to enhance the efficiency of the subsequent multivariate data analysis in the context of metabolic profiling of a toxicity study. We show that by processing the NMR data with the peak alignment using reduced set mapping (PARS) algorithm and the use of sparse representation of the data results in the information contained in the original NMR data being preserved with retained resolution but free of the problem of peak shifts. We can now describe a method for differential expression analysis of NMR spectra by using prior knowledge, i.e., the onset of dosing, a partitioning not possible to achieve using raw or bucketed data. In addition we also outline a scheme for soft removal of “biological noise” from the aligned data: exhaustive bio-noise subtraction (EBS). The result is a straightforward protocol for detection of peaks that appear as a consequence of the drug response. In other words, it is possible to elucidate peak origin, either from endogenous substances or from the administered drug/biomarkers. The partition of data originating from the normally regulating metabolome can, furthermore, be analyzed free of the superimposed biological noise. The proposed protocol results in enhanced interpretability of the processed data, i.e., a more refined metabolic trace, simplification of detection of consistent biomarkers, and a simplified search for metabolic end products of the administered drug.     

Hormonal and metabolic responses to overfeeding in three genotypes of ducks

Muscovy, Pekin and Mule duck are different in their body weight. To make a valid comparison in the lipid metabolism between these three genotypes, overfeeding was carried out by providing the animals with amounts of food in proportion to their body weight. Under these conditions, Muscovy ducks developed a strong liver steatosis, whereas it was not very pronounced in the Mule ducks and even less in the Pekin ducks. On the contrary, Pekin ducks showed a much marked extrahepatic fattening. At the beginning of overfeeding, there was a similarity in the three genotypes as regards the post-heparin lipoprotein-lipase (LPL) activity and the insulin and glucagon concentrations. After 10 days of overfeeding, the LPL activity dramatically fell in Muscovy and in Mule ducks, whereas it remained steady in Pekin ducks. Compared to values found at the beginning of the overfeeding period, plasma glucagon and insulin shown no evolution, except for the insulin of Pekin ducks which was dramatically higher. Those data suggest that high plasma insulin concentrations measured in Pekin ducks after 10 days of overfeeding can be responsible for the maintenance of the LPL activity, which favors the extrahepatic fattening to the detriment of liver steatosis.     

Sex‐ and strain‐specific effects of mitochondrial uncoupling on age‐related metabolic diseases in high‐fat diet‐fed mice

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria and may have evolved to protect cells against the production of damaging reactive oxygen species. Therefore, compounds that enhance mitochondrial uncoupling are potentially attractive anti‐aging therapies; however, chronic ingestion is associated with a number of unwanted side effects. We have previously developed a controlled‐release mitochondrial protonophore (CRMP) that is functionally liver‐directed and promotes oxidation of hepatic triglycerides by causing a subtle sustained increase in hepatic mitochondrial inefficiency. Here, we sought to leverage the higher therapeutic index of CRMP to test whether mild mitochondrial uncoupling in a liver‐directed fashion could reduce oxidative damage and improve age‐related metabolic disease and lifespan in diet‐induced obese mice. Oral administration of CRMP (20 mg/[kg‐day] × 4 weeks) reduced hepatic lipid content, protein kinase C epsilon activation, and hepatic insulin resistance in aged (74‐week‐old) high‐fat diet (HFD)‐fed C57BL/6J male mice, independently of changes in body weight, whole‐body energy expenditure, food intake, or markers of hepatic mitochondrial biogenesis. CRMP treatment was also associated with a significant reduction in hepatic lipid peroxidation, protein carbonylation, and inflammation. Importantly, long‐term (49 weeks) hepatic mitochondrial uncoupling initiated late in life (94–104 weeks), in conjugation with HFD feeding, protected mice against neoplastic disorders, including hepatocellular carcinoma (HCC), in a strain and sex‐specific manner. Taken together, these studies illustrate the complex variation of aging and provide important proof‐of‐concept data to support further studies investigating the use of liver‐directed mitochondrial uncouplers to promote healthy aging in humans.     

Potentiation of DOM-induced stimulus control by fluoxetine and citalopram: role of pharmacokinetics

The present investigation examined the interaction between 2,5-dimethoxy-4-methylamphetamine [DOM] and the selective serotonin reuptake inhibitor [SSRI] citalopram in rats trained with DOM [0.6 mg/kg; 75 min pretreatment time] as a discriminative stimulus. Pretreatment with citalopram at a dose of 1.0 mg/kg shifted the DOM dose response relationship to the left. Unlike previously tested SSRI's, the enhancement of DOM-induced stimulus control occurred in the absence of significant partial substitution by citalopram. DOM brain levels were measured using a GC-MS method both in the presence and absence of citalopram and fluoxetine in order to evaluate the pharmacokinetic contribution to the observed behavioral effect. The data indicated that fluoxetine but not citalopram significantly increased DOM brain levels. It is concluded that the effects of DOM as a discriminative stimulus are potentiated by the acute administration of citalopram and this effect is not mediated by additivity or pharmacokinetic mechanisms.     

不同来源肝细胞在体外降脂药物评价中药效反应的对比

目的:通过对比不同来源的人肝癌细胞系HepG2和原代大鼠肝细胞在体外降脂药物评价中药效反应,指导两种肝细胞在体外降脂药物评价中的实际应用。方法:用游离脂肪酸(油酸/棕榈酸,2:1)诱导HepG2细胞、原代大鼠肝细胞脂肪变性,并用100μmol·L-1苯扎贝特干预,检测细胞内甘油三酯(TG)、总胆固醇(TC)、活性氧(ROS)含量,细胞内脂滴数目、并检测细胞上清液中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果:FFA刺激使HepG2细胞和原代大鼠肝细胞脂质沉积(TG、脂滴)和氧化应激(ROS、MDA、SOD)水平上升。苯扎贝特对HepG2细胞1 mmol·L-1FFA造模组和原代大鼠肝细胞0.5 mmol·L-1FFA造模组脂质沉积和氧化应激水平改善显著;而HepG2细胞0.5 mmol·L-1FFA造模组和原代大鼠肝细胞1 mmol·L-1FFA造模组脂质沉积和氧化应激水平在苯扎贝特干预后变化不明显。结论:在相同FFA造模浓度,原代大鼠肝细胞病理特征变化更为明显;苯扎贝特对两种肝细胞在脂质沉积和氧化应激水平的作用也不完全相同。因而HepG2细胞和原代大鼠肝细胞在体外降脂药物评价中药效反应是不完全相同的。     

Oxysterols and redox signaling in the pathogenesis of non-alcoholic fatty liver disease

Abstract

Oxysterols are oxidized species of cholesterol coming from exogenous (e.g. dietary) and endogenous (in vivo) sources. They play critical roles in normal physiologic functions such as regulation of cellular cholesterol homeostasis. Most of biological effects are mediated by interaction with nuclear receptor LXRα, highly expressed in the liver as well as in many other tissues. Such interaction participates in the regulation of whole-body cholesterol metabolism, by acting as “lipid sensors”. Moreover, it seems that oxysterols are also suspected to play key roles in several pathologies, including cardiovascular and inflammatory disease, cancer, and neurodegeneration. Growing evidence suggests that oxysterols may contribute to liver injury in non-alcoholic fatty liver disease. The present review focuses on the current status of knowledge on oxysterols’ biological role, with an emphasis on LXR signaling and oxysterols’ physiopathological relevance in NAFLD, suggesting new pharmacological development that needs to be addressed in the near future.     

Mesenchymal stromal cells protect hepatocytes from lipotoxicity through alleviation of endoplasmic reticulum stress by restoring SERCA activity

The aim of this study was to investigate how mesenchymal stromal cells (MSCs) modulate metabolic balance and attenuate hepatic lipotoxicity in the context of non-alcoholic fatty liver disease (NAFLD). In vivo, male SD rats were fed with high-fat diet (HFD) to develop NAFLD; then, they were treated twice by intravenous injections of rat bone marrow MSCs. In vitro, HepG2 cells were cocultured with MSCs by transwell and exposed to palmitic acid (PA) for 24 hours. The endoplasmic reticulum (ER) stressor thapsigargin and sarco/ER Ca²⁺-ATPase (SERCA2)–specific siRNA were used to explore the regulation of ER stress by MSCs. We found that MSC administration improved hepatic steatosis, restored systemic hepatic lipid and glucose homeostasis, and inhibited hepatic ER stress in HFD-fed rats. In hepatocytes, MSCs effectively alleviated the cellular lipotoxicity. Particularly, MSCs remarkably ameliorated the ER stress and intracellular calcium homeostasis induced by either PA or thapsigargin in HepG2 cells. Additionally, long-term HFD or PA stimulation would activate pyroptosis in hepatocytes, which may contribute to the cell death and liver dysfunction during the process of NAFLD, and MSC treatment effectively ameliorates these deleterious effects. SERCA2 silencing obviously abolished the ability of MSCs against the PA-induced lipotoxicity. Conclusively, our study demonstrated that MSCs were able to ameliorate liver lipotoxicity and metabolic disturbance in the context of NAFLD, in which the regulation of ER stress and the calcium homeostasis via SERCA has played a key role.     

Ischemic preconditioning induces autophagy and limits necrosis in human recipients of fatty liver grafts, decreasing the incidence of rejection episodes

Whether ischemic preconditioning (IP) reduces ischemia/reperfusion (I/R) injury in human normal and fatty livers remains controversial. We compared two independent groups of liver donor transplants with versus without steatosis to evaluate IP consequences. Liver donors with (n=22) or without (n=28) steatosis either did or did not undergo IP before graft retrieval. Clinical data from the recipients, as well as histological and immunohistological characteristics of post-reperfusion biopsies were analyzed. Incidence of post-reperfusion necrosis was increased (10/10 versus 9/14, respectively; P<0.05) and the clinical outcome of recipients was worse for non-IP steatotic liver grafts compared with non-IP non-steatotic grafts. IP significantly lowered the transaminase values only in patients receiving a non-steatotic liver. An increased expression of beclin-1 and LC3, two pro-autophagic proteins, tended to decrease the incidence of necrosis (P=0.067) in IP steatotic livers compared with non-IP steatotic group. IP decreased the incidence of acute and chronic rejection episodes in steatotic livers (2/12 versus 6/10; P=0.07 and 2/12 versus 7/10; P<0.05, respectively), but not in non-steatotic livers. Thus, IP may induce autophagy in human steatotic liver grafts and reduce rejection in their recipients.     

Downregulation of adipose triglyceride lipase in the heart aggravates diabetic cardiomyopathy in db/db mice

Adipose triglyceride lipase (ATGL) was recently identified as a rate-limiting triglyceride (TG) lipase and its activity is stimulated by comparative gene identification-58 (CGI-58). Mutations in the ATGL or CGI-58 genes are associated with neutral lipid storage diseases characterized by the accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, is characterized by TG accumulation in coronary atherosclerotic lesions and in the myocardium. Recent reports showed that myocardial TG accumulation is significantly higher in patients with diabetes and is associated with impaired left ventricular diastolic function. Therefore, we investigated the roles of ATGL and CGI-58 in the development of myocardial steatosis in the diabetic state. Histological examination with oil red O staining showed marked lipid deposition in the hearts of diabetic fatty db/db mice. Cardiac triglyceride and diglyceride contents were greater in db/db mice than in db/+ control mice. Next, we determined the expression of genes and proteins that affect lipid metabolism, and found that ATGL and CGI-58 expression levels were decreased in the hearts of db/db mice. We also found increased expression of genes regulating triglyceride synthesis (sterol regulatory element-binding protein 1c, monoacylglycerol acyltransferases, and diacylglycerol acyltransferases) in db/db mice. Regarding key modulators of apoptosis, PKC activity, and oxidative stress, we found that Bcl-2 levels were lower and that phosphorylated PKC and 8-hydroxy-2′-deoxyguanosine levels were higher in db/db hearts. These results suggest that reduced ATGL and CGI-58 expression and increased TG synthesis may exacerbate myocardial steatosis and oxidative stress, thereby promoting cardiac apoptosis in diabetic mice.