ColE-type plasmid bearing blaOXA-232 increases persister cell formation.

OBJECTIVES: Bacterial persister cells are a sub-population of cells that are tolerant to high concentrations of antibiotics. In this study, we investigated the effect of plasmids bearing carbapenemase genes on persister cell formation. METHODS: Three plasmids, IncX3-type plasmid with blaNDM-1, IncN-type plasmid with blaKPC-2, and ColE-type plasmid with blaOXA-232, were transformed into Escherichia coli MG1655. For the ColE-type plasmid (pM5_OXA232), gene-deletion plasmids were constructed and transformed into the MG1655. Persister assays were performed against ciprofloxacin and amikacin, and expression levels of relA and spoT were measured for the wild-type E. coli and all transformants. RESULTS: Unlike the other two plasmids, transformation of ColE-type plasmid (pM5_OXA232) caused a significant increase in the formation of persister cells. Compared with transformants that harboured intact pM5_OXA232, transformants that harboured plasmids with deletions of gene(s), vbhA, hypothetical gene, or a mobile gene cassette showed decreased persister cell formation. Expression levels of relA and spoT exhibited patterns similar to those of persister cell formation rates, particularly against ciprofloxacin. CONCLUSION: In this study, we showed that a small ColE-type plasmid bearing blaOXA-232 has an effect on persister cell formation, possibly contributing to the dissemination of low-level carbapanemase.

Comparison of Virulence between Two Main Clones (ST11 and ST307) of Klebsiella pneumoniae Isolates from South Korea.

In this study, we investigate the characteristics of two main clones of carbapenemase-producing Klebsiella pneumoniae isolates from South Korea, ST11 and ST307, including carbapenem-susceptible isolates. Antibiotic susceptibility, serotype or wzi allelic type, the presence of virulence genes, and virulence with respect to serum resistance and macrophage internalization were determined for ST11 and ST307 isolates. ST11 isolates had a wide range of characteristics, including serotype and virulence, compared with those of homogeneous ST307 isolates. The wzi14 or K14 type had higher virulence than that of other serotypes among the ST11 isolates, and the homogeneous ST307 isolates showed similar virulence level as that of the wzi14-type ST11 isolates. Our data suggest that it is necessary to monitor not only the introduction and spread of a specific clone, but also its detailed serotype.

Two Distinct Genotypes of KPC-2-Producing Klebsiella pneumoniae Isolates from South Korea.

In this study, we investigated the characteristics of KPC-2-producing Klebsiella pneumoniae (KP-Kp) isolates from a hospital in South Korea. Among the 37 KP-Kp isolates, two main clones were identified-ST11 and ST307. ST11 isolates showed higher minimum inhibitory concentrations for carbapenems than ST307 isolates. All ST307 isolates were resistant to gentamicin and trimethoprim-sulfamethoxazole, but ST11 isolates were not. However, most tigecycline-resistant or colistin-resistant isolates belonged to ST11. The two KP-Kp clones showed different combinations of wzi and K serotypes. Plasmids from ST11 KP-Kp isolates exhibited diverse incompatibility types. Serum resistance and macrophage infection assays indicated that ST11 may be more virulent than ST307. The changes in the main clones of KP-Kp isolates over time as well as the different characteristics of these clones, including virulence, suggest the need for their continuous monitoring.

Antiobesity Effects of Short-Chain Chitosan in Diet-Induced Obese Mice.

Dietary chitosan is known for its antiobesity effects by combining with bile acid and lipid droplets. When the chitosan structure is broken into short chains, the fat-binding capacity increases. The aim of this study was to compare long-chain chitosan (LC) with short-chain chitosan (SC) for their antiobesity effects in high-fat diet (HFD)-induced obese C57BL/6J mice for 12 weeks. The body weights of mice in both chitosan groups were decreased, especially in the SC group compared with the LC group. Total white adipose tissue and visceral fat weights were also decreased in mice of the SC group more than those of the HFD group. Moreover, SC supplementation lowered plasma triglyceride (TG) and cholesterol levels, whereas LC only lowered plasma free fatty acid level. Fecal lipids were increased in mice of both LC and SC groups, and hepatic TG and cholesterol levels were decreased in both groups. SC lowered phosphatidate phosphohydrolase activity and elevated ß-oxidation in the liver. Furthermore, SC decreased the expression of the hepatic lipid-regulating genes, including fatty acid synthase, peroxisome proliferator-activated receptor (PPAR)γ1, and PPARγ2; and increased the expression of carnitine palmitoyl transferase 1α and peroxisome proliferator-activated receptor γ coactivator (PGC)1α genes. In conclusion, we demonstrated that long-term supplementation of SC can ameliorate body weight and lipid levels by increasing lipid excretion and regulating lipid metabolism, including some enzyme activities and gene expression levels, in HFD-induced obese mice.

Apigenin Ameliorates Dyslipidemia, Hepatic Steatosis and Insulin Resistance by Modulating Metabolic and Transcriptional Profiles in the Liver of High-Fat Diet-Induced Obese Mice.

Several in vitro and in vivo studies have reported the anti-inflammatory, anti-diabetic and anti-obesity effects of the flavonoid apigenin. However, the long-term supplementary effects of low-dose apigenin on obesity are unclear. Therefore, we investigated the protective effects of apigenin against obesity and related metabolic disturbances by exploring the metabolic and transcriptional responses in high-fat diet (HFD)-induced obese mice. C57BL/6J mice were fed an HFD or apigenin (0.005%, w/w)-supplemented HFD for 16 weeks. In HFD-fed mice, apigenin lowered plasma levels of free fatty acid, total cholesterol, apolipoprotein B and hepatic dysfunction markers and ameliorated hepatic steatosis and hepatomegaly, without altering food intake and adiposity. These effects were partly attributed to upregulated expression of genes regulating fatty acid oxidation, tricarboxylic acid cycle, oxidative phosphorylation, electron transport chain and cholesterol homeostasis, downregulated expression of lipolytic and lipogenic genes and decreased activities of enzymes responsible for triglyceride and cholesterol ester synthesis in the liver. Moreover, apigenin lowered plasma levels of pro-inflammatory mediators and fasting blood glucose. The anti-hyperglycemic effect of apigenin appeared to be related to decreased insulin resistance, hyperinsulinemia and hepatic gluconeogenic enzymes activities. Thus, apigenin can ameliorate HFD-induced comorbidities via metabolic and transcriptional modulations in the liver.

Time-dependent network analysis reveals molecular targets underlying the development of diet-induced obesity and non-alcoholic steatohepatitis.

Prolonged high-fat diet leads to the development of obesity and multiple comorbidities including non-alcoholic steatohepatitis (NASH), but the underlying molecular basis is not fully understood. We combine molecular networks and time course gene expression profiles to reveal the dynamic changes in molecular networks underlying diet-induced obesity and NASH. We also identify hub genes associated with the development of NASH. Core diet-induced obesity networks were constructed using Ingenuity pathway analysis (IPA) based on 332 high-fat diet responsive genes identified in liver by time course microarray analysis (8 time points over 24 weeks) of high-fat diet-fed mice compared to normal diet-fed mice. IPA identified five core diet-induced obesity networks with time-dependent gene expression changes in liver. These networks were associated with cell-to-cell signaling and interaction (Network 1), lipid metabolism (Network 2), hepatic system disease (Network 3 and 5), and inflammatory response (Network 4). When we merged these core diet-induced obesity networks, Tlr2, Cd14, and Ccnd1 emerged as hub genes associated with both liver steatosis and inflammation and were altered in a time-dependent manner. Further, protein-protein interaction network analysis revealed Tlr2, Cd14, and Ccnd1 were interrelated through the ErbB/insulin signaling pathway. Dynamic changes occur in molecular networks underlying diet-induced obesity. Tlr2, Cd14, and Ccnd1 appear to be hub genes integrating molecular interactions associated with the development of NASH. Therapeutics targeting hub genes and core diet-induced obesity networks may help ameliorate diet-induced obesity and NASH.

Time-course microarrays reveal early activation of the immune transcriptome and adipokine dysregulation leads to fibrosis in visceral adipose depots during diet-induced obesity.

BACKGROUND: Visceral white adipose tissue (WAT) hypertrophy, adipokine production, inflammation and fibrosis are strongly associated with obesity, but the time-course of these changes in-vivo are not fully understood. Therefore, the aim of this study was to establish the time-course of changes in adipocyte morphology, adipokines and the global transcriptional landscape in visceral WAT during the development of diet-induced obesity. RESULTS: C57BL/6 J mice were fed a high-fat diet (HFD) or normal diet (ND) and sacrificed at 8 time-points over 24 weeks. Excessive fat accumulation was evident in visceral WAT depots (Epidydimal, Perirenal, Retroperitoneum, Mesentery) after 2-4 weeks. Fibrillar collagen accumulation was evident in epidydimal adipocytes at 24 weeks. Plasma adipokines, leptin, resistin and adipsin, increased early and time-dependently, while adiponectin decreased late after 20 weeks. Only plasma leptin and adiponectin levels were associated with their respective mRNA levels in visceral WAT. Time-course microarrays revealed early and sustained activation of the immune transcriptome in epididymal and mesenteric depots. Up-regulated inflammatory genes included pro-inflammatory cytokines, chemokines (Tnf, Il1rn, Saa3, Emr1, Adam8, Itgam, Ccl2, 3, 4, 6, 7 and 9) and their upstream signalling pathway genes (multiple Toll-like receptors, Irf5 and Cd14). Early changes also occurred in fibrosis, extracellular matrix, collagen and cathepsin related-genes, but histological fibrosis was only visible in the later stages. CONCLUSIONS: In diet-induced obesity, early activation of TLR-mediated inflammatory signalling cascades by CD antigen genes, leads to increased expression of pro-inflammatory cytokines and chemokines, resulting in chronic low-grade inflammation. Early changes in collagen genes may trigger the accumulation of ECM components, promoting fibrosis in the later stages of diet-induced obesity. New therapeutic approaches targeting visceral adipose tissue genes altered early by HFD feeding may help ameliorate the deleterious effects of diet-induced obesity.

Long-term adaptation of global transcription and metabolism in the liver of high-fat diet-fed C57BL/6J mice.

SCOPE: This study investigated the global transcriptional and metabolic changes occurring at multiple time points over 24 wk in response to a high-fat diet (HFD). METHODS AND RESULTS: C57BL/6J mice were fed a HFD or normal diet (ND) over 24 wk. HFD-fed mice developed early clinical indicators of obesity-related co-morbidities including fatty liver, insulin resistance, hyperglycemia and hypercholesterolemia. Time-course microarray analysis at eight time points over 24 wk identified 332 HFD responsive genes as potential targets to counteract diet-induced obesity (DIO) and related co-morbidities. Glucose regulating enzyme activity and gene expression were altered early in the HFD-fed mice. Fatty acid (FA) and triglyceride (TG) accumulation in combination with inflammatory changes appear to be likely candidates contributing to hepatic insulin resistance. Cidea seemed to be one of representative genes related to these changes. CONCLUSION: Global transcriptional and metabolic profiling across multiple time points in liver revealed potential targets for nutritional interventions to reverse DIO. In future, new approaches targeting HFD responsive genes and hepatic metabolism could help ameliorate the deleterious effects of an HFD and DIO-related complication.

Differential effect of corn oil-based low trans structured fat on the plasma and hepatic lipid profile in an atherogenic mouse model: comparison to hydrogenated trans fat.

BACKGROUND: Trans fat are not desirable in many aspects on health maintenance. Low trans structured fats have been reported to be relatively more safe than trans fats. METHODS: We examined the effects of low trans structured fat from corn oil (LC), compared with high trans fat shortening, on cholesterol and fatty acid metabolism in apo E deficient mice which is an atherogenic animal model. The animals were fed a high trans fat (10% fat: commercial shortening (CS)) or a low trans fat (LC) diet for 12 weeks. RESULTS: LC decreased apo B and hepatic cholesterol and triglyceride concentration compared to the CS group but significantly increased plasma total cholesterol and triglyceride concentration and fecal lipids with a simultaneous increase in HDL-cholesterol level, apo A-I, and the ratio of HDL-cholesterol to total cholesterol (HTR). Reduction of hepatic lipid levels by inclusion of LC intake was observed alongside modulation of hepatic enzyme activities related to cholesterol esterification, fatty acid metabolism and fecal lipids level compared to the CS group. The differential effects of LC intake on the plasma and hepatic lipid profile seemed to be partly due to the fatty acid composition of LC which contains higher MUFA, PUFA and SFA content as well as lower content of trans fatty acids compared to CS. CONCLUSIONS: We suggest that LC may exert a dual effect on plasma and hepatic lipid metabolism in an atherogenic animal model. Accordingly, LC, supplemented at 10% in diet, had an anti-atherogenic effect on these apo E-/- mice, and increased fecal lipids, decreased hepatic steatosis, but elevated plasma lipids. Further studies are needed to verify the exact mode of action regarding the complex physiological changes and alteration in lipid metabolism caused by LC.

Comparison of hesperetin and its metabolites for cholesterol-lowering and antioxidative efficacy in hypercholesterolemic hamsters.

This study was performed to compare the hypolipidemic and antioxidant efficacy of hesperetin and its metabolites in hypercholesterolemic hamsters. The hamsters were fed a high-fat (10% coconut oil and 0.2% cholesterol, wt/wt) diet or a high-fat diet supplemented with hesperetin (0.02%) or hesperetin metabolites, 3,4-dihydroxyphenylpropionic acid (DHPP) (0.012%) and 3-methoxy-4-hydroxycinnamic acid (ferulic acid) (0.013%), for 12 weeks. Dietary DHPP and ferulic acid were found to have significantly decreased the levels of the plasma total cholesterol, non-high-density lipoprotein-cholesterol (HDL-C), apolipoprotein B, hepatic lipids, and cholesterol-regulating enzymes compared to the control group. In particular, ferulic acid was more potent with respect to raising HDL-C/total cholesterol ratio and paraoxonase levels while decreasing atherogenic index values. Hesperetin and its metabolites seemed to enhance antioxidant capacity by lowering the hydrogen peroxide and lipid peroxide (thiobarbituric acid-reactive substrates) levels. Among the hesperetin metabolites tested, the relative potency of ferulic acid for reducing the risks of atherosclerosis in hamsters was found to be greater.

Actions of ferulic acid and vitamin E on prevention of hypercholesterolemia and atherogenic lesion formation in apolipoprotein E-deficient mice.

This study was carried out to investigate whether dietary vitamin E and ferulic acid (FA) can exert possible interactions on preventions of hypercholesterolemia and atherogenic lesion formation in C57BL/65 apolipoprotein E-deficient (apo E(-/-)) mice. Four-week-old male apo E(-/-) mice were randomly divided into three groups and given one of three types of Western diets with various amounts of vitamin E (0.02%, 0%, or 0.2%) for 15 weeks. FA was added to vitamin E-free Western diet and vitamin E-rich Western diet at the 0.02% level. The plasma total cholesterol concentration was significantly lowered when FA was added to the vitamin E-free and vitamin E-rich Western diet as compared to the normal vitamin E Western diet (0.02% vitamin E), and this was accompanied with a decreased hepatic acyl-coenzyme A:cholesterol acyltransferase activity. The hepatic and erythrocyte thiobarbituric acid-reactive substances levels were significantly lowered when FA was added to the vitamin E-rich Western diet, which was attributable to increased activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and paraoxonase. Accordingly, vitamin E and/or FA are beneficial for prevention of hypercholesterolemia and atherogenesis in apo E(-/-) mice. In particular, dietary FA exhibited an anti-atherosclerotic property, and this effect was synergistically enhanced with the vitamin E supplement.

Low trans structured fat from flaxseed oil improves plasma and hepatic lipid metabolism in apo E(-/-) mice.

The objective of this study was to explicate the effects of feeding low trans structured fat from flaxseed oil (LF) on plasma and hepatic lipid metabolism involved in apo E(-/-) mice. The animals were fed a commercial shortening (CS), commercial low trans fat (CL) and LF diet based on AIN-76 diet (10% fat) for 12 weeks. LF supplementation exerted a significant suppression in hepatic lipid accumulation with the concomitant decrease in liver weight. The LF significantly lowered plasma total cholesterol and free fatty acid whereas it significantly increased HDL-C concentration and the HDL-C/total-C ratio compared to the CS group. Reduction of hepatic lipid levels in the LF group was related with the suppression of hepatic enzyme activities for fatty acid and triglyceride synthesis, and cholesterol regulating enzyme activity compared to the CS and CL groups. Accordingly, low trans structured fat from flaxseed oil is highly effective for improving hyperlipidemia and hepatic lipid accumulation in apo E(-/-) mice.

Hypolipidemic effect of dietary diacylglycerol oil in Sprague-Dawley rats fed a normal diet.

The present study compared the effects of dietary diacylglycerol (DG) and triacylglycerol (TG) oil on lipid metabolism in rats fed a 5% fat (AIN-76) diet for 6 weeks. The plasma and hepatic lipids, hepatic cholesterol-regulating enzyme activity, and hepatic and adipose tissue fatty acid metabolism enzyme activities were determined. Among plasma lipids, triglyceride, free fatty acid, and phospholipid concentrations were significantly lower in the DG group than in the TG group. A lower plasma TG level was accompanied by an increase in adipocyte lipoprotein lipase activity. The hepatic triglyceride level was significantly (P < .001) lowered in the DG group, which was attributable to an increased fatty acid oxidation enzyme (beta-oxidation) activity and a reduced fatty acid synthesis enzyme (glucose-6-phosphate dehydrogenase) activity. The plasma cholesterol concentration was significantly lower in the DG group and was accompanied by a lower hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. The DG oil used in this study was beneficial for enhancing lipid metabolism with apparent hypolipidemic effects.

Naringin time-dependently lowers hepatic cholesterol biosynthesis and plasma cholesterol in rats fed high-fat and high-cholesterol diet.

The flavonoid naringin was investigated for its differential effects on hepatic cholesterol regulation when supplemented for 3 weeks and 6 weeks. Sprague-Dawley rats were fed a high-fat and high-cholesterol diet with or without 0.02% naringin supplement for 3 or 6 weeks. Supplementation with naringin resulted in a significant decrease in the plasma cholesterol and triglyceride concentrations in the 6-week trial. Although high-density lipoprotein (HDL)-cholesterol was not altered in either trial, the HDL-cholesterol/total cholesterol ratio (in percent) was significantly higher, and the atherogenic index was significantly lower in the naringin-supplemented groups in the 6-week trial. The hepatic cholesterol content was also lowered by naringin supplementation only in the 6-week trial. The hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was lower in the rats supplemented with naringin for 6 weeks, while the hepatic acyl-coenzyme A:cholesterol acyltransferase activity was lower in both the 3-week and 6-week trials. Results indicate that supplementation with naringin for 3 weeks did not exhibit a hypolipidemic effect when given with a HFHC diet. Naringin can, however, be beneficial for lowering hepatic cholesterol biosynthesis and levels of plasma lipids when supplemented for 6 weeks in this animal model.