Kinetics and morphology of chemically induced popliteal lymph node reactions compared with antigen-, mitogen-, and graft-versus-host-reaction-induced responses

Changes in the popliteal lymph node (PLN) in mice evoked by a local graft-versus-host (GVH) reaction and by a single injection of various agents into the hind footpad were compared. The drug diphenylhydantoin induced similar weight changes in time as the GVH reaction. More vigorous and protracted reactions were induced by the drug nitrofurantoin and the contact sensitizer dinitrochlorobenzene, whereas the antigens lipopolysaccharide and sheep erythrocytes caused very moderate and short-lasting weight changes. Alterations of lymph node architecture upon injection of diphenylhydantoin resembled those observed during the GVH response. Some quantitative and qualitative differences were noted for nitrofurantoin, but clearly deviant morphological alterations were seen in response to lipopolysaccharide and sheep erythrocytes. The PLN reaction to dinitrochlorobenzene had features of both the GVH reaction and the antigen-induced responses. These findings support the concept that some drugs and chemicals may induce or exacerbate lymphoproliferative disorders by GVH-like mechanisms.     

Inhibition of intestinal cholesterol absorption decreases atherosclerosis but not adipose tissue inflammation

Adipose tissue inflammation is associated with insulin resistance and increased cardiovascular disease risk in obesity. We previously showed that addition of cholesterol to a diet rich in saturated fat and refined carbohydrate significantly worsens dyslipidemia, insulin resistance, adipose tissue macrophage accumulation, systemic inflammation, and atherosclerosis in LDL receptor-deficient (Ldlr^−/−) mice. To test whether inhibition of intestinal cholesterol absorption would improve metabolic abnormalities and adipose tissue inflammation in obesity, we administered ezetimibe, a dietary and endogenous cholesterol absorption inhibitor, to Ldlr^−/− mice fed chow or high-fat, high-sucrose (HFHS) diets without or with 0.15% cholesterol (HFHS+C). Ezetimibe blunted weight gain and markedly reduced plasma lipids in the HFHS+C group. Ezetimibe had no effect on glucose homeostasis or visceral adipose tissue macrophage gene expression in the HFHS+C fed mice, although circulating inflammatory markers serum amyloid A (SSA) and serum amyloid P (SSP) levels decreased. Nevertheless, ezetimibe treatment led to a striking (>85%) reduction in atherosclerotic lesion area with reduced lesion lipid and macrophage content in the HFHS+C group. Thus, in the presence of dietary cholesterol, ezetimibe did not improve adipose tissue inflammation in obese Ldlr^−/− mice, but it led to a major reduction in atherosclerotic lesions associated with improved plasma lipids and lipoproteins.     

Evidence for regulated monoacylglycerol acyltransferase expression and activity in human liver

Intrahepatic lipid accumulation is extremely common in obese subjects and is associated with the development of insulin resistance and diabetes. Hepatic diacylglycerol and triacylglycerol synthesis predominantly occurs through acylation of glycerol-3-phosphate. However, an alternative pathway for synthesizing diacylglycerol from monoacylglycerol acyltransferases (MGAT) could also contribute to hepatic glyceride pools. MGAT activity and the expression of the three genes encoding MGAT enzymes (MOGAT1, MOGAT2, and MOGAT3) were determined in liver biopsies from obese human subjects before and after gastric bypass surgery. MOGAT expression was also assessed in liver of subjects with nonalcoholic fatty liver disease (NAFLD) or control livers. All MOGAT genes were expressed in liver, and hepatic MGAT activity was readily detectable in liver lysates. The hepatic expression of MOGAT3 was highly correlated with MGAT activity, whereas MOGAT1 and MOGAT2 expression was not, and knockdown of MOGAT3 expression attenuated MGAT activity in a liver-derived cell line. Marked weight loss following gastric bypass surgery was associated with a significant reduction in MOGAT2 and MOGAT3 expression, which were also overexpressed in NAFLD subjects. These data suggest that the MGAT pathway is active and dynamically regulated in human liver and could be an important target for pharmacologic intervention for the treatment of obesity-related insulin resistance and NAFLD.     

Cytometric analysis for drug-induced steatosis in HepG2 cells

Drugs are capable of inducing hepatic lipid accumulation. When fat accumulates, lipids are primarily stored as triglycerides which results in steatosis and provides substrates for lipid peroxidation. An in vitro multiparametric flow cytometry assay was performed in HepG2 cells by using fluorescent probes to analyze cell viability (propidium iodide, PI), lipid accumulation (BODIPY493/503), mitochondrial membrane potential (tetramethyl rhodamine methyl ester, TMRM) and reactive oxygen species generation (ROS) (2′,7′-dihydrochlorofluorescein diacetate, DHCF-DA) as functional markers. All the measurements were restricted to live cells by gating the cells that excluded PI or those that exhibited the typical forward and side scatter features of live cells. The assay was qualified by analyzing a number of selected model drugs with a well documented induction of steatosis in vivo using different mechanisms as positive controls and several non-steatosic compounds as negative controls. For the cytometric screening assay, the concentrations tested were up to the corresponding IC₁₀ value determined by the MTT assay. Among the parameters analyzed, increased BODIPY fluorescence was the most sensitive and selective marker of drug-induced steatosis. However, a more consistent predictive approach was the combination of two endpoints: lipid accumulation and ROS generation. The assay correctly identified 100% of steatosis-positive and steatosis-negative compounds, and a high steatosis risk was predicted for amiodarone, doxycycline, tetracycline and valproate treatments at therapeutic doses. The results suggest that this cell-based assay may be a useful approach to identify the potential of drug candidates to induce steatosis.     

Screening and identification of differentially expressed genes in goose hepatocytes exposed to free fatty acid

The overaccumulation of triglycerides in hepatocytes induces hepatic steatosis; however, little is known about the mechanism of goose hepatic steatosis. The aim of this study was to define an experimental model of hepatocellular steatosis with TG overaccumulation and minimal cytotoxicity, using a mixture of various proportions of oleate and palmitate free fatty acids (FFAs) to induce fat‐overloading, then using suppressive subtractive hybridization and a quantitative PCR approach to identify genes with higher or lower expression levels after the treatment of cells with FFA mixtures. Overall, 502 differentially expressed clones, representing 21 novel genes and 87 known genes, were detected by SSH. Based on functional clustering, up‐ and down‐regulated genes were mostly related to carbohydrate and lipid metabolism, enzyme activity and signal transduction. The expression of 20 selected clones involved with carbohydrate and lipid metabolism pathways was further studied by quantitative PCR. The data indicated that six clones similar to the genes ChREBP, FoxO1, apoB, IHPK2, KIF1B, and FSP27, which participate in de novo synthesis of fatty acid and secretion of very low density lipoproteins, had significantly lower expression levels in the hepatocytes treated with FFA mixtures. Meanwhile, 13 clones similar to the genes DGAT‐1, ACSL1, DHRS7, PPARα, L‐FABP, DGAT‐2, PCK, ACSL3, CPT‐1, A‐FABP, PPARβ, MAT, and ALDOB had significantly higher expression levels in the hepatocytes treated with FFA mixtures. These results suggest that several metabolic pathways are altered in goose hepatocytes, which may be useful for further research into the molecular mechanism of goose hepatic steatosis. J. Cell. Biochem. 111: 1482–1492, 2010. © 2010 Wiley‐Liss, Inc.     

Angiotensin-converting enzyme inhibition reduces lipid deposits in myocardium and improves left ventricular function of obese zucker rats

Objective: Alterations in the renin angiotensin system, cardiac lipotoxicity, and left ventricular (LV) dysfunction have been reported in obese rats. The present study examined whether angiotensin‐converting enzyme inhibition could ameliorate lipid deposition and ventricular function in the myocardium of obese Zucker rats (OZRs). Research Methods and Procedures: For 6 months, rats were treated as follows: Group (G) 1, OZR, no treatment; G2, OZR + ramipril (R); G3, OZR + amlodipine (AML); and G4, lean Zucker rats. LV function was assessed by echocardiogram and lipid deposits in cardiomyocytes (LDCM) by light microscopy using Oil red O. Results: At the end of the experiment, both OZR + R and OZR + AML groups presented similar reduction in blood pressure in comparison with untreated OZR (p < 0.01). OZR with R presented lower insulin‐to‐glucose ratio and lower serum triglycerides and cholesterol when compared with both untreated OZR and OZR with AML (p < 0.01). Fractional shortening by echocardiogram was as follows: G1, 25.4 ± 3.8 (vs. G2 and G4, p < 0.05); G2, 37.2 ± 2.4; G3, 29.3 ± 4.4 (vs. G2 and G4, p < 0.05); and G4, 40.8 ± 2.3. Percentage LDCM was as follows: G1, 12.4 ± 2.7 (vs. G2 and G4, p < 0.05); G2, 0.8 ± 0.2; G3, 11.1 ± 2.1 (vs. G2 and G4, p < 0.05); and G4, 0.1 ± 0.1. There was a negative correlation between fractional shortening and LDCM percentage in OZR (r = ?0.93) and in OZR + AML (r = ?0.87). Discussion: AML reduced blood pressure significantly; however, it failed to modify both metabolic parameters and LDCM. In contrast, R showed a substantial reduction in LDCM, together with LV function preservation.     

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.