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Neutraceuticals

Author and Curator: Larry H Bernstein, MD, FCAP 

 

Introduction

This article is concerned with a very fast growing part of the food industry. Unfortunately, there have been such products that were sold that did not contain what was purported to provide the benefit. This segment of the medicinal industry is regulated as FOOD (food product), unlike pharmaceuticals. This means that the basic safety requirement is compliance with a labeling standard.  There are no clinical trials.  The incentive for the development in this sector is from 2,500 years of history in the use of plants for health benefits.  The new science that can improve the potency and the safety is the technology that has grown up to extract, refine and produce the active substance in pure form.  The methods used called Flow Chemistry has been covered recently by Anthony Melvin Crasso at his outstanding pharmaceutical site.  I shall list important articles that have been in the Pharmaceutical Intelligence site at the end of this discussion.

Antidiarrheal and antioxidant activities of chamomile (Matricaria recutita L.) decoction extract in rats

Hichem Sebai, Mohamed-Amine Jabri, Abdelaziz Souli, Kais Rtibi, et al.
J Ethnopharmacol 152(2014)327–332
http://dx.doi.org/10.1016/j.jep.2014.01.015

Ethnopharmacological relevance: Matricaria recutita L. (Chamomile) has been widely used in the Tunisian traditional medicine for the treatment of digestive system disorders. The present work aims to investigate the protective effects of chamomile decoction extract (CDE) against castor oil-induced diarrhea and oxidative stress in rats. Methods:The antidiarrheal activity was evaluated using castor oil-induced diarrhea method. In this respect, rats were divided into six groups: Control, Castor oil, Castor oil + Loperamide (LOP) and Castor oil + various doses of CDE. Animals were perorally (p.o.) pre-treated with CDE during 1 hand intoxicated for 2 or 4h by acute oral administration of castor oil.  Results: Our results showed that CDE produced a significant dose-dependent protection against castor oil-induced diarrhea and intestinal fluid accumulation. On the other hand, we showed that diarrhea was accompagnied by an oxidative stress status assessed by an increase of malondialdehyde (MDA) level and depletion of antioxidant enzyme activities as superoxide dismutase(SOD), catalase (CAT) and glutathione peroxidase (GPx). Castor oil also increased gastric and intestinal mucosa hydrogen peroxide (H2O2) and free iron levels. Importantly, we showed that chamomile pre-treatment abrogated all these biochemical alterations. Conclusion: These findings suggested that chamomile extract had a potent antidiarrheal and antioxidant properties in rats confirming their use in traditional medicine.

Anti-diarrheal activity of methanol extract of Santalum album L. in mice and gastrointestinal effect on the contraction of isolated jejunum in rats

Huimin Guo, Jingze Zhang, Wenyuan Gao, Zhuo Qu, Changxiao Liu
J Ethnopharmacol 54(2014)704–710
http://dx.doi.org/10.1016/j.jep.2014.04.043

Ethnopharmacological relevance: Santalum album L., namely Sandalwood, honoredas “Green Gold”, is a traditional Chinese herb which has the effects of antidiarrheal and antibacterial activity. But there is limited scientific study on its activity and mechanism in gastrointestinal disorders.
Materials and methods: in vivo, after intragastric administration, the methanol extract of Sandalwood (SE) (200, 400 and 800 mg/kg) were studied in castor oil-induced diarrhea mice. By the test of small intestinal hyperfunction induced by neostigmine, SE was studied on gastrointestinal transit including gastric emptying and small intestinal motility. Meanwhile, in vitro, the effects of SE (0.02, 0.05, 0.1, 0.2, 0.3, 0.4 mg/mL) on the isolated tissue preparations of rat jejunum were also investigated. The rat jejunum strips were precontacted with acetylcholine (Ach; 10-6M), 5-hydroxytryptamine (5-HT, 200 μM) or potassium chloride (KCl; 60mM) and tested in the presence of SE. In addition, the possible myogenic effect was analyzed in the pretreatment of the jejunum preparations with SE or verapamilin Ca 2+-free high-K+ (60mM) solution containing EDTA. Results: At doses of 200, 400 and 800 mg/kg, SE showed significant anti-diarrheal activity against castor oil-induced diarrhea as compared with the control. At the same doses, it also inhibited the gastric emptying and small intestinal motility in the mice of which small intestinal hyperfunction induced by neostigmine. It caused inhibitory effects on the spontaneous contraction of rat-isolated jejunum in dose-dependent manner ranging from 0.02 to 0.4 mg/mL, and it also relaxed the Ach-induced, 5-HT-induced andK+-induced contractions. SE shifted the Ca 2+concentration–response curves to right, similar to that caused by verapamil (0.025mM).
Conclusions: These findings indicated that SE played a spasmolytic role in gastrointestinal motility which was probably mediated through inhibition of muscarinic receptors, 5-HT receptors and calcium influx. All these results provide pharmacological basis for its clinical use in gastrointestinal tract.

Apocynin attenuates isoproterenol-induced myocardial injury and fibrogenesis

Li Liu, Jingang Cui, Qinbo Yang, Chenglin Jia, Minqi Xiong, et al.
Biochem Biophys Res Commun 449(2014)55–61
http://dx.doi.org/10.1016/j.bbrc.2014.04.157

Oxidative stress is mechanistically implicated in the pathogenesis of myocardial injury and the subsequent fibrogenic tissue remodeling. Therapies targeting oxidative stresss in the process of myocardial fibrogenesis are still lacking and thus remain as an active research area in myocardial injury management. The current study evaluated the effects of a NADPH oxidase inhibitor, apocynin, on the production of reactive oxygen species and  the development of myocardial fibrogenesis in isoproterenol (ISO)-induced myocardial injury mouse model. The results revealed a remarkable effect of apocynin on attenuating the development of myocardial necrotic lesions, inflammation and fibrogenesis. Additionally, the protective effects of apocynin against myocardial injuries were associated with suppressed expression of an array of genes implicated in inflammatory and fibrogenic responses. Our study thus provided for the first time the histopathological and molecular evidence supporting the therapeutic value of apocynin against the development of myocardial injuries, in particular, myocardial fibrogenesis, which will benefit the mechanism-based drug development targeting oxidative stress in preventing and/or treating related myocardial disorders.

Anti-tumor effect of Shu-gan-Liang-Xue decoction in breast cancer is related to the inhibition of aromatase and steroid sulfatase expression

Ning Zhou, Shu-Yan Han, Fei Zhou, Ping-ping Li
J Ethnopharmacol 154 (2014) 687–695
http://dx.doi.org/10.1016/j.jep.2014.04.045

Ethnopharmacological relevance: Shu-Gan-Liang-Xue Decoction (SGLXD), a traditional Chinese herbal formula used to ameliorate the hot flushes in breast cancer patients, was reported to have anti-tumor effect on breast cancer. Estrogen plays a critical role in the genesis and evolution of breast cancer. Aromatase and steroid sulfatase (STS) are key estrogen synthesis enzymes that predominantly contribute to the high local hormone concentrations. The present study was to evaluate the anti-tumor effect of SGLXD on estrogen receptor (ER) positive breast cancer celllineZR-75-1, and to investigate its underlying mechanisms both in vitro and in vivo.
Materials and methods: The anti-tumor activity of SGLXD in vitro was investigated using the MTT assay. The in vivo anti-tumor effect of SGLXD was evaluated in non-ovariectomized and ovariectomized athymic nude mice. The effect of SGLXD on enzymatic activity of aromatase and STS was examined using the dual-luciferase  reporter (DLR) based on bioluminescent measurements. Aromatase and STS protein level were assessed using Western blot assay. Results: SGLXD showed dose-dependent inhibitory effect on the proliferation of ZR-75-1 cells with IC50 value of 3.40 mg/mL. It also suppressed the stimulating effect on cell proliferation of testosterone and estrogen sulfates (E1S). Oral administration of 6 g/kg of SGLXD for 25 days resulted in a reduction in tumor volume in non-ovariectomized and ovariectomized nude mice. The bioluminescent measurements confirmed that SGLXD has a dual-inhibitory effect on the activity of aromatase and STS. Western blot assay demonstrated that the treatment of SGLXD resulted in a decrease in aromatase and STS protein levels both in vitro and in vivo. Conclusion: Our results suggested that SGLXD showed anti-tumor effect on breast cancer cells both in vitro and in vivo. The anti-tumor activity of SGLXD is related to inhibition of aromatase and STS via decreasing their expression. SGLXD may be considered as a novel treatment for ER positive breast cancer.

Cardioprotective effect of embelin on isoproterenol-induced myocardial injury in rats: Possible involvement of mitochondrial dysfunction and apoptosis

Bidya Dhar Sahu, Harika Anubolu, Meghana Koneru, et al.
Life Sciences 107 (2014) 59–67
http://dx.doi.org/10.1016/j.lfs.2014.04.035

Aims: Preventive and/or therapeutic interventions using natural products for ischemic heart disease have gained considerable attention world wide. This study investigated the cardioprotective effect and possible mechanism of embelin, a major constituent of EmbeliaribesBurm, using isoproterenol (ISO)-induced myocardial infarction model in rats.
Materials and methods: Rats were pretreated for three days with embelin (50mg/kg,p.o) before inducing myocardial injury by administration of ISO (85mg/kg) subcutaneously at aninterval of 24h for 2 consecutive days. Serum was analyzed for cardiac specific injury biomarkers, lipids and lipoprotein content. Heart tissues were isolated and were used for histopathology, antioxidant and mitochondrial respiratory enzyme activity assays and western blot analysis. Key findings: Results showed that pretreatment with embelin significantly decreased the elevated levels of serum specific cardiac injury biomarkers (CK-MB, LDH and AST), serum levels of lipids and lipoproteins and histopathological changes when compared to ISO-induced controls. Exploration of the underlying mechanisms of embelin action revealed that embelin pretreatment restored the myocardial mitochondrial respiratory enzyme activities (NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and mitochondrial redox activity), strengthened antioxidant status and attenuated ISO-induced myocardial lipid peroxidation. Immunoblot analysis revealed that embelin interrupted mitochondria dependent apoptotic damage by increasing the myocardial expression of Bcl-2 and downregulating the expression of Bax, cytochrome c, cleaved-caspase-3&9 and PARP. Histopathology findings further strengthened the cardioprotective findings of embelin. Significance: Result suggested that embelin may have a potential benefit in preventing ischemic heart diseasel like myocardial infarction.

Effect of Matricaria chamomilla L. flower essential oil on the growth and ultrastructure of Aspergillus niger vanTieghem

Marziyeh Tolouee, Soheil Alinezhad, Reza Saberi, Ali Eslamifar, et al.
Intl J Food Microbiol 139 (2010) 127–133
http://dx.doi.org:/10.1016/j.ijfoodmicro.2010.03.032

The antifungal activity of Matricaria chamomilla L. flower essential oil was evaluated against Aspergillus niger with the emphasis on the plant’s mode of action at the electron microscopy level. A total of 21 compounds were identified in the plant oil using gaschromatography/massspectrometry(GC/MS) accounting for 92.86% of the oil composition. The main compounds identified were α-bisabolol (56.86%), trans-trans-farnesol (15.64%), cis-β-farnesene (7.12%), guaiazulene (4.24%), α-cubebene (2.69%), α-bisabololoxideA (2.19%) and chamazulene (2.18%). In the bioassay, A.niger was cultured on Potato Dextrose Broth medium in 6-well microplates in the presence of serial two fold concentrations of plant oil (15.62 to 1000 µg/mL) for 96h at 28°C. Based on the results obtained, A. niger growth was inhibited dose dependently with a maximum of ∼92.50% at the highest oil concentration. A marked retardation in conidial production by the fungus was noticed in relation to the inhibition of hyphal growth. The main changes of hyphae observed by transmission electron microscopy were disruption of cytoplasmic membranes and intracellular organelles, detachment of plasma membrane from the cell wall, cytoplasm depletion, and completed is organization of hyphal compartments. In scanning electron microscopy, swelling and deformation of hyphal tips, formation of short branches, and collapse of entire hyphae were the major changes observed. Morphological alterations might be due to the effect on cell permeability through direct interaction of M. chamomilla essential oil with the fungal plasma membrane. These findings indicate the potential of M. chamomilla L. essential oil in preventing fungal contamination and subsequent deterioration of stored food and other susceptible materials.

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Diet and Diabetes

Writer and Curator: Larry H Bernstein, MD, FCAP 

 

Bile acid signaling in lipid metabolism: Metabolomic and lipidomic analysis of lipid and bile acid markers linked to anti-obesity and anti-diabetes in mice

Yunpeng Qi, Changtao Jiang, Jie Cheng, Kristopher W. Krausz, et al.

Biochimica et Biophysica Acta 1851 (2015) 19–29

http://dx.doi.org/10.1016/j.bbalip.2014.04.008

Bile acid synthesis is the major pathway for catabolism of cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme in the bile acid biosynthetic pathway in the liver and plays an important role in regulating lipid, glucose and energy metabolism. Transgenic mice overexpressing CYP7A1 (CYP7A1-tg mice) were resistant to high fat diet (HFD)-induced obesity, fatty liver, and diabetes. However the mechanism of resistance to HFD-induced obesity of CYP7A1-tg mice has not been determined. In this study, metabolomic and lipidomic profiles of CYP7A1-tg mice were analyzed to explore the metabolic alterations in CYP7A1-tg mice that govern the protection against obesity and insulin resistance by using ultra-performance liquid chromatography-coupled with electrospray ionization quadrupole time-of-flight mass spectrometry combined with multivariate analyses. Lipidomics analysis identified seven lipid markers including lysophosphatidylcholines, phosphatidylcholines, sphingomyelins and ceramides that were significantly decreased in serum of HFD-fed CYP7A1-tgmice.Metabolomics analysis identified 13metabolites in bile acid synthesis including taurochenodeoxy-cholic acid, taurodeoxycholic acid, tauroursodeoxycholic acid, taurocholic acid, and tauro-β-muricholic acid (T-β-MCA) that differed between CYP7A1-tg and wild-type mice. Notably, T-β-MCA, an antagonist of the farnesoid X receptor (FXR) was significantly increased in intestine of CYP7A1-tg mice. This study suggests that reducing 12α-hydroxylated bile acids and increasing intestinal T-β-MCA may reduce high fat diet-induced increase of phospholipids, sphingomyelins and ceramides, and ameliorate diabetes and obesity. This article is part of a Special Issue entitled Linking transcription to physiology in lipidomics.

Bile acid synthesis is the major pathway for catabolism of cholesterol to bile acids. In the liver, cholesterol 7α-hydroxylase (CYP7A1) is the first and rate-limiting enzyme of the bile acid biosynthetic pathway producing two primary bile acids, cholic acid (CA, 3α, 7α, 12α-OH) and chenodeoxycholic acid (CDCA, 3α, 7α-OH) in humans. Sterol-12α hydroxylase (CYP8B1) catalyzes the synthesis of CA. In mice, CDCA is converted to α-muricholic acid (α-MCA: 3α, 6β, 7α-OH) and β-muricholic acid (β-MCA: 3α, 6β, 7β-OH). Bile acids are conjugated to taurine or glycine, secreted into the bile and stored in the gallbladder. After a meal, bile acids are released into the gastrointestinal tract. In the intestine, conjugated bile acids are first de-conjugated and then 7α-dehydroxylase activity in the gut flora converts CA to deoxycholic acid (DCA: 3α, 12α), and CDCA to lithocholic acid (LCA: 3α), two major secondary bile acids in humans.

In humans, most bile acids are glycine or taurine-conjugated and CA, CDCA and DCA are the most abundant bile acids. In mice, most bile acids are taurine-conjugated and CA and α- and β-MCAs are the most abundant bile acids. Bile acids facilitate absorption of dietary fats, steroids, and lipid soluble vitamins into enterocytes and are transported via portal circulation to the liver for metabolism and distribution to other tissues and organs. About 95% of bile acids are reabsorbed in the ileum and transported to the liver to inhibit CYP7A1 and bile acid synthesis. Enterohepatic circulation of bile acids provides a negative feedback mechanism to maintain bile acid homeostasis. Alteration of bile acid synthesis, secretion and transport causes cholestatic liver diseases, gallstone diseases, fatty liver disease, diabetes and obesity.

 Bile acid synthesis

 

Bile acid synthesis. In the classic bile acid synthesis pathway, cholesterol is converted to cholic acid (CA, 3α, 7α, 12α) and chenodeoxycholic acid (CDCA, 3α, 7α). CYP7A1 is the rate-limiting enzyme and CYP8B1 catalyzes the synthesis of CA. In mouse liver, CDCA is converted to α-muricholic acid (α-MCA, 3α, 6β, 7α) and β-MCA (3α, 6β, 7β). Most bile acids in mice are taurine (T)-conjugated and secreted into bile. In the intestine, gut bacteria de-conjugate bile acids and then remove the 7α-hydroxyl group from CA and CDCA to form secondary bile acids deoxycholic acid (DCA, 3α, 12α) and lithocholic acid (LCA, 3α), respectively. T-α-MCA and T-β-MCA are converted to T-hyodeoxycholic acid (THDCA, 3α, 6α), T-ursodeoxycholic acid (TUDCA, 3α, 7β), T-hyocholic acid (THCA, 3α, 6α, 7α) and T-murideoxycholic acid (TMDCA, 3α, 6β). These secondary bile acids are reabsorbed and circulated to liver to contribute to the bile acid pool. Secondary bile acids ω-MCA (3α, 6α, 7β) and LCA are excreted into feces.

Two FXR-dependent mechanisms are known to inhibit bile acid synthesis.  In the liver bile acid-activated FXR induces a negative receptor small heterodimer partner (SHP) to inhibit trans-activation activity of hepatic nuclear factor 4α(HNF4α) and liver receptor homologue-1 (LRH-1) that bind to the bile acid response element in the CYP7A1 and CYP8B1 gene promoters (Fig. 2, Pathway 1). In the intestine, bile acids activate FXR to induce fibroblast growth factor (mouse FGF15, or human FGF19), which activates hepatic FGF receptor 4 (FGFR4) and cJun N-terminal kinase 1/2 (JNK1/2) and extracellular-regulated kinase (ERK1/2) signaling of mitogen-activated protein kinase (MAPK) pathways to inhibit trans-activation of CYP7A1/CYP8B1 gene by HNF4α (Pathway 2). Several FXR-independent cell-signaling pathways have been reported and are shown as Pathway 3 (Fig. 2). Conjugated bile acids are known to activate several protein kinase Cs (PKC) and growth factor receptors, epidermal growth factor receptor (EGFR), and insulin receptor (IR) signaling to inhibit CYP7A1/CYP8B1 and bile acid synthesis via activating the ERK1/2, p38 and JNK1/2 pathways.

 

Bile acid signaling pathways. Bile acids activate FXR, TGR5 and cell signaling pathways to inhibit CYP7A1 and CYP8B1 gene transcription.

1) Hepatic FXR/SHP pathway: bile acid activated-FXR induces SHP, which inhibits HNF4α and LRH-1 trans-activation of CYP7A1 and CYP8B1 gene transcription in hepatocytes. Bile acid response element binds HNF4α and LRH-1.

2) Intestinal FXR/FGF19/FGFR4 pathway: in the intestine, FXR induces FGF15 (mouse)/FGF19 (human), which is secreted into portal circulation to activate FGF receptor 4 (FGFR4) in hepatocytes. FGFR4 signaling stimulates JNK1/2 and ERK1/2 pathways of MAPK signaling to inhibit CYP7A1 gene transcription by phosphorylation and inhibition of HNF4α binding activity.

3) FXR-independent signaling pathways: Conjugated bile acids activate PKCs,which activate the MAPK pathways to inhibit CYP7A1. Bile acids also activate insulin receptor (IR) signaling IRS/PI3K/PDK1/AKT, possibly via activation of epidermal growth factor receptor (EGFR) signaling, MAPKs (MEK, MEKK), to inhibit CYP7A1 gene transcription. The secondary bile acid TLCA activates TGR5 signaling in Kupffer cells. TGR5 signaling may regulate CYP7A1 by an unknown mechanism. TCA activates sphingosine-1-phosphate (S1P) receptor 2 (S1PR2), which may activate AKT and ERK1/2 to inhibit CYP7A1. S1P kinase 1 (Sphk1) phosphorylates sphingosine (Sph) to S-1-P, which activates S1PR2. On the other hand, nuclear SphK2 interacts with and inhibits histone deacetylase (HDAC1/2) and may induce CYP7A1. The role of S1P, SphK2, and S1PR2 signaling in regulation of bile acid synthesis is not known.

 

When challenged with an HFD, CYP7A1-tg mice had lower body fat mass and higher lean mass compared to wild-type mice. As a platform for comprehensive and quantitative description of the set of lipid species, lipidomics was used to investigate the mechanism of this phenotype. By use of an unsupervised PCA model with the cumulative R2X 0.677 for serum and 0.593 for liver, CYP7A1-tg and wild-type mice were clearly separated based on the scores plot (Supplementary Fig. S2), indicating that these two groups have distinct lipidomic profiles. Supervised PLS-DA models were then established to maximize the difference of metabolic profiles between CYP7A1-tg and wild-type groups as well as to facilitate the screening of lipid marker metabolites (Fig. 3).

PLS-DA analysis of CYP7A1-tg and wild-type (WT)mice challenged with HFD. Based on the score plots, distinct lipidomic profiles of male CYP7A1-tg and wild-type groups were shown for serum (A) and liver samples (B). Based on the loading plots (C for serum and D for liver) the most significant ions that led to the separation between CYP7A1-tg and wild-type groups were obtained and identified as follows: 1. LPC16:0; 2. LPC18:0; 3. LPC18:1; 4. LPC 18:2; 5. PC16:0-20:4; 6. PC16:0-22:6; 7. SM16:0. (not shown)

Fig. 5. OPLS-DA highlighted thirteen markers in bile acid pathway that contribute significantly to the clustering of CYP7A1-tg and wild-type (WT) mice. Ileum bile acids are shown. (not shown)

(A) In the score plot, female CYP7A1-tg andWTmicewere well separated;

(B) using a statistically significant thresholds of variable confidence approximately 0.75 in the S-plot, a number of ions were screened out as potential markers, which were later identified as 13 bile acid metabolites, including α-MCA, TCA, CDCA, and TCDCA etc.

Our recent study of CYP7A1-tg mice revealed that increased CYP7A1 expression and enlarged bile acid pool resulted in significant improvement of lipid homeostasis and resistance to high-fed diet-induced hepatic steatosis, insulin resistance, and obesity in CYP7A1-tg mice. In this study, metabolomics and lipidomics were employed to characterize the metabolic profiles of CYP7A1-tg mice and to provide new insights into the critical role of bile acids in regulation of lipid metabolism and metabolic diseases. Lipidomics analysis of serum lipid profiles of high fat diet-fed CYP7A1-tg identified 7 lipidomic markers that were reduced in CYP7A1-tg mice compared to wild type mice. Metabolomics analysis identified 13 bile acid metabolites that were altered in CYP7A1-tg mice. In CYP7A1-tg mice, TCA and TDCA were reduced, whereas T-β-MCA was increased in the intestine compared to that of wild type mice. The decrease of serum LPC, PC, SM and CER, and 12α-hydroxylated bile acids, and increase of T-β-MCA may contribute to the resistance to diet-induced obesity and diabetes in CYP7A1-tg mice (Fig. 8).

The present metabolomics and lipidomics analysis revealed that even upon challenging with HFD, CYP7A1-tg mice had reduced lipid levels including LPC, PC, SM and CER. Metabolomics studies of human steatotic liver tissues and HFD-fed mice showed that serum and liver LPC and PC and other lipids levels were increased compared with non-steatotic livers, suggesting altered lipid metabolism contributes to non-alcoholic fatty liver disease (NAFLD). In HFD-fed CYP7A1-tg mice, reduced serum PC, LPC, SM and CER levels suggest a role for bile acids in maintaining phospholipid homeostasis to prevent NAFLD. SMs are important membrane phospholipids that interact with cholesterol in membrane rafts and regulate cholesterol distribution and homeostasis. A role for SM and CER in the pathogenesis of insulin resistance, diabetes and obesity and development of atherosclerosis has been reported. CER has a wide range of biological functions in cellular signaling such as activating protein kinase C and c-Jun N-terminal kinase (JNK), induction of β-cell apoptosis and insulin resistance. CER increases reactive oxidizing species and activates the NF-κB pathway, which induces proinflammatory cytokines, diabetes and insulin resistance. CER is synthesized from serine and palmitoyl-CoA or hydrolysis of SM by acid sphingomyelinase (ASM). HFD is known to increase CER and SM in liver. The present observation of decreased SM and CER levels in HFD-fed CYP7A1-tg mice indicated that bile acids might reduce HFD-induced increase of SM and CER. DCA activates an ASM to convert SM to CER, and Asm−/− hepatocytes are resistant to DCA induction of CER and activation of the JNK pathway [65]. In CYP7A1-tg mice, enlarged bile acid pool inhibits CYP8B1 and reduces CA and DCA levels. Thus, decreasing DCA may reduce ASM activity and SM and CER levels, and contribute to reducing inflammation and improving insulin sensitivity in CYP7A1-tg mice. It has been reported recently that in diabetic patients, serum 12α-hydroxylated bile acids are increased and correlated to insulin resistance [66].

Fig. 8. Mechanisms of anti-diabetic and anti-obesity function of bile acids in CYP7A1-tg mice. In CYP7A1-tg mice, overexpressing CYP7A1 increases bile acid pool size and reduces cholic acid by inhibiting CYP8B1. Lipidomics analysis revealed decreased serum LPC, PC, SM and CER. These lipidomic markers are increased in hepatic steatosis and NAFLD. Bile acids may reduce LPC, PC, SM and CER levels and protect against high fat diet-induced insulin resistance and obesity in CYP7A1-tgmice. Metabolomics analysis showed decreased intestinal TCA and TDCA and increased intestinal T-β-MCA in CYP7A1-tgmice.High fat diets are known to increase CA synthesis and intestinal inflammation. It is proposed that decreasing CA and  DCA synthesis may increase intestinal T-β-MCA,which antagonizes FXR signaling to increase bile acid synthesis and prevent high fat diet-induced insulin resistance and obesity. (not shown)

In conclusion,metabolomics and lipidomicswere employed to characterize the metabolic profiles of CYP7A1-tg mice, aiming to provide new insights into the mechanism of bile acid signaling in regulation of lipid metabolism and maintain lipid homeostasis. A number of lipid and bile acid markers were unveiled in this study. Decreasing of lipid markers, especially SM and CER may explain the improved insulin sensitivity and obesity in CYP7A1-tg mice. Furthermore, this study uncovered that enlarged bile acid pool size and altered bile acid composition may reduce de-conjugation by gut microbiota and increase tauroconjugated muricholic acids, which partially inhibit intestinal FXR signaling without affecting hepatic FXR signaling. This study is significant in applying metabolomics for diagnosis of lipid biomarkers for fatty liver diseases, obesity and diabetes. Increasing CYP7A1 activity and bile acid synthesis coupled to decreasing CYP8B1 and 12α-hydroxylated bile acids may be a therapeutic strategy for treating diabetes and obesity.

 

Bile acids are nutrient signaling hormones

Huiping Zhou, Phillip B. Hylemon
Steroids 86 (2014) 62–68
http://dx.doi.org/10.1016/j.steroids.2014.04.016

Bile salts play crucial roles in allowing the gastrointestinal system to digest, transport and metabolize nutrients. They function as nutrient signaling hormones by activating specific nuclear receptors (FXR, PXR, Vitamin D) and G-protein coupled receptors [TGR5, sphingosine-1 phosphate receptor 2 (S1PR2), muscarinic receptors]. Bile acids and insulin appear to collaborate in regulating the metabolism of nutrients in the liver. They both activate the AKT and ERK1/2 signaling pathways. Bile acid induction of the FXR-a target gene, small heterodimer partner (SHP), is highly dependent on the activation PKCf, a branch of the insulin signaling pathway. SHP is an important regulator of glucose and lipid metabolism in the liver. One might hypothesize that chronic low grade inflammation which is associated with insulin resistance, may inhibit bile acid signaling and disrupt lipid metabolism. The disruption of these signaling pathways may increase the risk of fatty liver and non-alcoholic fatty liver disease (NAFLD). Finally, conjugated bile acids appear to promote cholangiocarcinoma growth via the activation of S1PR2.

 

In the past, bile salts were considered to be just detergent molecules that were required for the solubilization of cholesterol in the gall bladder, promoting the digestion of dietary lipids and stimulating the absorption of lipids, cholesterol and fat-soluble vitamins in the intestines. Bile salts were also known to stimulate bile flow, promote cholesterol secretion from the liver, and have antibacterial properties. However, in 1999, three independent laboratories reported that bile acids were natural ligands for the farnesoid X receptor (FXR-α) . The recognition that bile acids activated specific nuclear receptors started a renaissance in the field of bile acid research. Since 1999, bile acids have been reported to activate other nuclear receptors (pregnane X receptor, vitamin D receptor), G protein coupled receptors [TGR5, sphingosine-1-phosphate receptor 2 (S1PR2), muscarinic receptor 2 (M2)] and cell signaling pathways (JNK1/2, AKT, and ERK1/2). Deoxycholic acid (DCA), a secondary bile acid, has also been reported to activate the epidermal growth factor receptor (EGFR). It is now clear that bile acids function as hormones or nutrient signaling molecules that help to regulate glucose, lipid, lipoprotein, and energy metabolism as well as inflammatory responses.

Bile acids are synthesized from cholesterol in liver hepatocytes, conjugated to either glycine or taurine and actively secreted via ABC transporters on the canalicular membrane into biliary bile. Conjugated bile acids are often referred to as bile salts. Bile acid synthesis represents a major output pathway of cholesterol from the body. Bile acids are actively secreted from hepatocytes via the bile salt export protein (BSEP, ABCB11) along with phospholipids by ABCB4 and cholesterol by ABCG5/ABCG8 in a fairly constant ratio under normal conditions. Bile acids are detergent molecules and form mixed micelles with cholesterol and phospholipids, which help to keep cholesterol in solution in the gall bladder. Eating stimulates the gall bladder to contract, emptying its contents into the small intestines. Bile salts are crucial for the solubilization and absorption of cholesterol and lipids as well as lipid soluble vitamins (A, D, E, and K). They activate pancreatic enzymes and form mixed micelles with lipids in the small intestines, promoting their absorption. Bile acids are efficiently recovered from the intestines, primarily the ileum, by the apical sodium dependent transporter (ASBT). Bile acids are secreted from ileocytes, on the basolateral side, by the organic solute OSTα/OSTβ transporter. Secondary bile acids, formed by 7α-dehydroxylation of primary bile acids by anaerobic gut bacteria, can be passively absorbed from the large bowel or secreted in the feces. Absorbed bile acids return to the liver via the portal blood where they are actively transported into hepatocytes primarily via the sodium taurocholate cotransporting polypeptide (NTCP, SLC10A1). Bile acids are again actively secreted from the hepatocytes into the bile, stimulating bile flow and the secretion of cholesterol and phospholipids. Bile acids undergo enterohepatic circulation several times each day (Fig. 1). During their enterohepatic circulation approximately 500–600 mg/day are lost via fecal excretion and must be replaced by new bile acid synthesis in the liver. The bile acid pool size is tightly regulated as excess bile acids can be highly toxic to mammalian cells.

Enterohepatic circulation of bile acids

 

Enterohepatic circulation of bile acids. Bile acids are synthesized and conjugated mainly to glycine or taurine in hepatocytes. Bile acids travel to the gall bladder for storage during the fasting state. During digestion, bile acids travel to the duodenum via the common bile duct. 95% of the bile acids delivered to the duodenum are absorbed back into blood within the ileum and circulate back to the liver through the portal vein. 5% of bile acids are lost in feces.

There are two pathways of bile acid synthesis in the liver, the neutral pathway and the acidic pathway (Fig. 2). The neutral pathway is believed to be the major pathway of bile acid synthesis in humans under normal physiological conditions. The neutral pathway is initiated by cholesterol 7α-hydroxylase (CYP7A1), which is the rate-limiting step in this biochemical pathway. CYP7A1 is a cytochrome P450 monooxygenase, and the gene encoding this enzyme is highly regulated by a feed-back repressive mechanism involving the FXR-dependent induction of fibroblast growth factor 15/19 (FGF15/19) by bile acids in the intestines. FGF15/19 binds to the fibroblast growth factor receptor 4 (FGFR4)/β-Klotho complex in hepatocytes activating both the JNK1/2 and ERK1/2 signaling cascades. Activation of the JNK1/2 pathway has been reported to down-regulate CYP7A1 mRNA in hepatocytes. FGFR4 and β-Klotho mice have increased levels of CYP7A1 and upregulated bile acid synthesis. Moreover, treatment of FXR mice with a specific FXR agonist failed to repress CYP7A1 in the liver. These results support an important role of FGF15, synthesized in the intestines by activation of FXR, in the regulation of CYP7A1 and bile acid synthesis in the liver. CYP7A1 has also been reported to be down-regulated by glucagon and pro-inflammatory cytokines and up-regulated by glucose and insulin during the postprandial period.

Fig. 2. (not shown) Biosynthetic pathways of bile acids. Two major pathways are involved in bile acid synthesis. The neutral (or classic) pathway is controlled by cholesterol 7α-hydroxylase (CYP7A1) in the endoplasmic reticulum. The acidic (or alternative) pathway is controlled by sterol 27-hydroxylase (CYP27A1) in mitochondria. The sterol 12α-hydroxylase (CYP8B1) is required to synthesis of cholic acid (CA). The oxysterol 7α-hydroxylase (CYP7B1) is involved in the formation of chenodeoxycholic acid (CDCA) in acidic pathway. The neutral pathway is also able to form CDCA by CYP27A1.

The neutral pathway of bile acid synthesis produces both cholic acid (CA) and chenodeoxycholic acid (CDCA) (Fig. 2). The ratio of CA and CDCA is primarily determined by the activity of sterol 12α-hydroxylase (CYP8B1). The gene encoding CYP8B1 is also highly regulated by bile acids. Bile acids induce the gene encoding small heterodimer partner (SHP) in the liver via activation of the farnesoid X receptor (FXR-α). SHP is an orphan nuclear receptor without a DNA binding domain. It interacts with several transcription factors, including hepatocyte nuclear factor 4 (HNF4α) and liver-related homolog-1 (LRH-1), and acts as a dominant negative protein to inhibit transcription. In this regard, a liver specific knockout of LRH-1 completely abolished the expression of CYP8B1, but had little effect on CYP7A1. These results suggest that the interaction of SHP with LRH-1, caused by bile acids, may be the key regulator of hepatic CYP8B1 and the ratio of CA/CDCA. The acidic or alternative pathway of bile acid synthesis is initiated in the inner membrane of mitochondria by sterol 27-hydroxylase (CYP27A1). This enzyme also has low sterol 25-hydroxylase activity. CYP27A1 is capable of further oxidizing the 27-hydroxy group to a carboxylic acid. Unlike, CYP7A1, CYP27A1 is widely expressed in various tissues in the body where it may produce regulatory oxysterols. Even though CYP27A1 is the initial enzyme in the acidic pathway of bile acid synthesis, it may not be the rate limiting step. The inner mitochondrial membrane is very low in cholesterol content. Hence, cholesterol transport into the mitochondria appears to be the rate limiting step.

The acidic pathway of bile acid synthesis is now being viewed as an important pathway for generating regulatory oxysterols. For example, 25-hydroxy-cholesterol and 27-hydroxycholesterol are natural ligands for the liver X receptor (LXR), which is involved in regulating cholesterol and lipid metabolism. Moreover, recent studies report that 25-hydroxycholesterol, formed by CYP27A1, can be converted into 5-cholesten-3β-25-diol-3-sulfate in the liver. The sulfated 25-hydroxycholesterol is a regulator of inflammatory responses, lipid metabolism and cell proliferation, and is located in the liver. Recent evidence suggests that sulfated 25-hydroxycholesterol is a ligand for peroxisome proliferator-activated receptor gamma (PPARc), which is a major regulator of inflammation and lipid metabolism. The 7α-hydroxylation of oxysterols is catalyzed by oxysterol 7α-hydroxylase (CYP7B1). This biotransformation allows some of these oxysterols to be converted to bile acids. Finally, oxysterols generated in extrahepatic tissues can be transported to the liver and metabolized into bile acids.

Bile acids can activate several different nuclear receptors (FXR, PXR and Vitamin D) and GPCRs (TGR5, S1PR2, and [M2] Muscarinic receptor). The ability of different bile acids to activate FXR-α occurs in the following order CDCA > LCA = DCA > CA; for the pregnane X receptor (PXR) LCA > DCA > CA and the vitamin D receptor, 3-oxo-LCA > LCA > DCA > CA. LCA is the best activator of PXR and the vitamin D receptor which correlates with the hydrophobicity and toxicity of this bile acid toward mammalian cells. Activation of PXR and the vitamin D receptor induces genes encoding enzymes which metabolize LCA into a more hydrophilic and less toxic metabolite. These nuclear receptors appear to function in the protection of cells from hydrophobic bile acids. In contrast, FXR-α appears to play a much more extensive role in the body by regulating bile acid synthesis, transport, and enterohepatic circulation. Moreover, FXR-α also participates in the regulation of glucose, lipoprotein and lipid metabolism in the liver as well as a suppressor of inflammation in the liver and intestines.

TGR5, also referred to as membrane-type bile acid receptor (MBAR), was the first GPCR to be reported to be activated by bile acids in the order LCA > DCA > CDCA > CA. TGR5 is a Gas type receptor which activates adenyl cyclase activity increasing the rate of the synthesis of c-AMP. TGR5 is widely expressed in human tissues, including: intestinal neuroendocrine cells, gall bladder, spleen, brown adipose tissue, macrophages and cholangiocytes, but not hepatocytes. TGR5 may play a role in various physiological processes in the body. TGR5 appears to be important in regulating energy metabolism. It has been postulated that bile acids may activate TGR5 in brown adipose tissue, activating type 2-iodothyroxine deiodinase and leading to increased levels of thyroid hormone and stimulation of energy metabolism. Moreover, TGR5 has been reported to promote the release of glucagon-like peptide-1 release from neuroendocrine cells, which increases insulin release in the pancreas. These results suggest that TGR5 may play a role in glucose homeostasis in the body. TGR5 is a potential target for drug development for treating type 2 diabetes and other metabolic disorders.

Interrelationship between sphingosine 1-phosphate receptor 2 and the insulin signaling pathway

 

Interrelationship between sphingosine 1-phosphate receptor 2 and the insulin signaling pathway in regulating hepatic nutrient metabolism. S1PR2, sphingosine 1-phosphate receptor 2; Src, Src Kinase; EGFR, epidermal growth factor receptor; PPARa, peroxisome proliferator-activated receptor alpha; NTCP, Na+/taurocholate cotransporting polypeptide; BSEP, bile salt export pump; PC, phosphotidylcholine; PECK, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; PDK1, phosphoinositide-dependent protein kinase 1; AKT, protein kinase B; SREBP, sterol regulatory element-binding protein; PKCf, protein kinase C zeta; FXR, farnesoid X receptor; SHP, small heterodimeric partner; MDR3, phospholipid transporter (ABCB4); GSK3b, glycogen synthase kinase 3 beta.

 

Both unconjugated and conjugated bile acids activate the insulin signaling (AKT) and ERK1/2 pathways in hepatocytes. Interesting, insulin and bile acids both activated glycogen synthase activity to a similar extent in primary rat hepatocytes. Moreover, the addition of both insulin and bile acids to the culture medium resulted in an additive effect on activation of glycogen synthase activity in primary hepatocytes. Infusion of taurocholate (TCA) into the chronic bile fistula rat rapidly activated the AKT and ERK1/2 signaling pathway and glycogen synthase activity. In addition, there was a rapid down-regulation of the gluconeogenic genes, PEP carboxykinase (PEPCK) and glucose-6-phophatase (G-6-Pase) and a marked up-regulation of SHP mRNA in these sample livers. These results suggest that TCA functions much like insulin to regulate hepatic glucose metabolism both in vitro and in vivo.

It has been reported that PKCζ phosphorylates FXR-α and may allow for its activation of target gene expression. In contrast, phosphorylation of FXR-α by AMPK inhibits the ability of FXR to induce target genes. PKCζ has been reported to be important for the translocation of the bile acid transporters NTCP (SLC10A1) and BSEP (ABC B11) to the basolateral and canalicular membranes, respectively. Finally, it has been recently reported that PKCζ phosphorylates SHP allowing both to translocate to the nucleus and down-regulate genes via epigenetic mechanisms. In total, these results all suggest that the insulin signaling pathway is an important regulator of FXR-α activation and bile acid signaling in the liver.

The activation of the insulin signaling pathway and FXR-α appear to collaborate in the coordinate regulation of glucose, bile acid and lipid metabolism in the liver. SHP, an FXR target gene, is an important pleotropic regulator of multiple metabolic pathways in the liver (Fig. 3). The S1PR2 appears to be an important regulator of hepatic lipid metabolism as S1PR2 mice rapidly (2 weeks) develop overt fatty livers on a high fat diet as compared to wild type mice (unpublished data). It is well established that inflammation and the synthesis of inflammatory cytokines i.e. TNFα inhibit insulin signaling by activation of the JNK1/2 signaling pathway, which phosphorylates insulin receptor substrate 1. Inflammation is believed to be an important factor in the development of type 2 diabetes and fatty liver disease. A Western diet is correlated with low grade chronic inflammation and insulin resistance. Inhibition of the insulin signaling pathway may decrease the ability of bile acids to activate FXR-α, induce SHP and other FXR target genes, leading to an increased risk of fatty liver and non-alcoholic fatty liver disease (NAFLD).

There appears to be extensive interplay between bile salts and insulin signaling in the regulation of nutrient metabolism in both the intestines and liver. Bile salts play a key role in the solubilization and absorption of nutrients from the intestines. The absorption of nutrients stimulates the secretion of insulin from the pancreas. Moreover, bile acids may also stimulate the secretion of insulin by activating TGR5 in intestinal neuroendocrine cells resulting in the secretion of glucagon-like peptide-1. In the liver, bile salts and insulin both activate the AKT and ERK1/2 signaling pathways which yields a stronger signal than either alone. The activation of PKCζ, a branch of the insulin signaling pathway, is required for the optimal induction of FXR target genes and the regulation of the cellular location of bile acid transporters

 

Fruit and vegetable consumption and risk of type 2 diabetes mellitus: A dose-response meta-analysis of prospective cohort studies

  1. Wu, D. Zhang, X. Jiang, W. Jiang
    Nutrition, Metabolism & Cardiovascular Diseases (2015) 25, 140-147
    http://dx.doi.org/10.1016/j.numecd.2014.10.004

Background and aims: We conducted a dose-response meta-analysis to summarize the evidence from prospective cohort studies regarding the association of fruit and vegetable consumption with risk of type 2 diabetes mellitus (T2DM). Methods and results: Pertinent studies were identified by searching Embase and PubMed through June 2014. Study-specific results were pooled using a random-effect model. The dose-response relationship was assessed by the restricted cubic spline model and the multivariate random-effect meta-regression. We standardized all data using a standard portion size of 106 g. The Relative Risk (95% confidence interval) [RR (95% CI)] of T2DM was 0.99 (0.98-1.00) for every 1 serving/day increment in fruit and vegetable (FV) (P < 0.18), 0.98 (0.95-1.01) for vegetable (P < 0.12), and 0.99 (0.97-1.00) for fruit (P < 0.05). The RR (95%CI) of T2DM was 0.99 (0.97-1.01), 0.98 (0.96-1.01), 0.97 (0.93-1.01), 0.96 (0.92-1.01), 0.96 (0.91-1.01) and 0.96 (0.91-1.01) for 1, 2, 3, 4, 5 and 6 servings/day of FV (P for non-linearity < 0.44). The T2DM risk was 0.96 (0.95-0.99), 0.94 (0.90-0.98), 0.94 (0.89-0.98), 0.96 (0.91-1.01), 0.98 (0.92-1.05) and 1.00 (0.93-1.08) for 1, 2, 3, 4, 5 and 6 servings/day of vegetable (P for non-linearity < 0.01). The T2DM risk was 0.95 (0.93-0.97), 0.91 (0.89-0.94), 0.88 (0.85-0.92), 0.92 (0.88-0.96) and 0.96 (0.92-1.01) for 0.5, 1, 2, 3 and 4 servings/day of fruit (P for non-linearity < 0.01). Conclusions: Two-three servings/day of vegetable and 2 servings/day of fruit conferred a lower risk of T2DM than other levels of vegetable and fruit consumption, respectively.

dose-response analysis between total fruit and vegetable consumption and risk of type 2 diabetes mellitus

 

The dose-response analysis between total fruit and vegetable consumption and risk of type 2 diabetes mellitus. The solid line and the long dash line represent the estimated relative risk and its 95% confidence interval.

 

Healthy behaviours and 10-year incidence of diabetes: A population cohort study

G.H. Long , I. Johansson , O. Rolandsson , …, E. Fhärm, L.Weinehall, et al.
Preventive Medicine 71 (2015) 121–127
http://dx.doi.org/10.1016/j.ypmed.2014.12.013

Objective. To examine the association between meeting behavioral goals and diabetes incidence over 10 years in a large, representative Swedish population. Methods. Population-based prospective cohort study of 32,120 individuals aged 35 to 55 years participating in a health promotion intervention in Västerbotten County, Sweden (1990 to 2013). Participants underwent an oral glucose tolerance test, clinical measures, and completed diet and activity questionnaires. Poisson regression quantified the association between achieving six behavioral goals at baseline – body mass index (BMI) < 25 kg/m2, moderate physical activity, non-smoker, fat intake  < 30% of energy, fibre intake ≥15 g/4184 kJ and alcohol intake ≤ 20 g/day – and diabetes incidence over 10 years. Results. Median interquartile range (IQR) follow-up time was 9.9 (0.3) years; 2211 individuals (7%) developed diabetes. Only 4.4% of participants met all 6 goals (n = 1245) and compared to these individuals, participants meeting 0/1 goals had a 3.74 times higher diabetes incidence (95% confidence interval (CI) = 2.50 to 5.59), adjusting for sex, age, calendar period, education, family history of diabetes, history of myocardial infarction and long-term illness. If everyone achieved at least four behavioral goals, 14.1% (95% CI: 11.7 to 16.5%) of incident diabetes cases might be avoided. Conclusion. Interventions promoting the achievement of behavioral goals in the general population could significantly reduce diabetes incidence.

 

Long term nutritional intake and the risk for non-alcoholic fatty liver disease (NAFLD): A population based study

Shira Zelber-Sagi, Dorit Nitzan-Kaluski, Rebecca Goldsmith, et al.
Journal of Hepatology 47 (2007) 711–717
http://dx.doi.org:/10.1016/j.jhep.2007.06.020

Background/Aims: Weight loss is considered therapeutic for patients with NAFLD. However, there is no epidemiological evidence that dietary habits are associated with NAFLD. Dietary patterns associated with primary NAFLD were investigated. Methods: A cross-sectional study of a sub-sample (n = 375) of the Israeli National Health and Nutrition Survey. Exclusion criteria were any known etiology for secondary NAFLD. Participants underwent an abdominal ultrasound, biochemical tests, dietary and anthropometric evaluations. A semi-quantitative food-frequency questionnaire was administered. Results: After exclusion, 349 volunteers (52.7% male, mean age 50.7 ± 10.4, 30.9% primary NAFLD) were included. The NAFLD group consumed almost twice the amount of soft drinks (P = 0.03) and 27% more meat (P < 0.001). In contrast, the NAFLD group consumed somewhat less fish rich in omega-3 (P = 0.056). Adjusting for age, gender, BMI and total calories, intake of soft drinks and meat was significantly associated with an increased risk for NAFLD (OR = 1.45, 1.13–1.85 95% CI and OR = 1.37, 1.04–1.83 95% CI, respectively). Conclusions: NAFLD patients have a higher intake of soft drinks and meat and a tendency towards a lower intake of fish rich in omega-3. Moreover, a higher intake of soft drinks and meat is associated with an increased risk of NAFLD, independently of age, gender, BMI and total calories.

 

The association between types of eating behavior and dispositional mindfulness in adults with diabetes. Results from Diabetes MILES. The Netherlands

Sanne R. Tak, Christel Hendrieckx, Giesje Nefs, Ivan Nyklícek, et al.
Appetite 87 (2015) 288–295
http://dx.doi.org/10.1016/j.appet.2015.01.006

Although healthy food choices are important in the management of diabetes, making dietary adaptations is often challenging. Previous research has shown that people with type 2 diabetes are less likely to benefit from dietary advice if they tend to eat in response to emotions or external cues. Since high levels of dispositional mindfulness have been associated with greater awareness of healthy dietary practices in students and in the general population, it is relevant to study the association between dispositional mindfulness and eating behavior in people with type 1 or 2 diabetes. We analyzed data from Diabetes MILES – The Netherlands, a national observational survey in which 634 adults with type 1 or 2 diabetes completed the Dutch Eating Behavior Questionnaire (to assess restrained, external and emotional eating behavior) and the Five Facet Mindfulness Questionnaire-Short Form (to assess dispositional mindfulness), in addition to other psychosocial measures. After controlling for potential confounders, including  demographics, clinical variables and emotional distress, hierarchical linear regression analyses showed that higher levels of dispositional mindfulness were associated with eating behaviors that were more restrained (β = 0.10) and less external (β = −0.11) and emotional (β = −0.20). The mindfulness subscale ‘acting with awareness’ was the strongest predictor of both external and emotional eating behavior, whereas for emotional eating, ‘describing’ and ‘being non-judgmental’ were also predictive. These findings suggest that there is an association between dispositional mindfulness and eating behavior in adults with type 1 or 2 diabetes. Since mindfulness interventions increase levels of dispositional mindfulness, future studies could examine if these interventions are also effective in helping people with diabetes to reduce emotional or external eating behavior, and to improve the quality of their diet.

 

Soft drink consumption is associated with fatty liver disease independent of metabolic syndrome

Ali Abid, Ola Taha, William Nseir, Raymond Farah, Maria Grosovski, Nimer Assy
Journal of Hepatology 51 (2009) 918–924
http://dx.doi.org:/10.1016/j.jhep.2009.05.033

Background/Aims: The independent role of soft drink consumption in non-alcoholic fatty liver disease (NAFLD) patients remains unclear. We aimed to assess the association between consumption of soft drinks and fatty liver in patients with or without metabolic syndrome. Methods: We recruited 31 patients (age: 43 ± 12 years) with NAFLD and risk factors for metabolic syndrome, 29 patients with NAFLD and without risk factors for metabolic syndrome, and 30 gender- and age-matched individuals without NAFLD. The degree of fatty infiltration was measured by ultrasound. Data on physical activity and intake of food and soft drinks were collected during two 7-day periods over 6 months using a food questionnaire. Insulin resistance, inflammation, and oxidant–antioxidant markers were measured.
Results: We found that 80% of patients with NAFLD had excessive intake of  soft drink beverages (>500 cm3/day) compared to 17% of healthy controls (p < 0.001). The NAFLD group consumed five times more carbohydrates from soft drinks compared to healthy controls (40% vs. 8%, p < 0.001). Seven percent of patients consumed one soft drink per day, 55% consumed two or three soft drinks per day, and 38% consumed more than four soft drinks per day for most days and for the 6-month period. The most common soft drinks were Coca-Cola (regular: 32%; diet: 21%) followed by fruit juices (47%). Patients with NAFLD with metabolic syndrome had similar malonyldialdehyde, paraoxonase, and C-reactive protein (CRP) levels but higher homeostasis model assessment (HOMA) and higher ferritin than NAFLD patients without metabolic syndrome (HOMA: 8.3 ± 8 vs. 3.7 ± 3.7 mg/dL, p < 0.001; ferritin: 186 ± 192 vs. 87 ± 84 mg/dL, p < 0.01). Logistic regression analysis showed that soft drink consumption is a strong predictor of fatty liver (odds ratio: 2.0; p < 0.04) independent of metabolic syndrome and CRP level. Conclusions: NAFLD patients display higher soft drink consumption independent of metabolic syndrome diagnosis. These findings might optimize NAFLD risk stratification.

 

Dietary predictors of arterial stiffness in a cohort with type 1 and type 2 diabetes

K.S. Petersen, J.B. Keogh, P.J. Meikle, M.L. Garg, P.M. Clifton
Atherosclerosis 238 (2015) 175-181
http://dx.doi.org/10.1016/j.atherosclerosis.2014.12.012

Objective: To determine the dietary predictors of central blood pressure, augmentation index and pulse wave velocity (PWV) in subjects with type 1 and type 2 diabetes. Methods: Participants were diagnosed with type 1 or type 2 diabetes and had PWV and/or pulse wave analysis performed. Dietary intake was measured using the Dietary Questionnaire for Epidemiological Studies Version 2 Food Frequency Questionnaire. Serum lipid species and carotenoids were measured, using liquid chromatography electrospray ionization- tandem mass spectrometry and high performance liquid chromatography, as biomarkers  of dairy and vegetable intake, respectively. Associations were determined using linear regression adjusted for potential confounders. Results: PWV (n = 95) was inversely associated with reduced fat dairy intake (β = -0.01; 95% CI -0.02, -0.01; p = 0 < 0.05) in particular yoghurt consumption (β = 0.04; 95% CI -0.09, -0.01; p = 0 < 0.05) after multivariate adjustment. Total vegetable consumption was negatively associated with PWV in the whole cohort after full adjustment (β =0.04; 95% CI -0.07, -0.01; p < 0.05). Individual lipid species, particularly those containing 14:0, 15:0, 16:0, 17:0 and 17:1 fatty acids, known to be of ruminant origin, in lysophosphatidylcholine, cholesterol ester, diacylglycerol, phosphatidylcholine, sphingomyelin and triacylglycerol classes were positively associated with intake of full fat dairy, after adjustment for multiple comparisons. However, there was no association between serum lipid species and PWV. There were no dietary predictors of central blood pressure or augmentation index after multivariate adjustment. Conclusion: In this cohort of subjects with diabetes reduced fat dairy intake and vegetable consumption were inversely associated with PWV. The lack of a relationship between serum lipid species and PWV suggests that the fatty acid composition of dairy may not explain the beneficial effect.

In this cohort with type 1 and type 2 diabetes there was an inverse association between reduced fat dairy intake, in particular yoghurt consumption, and PWV, which persisted after multivariate adjustment. Serum lipid species, known to be of ruminant origin, were positively associated with full fat dairy consumption; however there was no association between these lipid species and PWV. In addition, higher vegetable intake was also associated with lower PWV. There were no dietary predictors of central blood pressure or augmentation index identified in this cohort.

In this study there was no relationship between augmentation index and PWV, which has been previously reported. Augmentation index is not a direct measure of arterial stiffness and is influenced by the timing and magnitude of the wave reflection. In contrast, PWV is a robust measure of arterial stiffness as it is determined by measuring the velocity of the waveform between the carotid and femoral arteries. Previously, it has been shown that in a population with diabetes PWV was elevated compared with healthy controls, however augmentation index was not different. Lacy et al.  concluded that augmentation index is not a reliable measure of arterial stiffness in people with diabetes. This may explain why we did not see an association between augmentation index and dietary intake, despite seeing correlations with PWV.

 

Curcumin ameliorates diabetic nephropathy by inhibiting the activation of the SphK1-S1P signaling pathway

Juan Huang, Kaipeng Huang, Tian Lan, Xi Xie, .., Peiqing Liu, Heqing Huang
Molecular and Cellular Endocrinology 365 (2013) 231–240
http://dx.doi.org/10.1016/j.mce.2012.10.024

Curcumin, a major polyphenol from the golden spice Curcuma longa commonly known as turmeric, has been recently discovered to have renoprotective effects on diabetic nephropathy (DN). However, the mechanisms underlying these effects remain unclear. We previously demonstrated that the sphingosine kinase 1-sphingosine 1-phosphate (SphK1-S1P) signaling pathway plays a pivotal role in the pathogenesis of DN. This study aims to investigate whether the renoprotective effects of curcumin on DN are associated with its inhibitory effects on the SphK1-S1P signaling pathway. Our results demonstrated that the expression and activity of SphK1 and the production of S1P were significantly down-regulated by curcumin in diabetic rat kidneys and glomerular mesangial cells (GMCs) exposed to high glucose (HG). Simultaneously, SphK1-S1P-mediated fibronectin (FN) and transforming growth factor-beta 1 (TGF-b1) overproduction were inhibited. In addition, curcumin dose dependently reduced SphK1 expression and activity in GMCs transfected with SphKWT and significantly suppressed the increase in SphK1-mediated FN levels. Furthermore, curcumin inhibited the DNA-binding activity of activator protein 1 (AP-1), and c-Jun small interference RNA (c-Jun-siRNA) reversed the HG-induced up-regulation of SphK1. These findings suggested that down-regulation of the SphK1-S1P pathway is probably a novel mechanism by which curcumin improves the progression of DN. Inhibiting AP-1 activation is one of the therapeutic targets of curcumin to modulate the SphK1-S1P signaling pathway, thereby preventing diabetic renal fibrosis.

The creation of the STZ-induced DN model relies on the level and continuous cycle of high blood glucose in vivo. Long-term hyperglycemia induces significant structural changes in the kidney, including glomerular hypertrophy, GBM thickening, and later glomerulosclerosis and tubulointerstitial fibrosis, leading to microalbuminuria and elevated Cr levels. These effects usually occur at around 8–12 weeks after diabetes formation. In the current study, the experimental diabetic model was induced by a single intraperitoneal injection of STZ (60 mg/kg). When the experiment was terminated at 12 weeks, FBG, KW/BW, BUN, Cr, and UP 24 h were significantly increased and body weight was remarkably decreased in the STZ-induced diabetic rats compared with those in the normal control group. Furthermore, PAS staining of the kidneys revealed the induction of glomerular hypertrophy, mesangial matrix expansion, and increased regional adhesion of the glomerular tuft to the Bowman’s capsule in the diabetic rats. This finding indicated the emergence of the diabetic renal injury model characterized by renal hypertrophy, glomerulus damage, and renal dysfunction. As the limited water solubility of curcumin, various methods such as heat treatment, mild alkali and sodium carboxymethyl cellulose are used to increase the solubility of curcumin before administration. Based on our previous study, we employed 1% sodium carboxymethyl cellulose as the vehicle to solubilize curcumin. Compared with the diabetic group, curcumin treatment slightly reduced FBG level and significantly decreased KW/BW, BUN, Cr, and UP 24 h. Moreover, curcumin remarkably improved glomerular pathological changes in the diabetic kidneys. Consistent with previous studies, the current results demonstrated that curcumin prominently ameliorated renal function and renal parenchymal alterations in the diabetic renal injury model. Previous studies revealed that the amelioration of renal dysfunction in diabetes by curcumin was partly related to its function in inhibiting inflammatory injury. Based on these findings, the current experiment further explored whether the renoprotective effects of curcumin are associated with the regulation of the SphK1-S1P signaling pathway.

S1P is a polar sphingolipid metabolite acting as an extracellular mediator and an intracellular second messenger. Ample evidence proves that S1P participates in cell growth, proliferation, migration, adhesion, molecule expression, and angiogenesis. The formation of S1P is catalyzed by SphK1. Recently, the SphK1-S1P signaling pathway has gained considerable attention because of its potential involvement in the progression of DN. Hyperglycemia, AGE, and oxidative stress can activate SphK1 and can increase the intracellular level of S1P. Geoffroy et al. (2004) reported that the treatment of cells with low AGE concentration increases SphK activity and S1P production, thereby and S1P content were significantly increased simultaneously with the up-regulated expression of FN and TGF-β1 (mRNA and protein) in the diabetic rat kidneys. These findings indicated the activation of the SphK1-S1P signaling pathway and the appearance of pathological alterations, including ECM accumulation. After curcumin treatment for 12 weeks, elevations of the said indexes were significantly inhibited. HG remarkably activated the SphK1-S1P signaling pathway and increased FN and TGF-β1 expressions in GMCs. Curcumin dramatically suppressed the SphK1-S1P pathway as well as FN and TGF-β1 levels in a dose-dependent manner. Overall, these results indicated that curcumin ameliorated the pathogenic progression of DN by inhibiting the activation of the SphK1-S1P signaling pathway, resulting in the down-regulation of TGF-β1 and the subsequent reduction of ECM accumulation.

SphK1 expression is mediated by a novel AP-1 element located within the first intron of the human SphK1 gene. AP-1 sites are also found in rat SphK1 promoter from NCBI. Numerous studies indicated that curcumin can inhibit the activity of AP-1 and is widely used as an AP-1 inhibitor. Therefore, further elucidating the link between the inhibition of the SphK1-S1P signaling pathway by curcumin and the suppression of AP-1 activity is important. The data showed that treatment with c-Jun-siRNA significantly down-regulated the basal levels of SphK1 expression. Thus, inhibiting AP-1 activity is one of the therapeutic targets of curcumin in modulating the SphK1-S1P signaling pathway, thereby inhibiting diabetic renal fibrosis.

In summary, curcumin inhibited SphK1 expression and activity, reduced S1P content, and effectively inhibited increased FN and TGF-β1 expressions mediated by the SphK1-S1P signaling pathway. Moreover, the inhibitory effect of curcumin on SphK1-S1P was independent of its hypoglycemic and anti-oxidant roles and might be closely related to the inhibition of AP-1 activity. Our findings suggested that the SphK1-S1P pathway might be a novel mechanism by which curcumin attenuates renal fibrosis and ameliorates DN. In addition, the present study provides further experimental evidence for the clinical application and new drug exploration of curcumin.

 

Antidiabetic Activity of Hydroalcoholic Extracts of Nardostachys jatamansi in Alloxan-induced Diabetic Rats

  1. A. Aleem, B. Syed Asad, Tasneem Mohammed, et al.
    British Journal of Medicine & Medical Research 4(28): 4665-4673, 2014

A review of literature indicates that diabetes mellitus was fairly well known and well conceived as an entity in India with complications like angiopathy, retinopathy, nephropathy, and causing neurological disorders. The antidiabetic study was carried out to estimate the anti-hyperglycemic potential of Nardostachys Jatamansi rhizome’s hydroalcoholic extracts in alloxan induced diabetic rats over a period of two weeks. The hydroalcoholic extract HAE1 at a dose (500mg/kg) exhibited significant antihyperglycemic activity than extract HAE2 at a dose (500mg/kg) in diabetic rats. The hydroalcoholic extracts showed improvement in different parameters associated with diabetes, like body weight, lipid profile and biochemical parameters. Extracts also showed improvement in regeneration of β-cells of pancreas in diabetic rats. Histopath-ological studies strengthen the healing of pancreas by hydroalcoholic extracts (HAE1& HAE2) of Nardostachys Jatamansi, as a probable mechanism of their antidiabetic activity.
Metabolic syndrome and serum carotenoids : findings of a cross-sectional study in Queensland, Australia

Coyne, T, Ibiebele, T,… McClintock, C and Shaw, J
Brit J Nutrition: Int J Nutr Sci 2009; 102(11). pp. 1668-1677
Several components of the metabolic syndrome, particularly diabetes and cardiovascular disease, are known to be oxidative stress-related conditions and there is research to suggest that antioxidant nutrients may play a protective role in these conditions. Carotenoids are compounds derived primarily from plants and several have been shown to be potent antioxidant nutrients. The aim of this study was to examine the associations between metabolic syndrome status and major serum carotenoids in adult Australians. Data on the presence of the metabolic syndrome, based on International Diabetes Federation criteria, were collected from 1523 adults aged 25 years and over in six randomly selected urban centers in Queensland, Australia, using a cross sectional study design. Weight, height, BMI, waist circumference, blood  pressure, fasting and 2-hour blood glucose and  lipids were determined, as well as five serum carotenoids. Mean serum alpha-carotene, beta-carotene and the sum of the five carotenoid concentrations were significantly lower (p<0.05) in persons with the metabolic syndrome (after adjusting for age, sex, education, BMI status, alcohol intake, smoking, physical activity status and vitamin/mineral use) than persons without the syndrome. Alpha, beta and total carotenoids also decreased significantly (p<0.05) with increased number of components of the metabolic syndrome, after adjusting for these confounders. These differences were significant among former smokers and non-smokers, but not in current smokers. Low concentrations of serum alpha-carotene, beta carotene and the sum of five carotenoids appear to be associated with metabolic syndrome status. Additional research, particularly longitudinal studies, may help to determine if these associations are causally related to the metabolic syndrome, or are a result of the pathologies of the syndrome.

Although there is no universal definition of the metabolic syndrome, it is generally described as a constellation of pathologies or anthropometric conditions, which include central obesity, glucose intolerance, lipid abnormalities, and hypertension. It is, however, universally accepted that the presence of the metabolic syndrome is associated with increased risk of type 2 diabetes and cardiovascular disease. The prevalence of the metabolic syndrome in developed countries varies widely depending upon definitions used and age ranges included, but is estimated to be 24% among adults 20 years and over in the US. Given the impending worldwide epidemic of obesity, diabetes and cardiovascular disease, strategies aimed at greater understanding of the pathology of the syndrome, as well as strategies aimed at preventing or treating persons with the syndrome are urgently required.

Few studies have investigated associations of antioxidant nutrients and the metabolic syndrome. Ford and colleagues reported lower levels of several carotenoids and vitamins C and E among those with metabolic syndrome present compared with those without the syndrome in the Third National Health and Nutrition Examination Survey. Sugiura et al.  suggested that several carotenoids may exert a protective effect against the development of the metabolic syndrome, especially among current smokers. Confirming these findings in another population may add strength to these associations.

Our study showed significantly lower concentrations of β-carotene, α-carotene and the sum of the five carotenoids among those with the metabolic syndrome present compared to those without. We also found decreasing concentrations of all the carotenoids tested as the number of the metabolic syndrome components increased. These findings are consistent with data reported by Ford et al. from the third 262 National Health and Nutrition Examination Survey (NHANES III). In the NHANES III study, significantly lower concentrations of all the carotenoids, except lycopene, were found among persons with the metabolic syndrome compared with those without, after adjusting for  confounding factors similar to those in our study.

 

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Diet and Cholesterol

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

Introduction

We are all familiar with the conundrum of diet and cholesterol.  As previously described, cholesterol is made by the liver. It is the backbone for the synthesis of sex hormones, corticosteroids, bile, and vitamin D. It is also under regulatory control, and that is not fully worked out, but it has health consequences. The liver is a synthetic organ that is involved with glycolysis, gluconeogenesis, cholesterol synthesis, and unlike the heart and skeletal muscles – which are energy transducers – the liver is anabolic, largely dependent on NADPH.  The mitochondria, which are associated with aerobic metabolism, respiration, are also rich in the liver.  The other part of this story is the utilization of lipids synthesized by the liver in the vascular endothelium.  The vascular endothelium takes up and utilizes/transforms cholesterol, which is involved in the degenerative development of pathogenic plaque.  Plaque is associated with vascular rigidity, rupture and hemorrhage, essential in myocardial inmfarction. What about steroid hormones?  There is some evidence that sex hormone differences may be a factor in coronary vascular disease and cardiac dysfunction.  The evidence that exercise is beneficial is well established, but acute coronary events can occur during exercise.  WE need food, and food is at the center of the discussion – diet and cholesterol.  The utilization of food varies regionally, and is dependent on habitat.  But it is also strongly influence by culture.  We explore this further in what follows.

A high fat, high cholesterol diet leads to changes in metabolite patterns in pigs – A metabolomic study

Jianghao Sun, Maria Monagas, Saebyeol Jang, Aleksey Molokin, et al.
Food Chemistry 173 (2015) 171–178
http://dx.doi.org/10.1016/j.foodchem.2014.09.161

Non-targeted metabolite profiling can identify biological markers of dietary exposure that lead to a better understanding of interactions between diet and health. In this study, pigs were used as an animal model to discover changes in metabolic profiles between regular basal and high fat/high cholesterol diets. Extracts of plasma, fecal and urine samples from pigs fed high fat or basal regular diets for 11 weeks were analysed using ultra-high performance liquid chromatography with high-resolution mass spectrometry (UHPLC–HRMS) and chemometric analysis. Cloud plots from XCMS online were used for class separation of the most discriminatory metabolites. The major metabolites contributing to the discrimination were identified as bile acids (BAs), lipid metabolites, fatty acids, amino acids and phosphatidic acid (PAs), phosphatidylglycerol (PGs), glycerophospholipids (PI), phosphatidylcholines (PCs) and tripeptides. These results suggest the developed approach can be used to identify biomarkers associated with specific feeding diets and possible metabolic disorders related to diet.

Nutritional metabolomics is a rapidly developing sub-branch of metabolomics, used to profile small-molecules to support integration of diet and nutrition in complex bio-systems research. Recently, the concept of ‘‘food metabolome’’ was introduced and defined as all metabolites derived from food products. Chemical components in foods are absorbed either directly or after digestion, undergo extensive metabolic modification in the gastrointestinal tract and liver and then appear in the urine and feces as final metabolic products. It is well known that diet has a close relationship with the long-term health and well-being of individuals. Hence, investigation of the ‘‘food metabolome’’ in biological samples, after feeding specific diets, has the potential to give objective information about the short- and long-term dietary intake of individuals, and to identify potential biomarkers of certain dietary patterns. Previous studies have identified potential biomarkers after consumption of specific fruits, vegetables, cocoa, and juices. More metabolites were revealed by using metabolomic approaches compared with the detection of pre-defined chemicals found in those foods.

Eating a high-fat and high cholesterol diet is strongly associated with conditions of obesity, diabetes and metabolic syndrome, that are increasingly recognized as worldwide health concerns. For example, a high fat diet is a major risk factor for childhood obesity, cardiovascular diseases and hyperlipidemia. Little is known on the extent to which changes in nutrient content of the human diet elicit changes in metabolic profiles. There are several reports of metabolomic profiling studies on plasma, serum, urine and liver from high fat-diet induced obese mice, rats and humans. Several potential biomarkers of obesity and related diseases, including lysophosphatidylcholines (lysoPCs), fatty acids and branched-amino acids (BCAAs) have been reported.

To model the metabolite response to diet in humans, pigs were fed a high fat diet for 11 weeks and the metabolite profiles in plasma, urine and feces were analyzed. Non-targeted ultra high performance liquid chromatography tandem with high resolution mass spectrometry (UHPLC–MS) was utilized for metabolomics profiling. Bile acids (BAs), lipid metabolites, fatty acids, amino acids and phosphatidic acid (PAs), phosphatidylglycerol (PGs), glycerophospholipids (PI), phosphatidylcholines (PCs), tripeptides and isoflavone conjugates were found to be the final dietary metabolites that differentiated pigs fed a high-fat and high cholesterol diet versus a basal diet. The results of this study illustrate the capacity of this metabolomic profiling approach to identify new metabolites and to recognize different metabolic patterns associated with diet.

Body weight, cholesterol and triglycerides were measured for all the pigs studied. There was no significant body weight gain between pigs fed diet A and diet B after 11 weeks of treatment. The serum cholesterol and triglyceride levels were significantly higher in pigs fed with diet B compared with the control group at the end of experiment.

Plasma, urine and fecal samples were analyzed in both positive and negative ionization mode. To obtain reliable and high-quality metabolomic data, a pooled sample was used as a quality control (QC) sample to monitor the run. The QC sample (a composite of equal volume from 10 real samples) was processed as real samples and placed in the sample queue to monitor the stability of the system. All the samples were submitted in random for analysis. The quantitative variation of the ion features across the QC samples was less than 15%. The ion features from each possible metabolite were annotated by XCMS online to confirm the possible fragment ions, isotopic ions and possible adduct ions. The reproducibility of the chromatography was determined by the retention time variation profiles that were generated by XCMS. The retention time deviation was less than 0.3 min for plasma samples, less than 0.3 min for fecal samples, and less than 0.2 min for urine samples, respectively. On the basis of these results of data quality assessment, the differences between the test samples from different pigs proved more likely to reflect varied metabolite profiles rather than analytical variation. The multivariate analysis results from the QC sample showed the deviation of the analytical system was acceptable.
Good separation can be observed between pigs on the two diets, which is also reflected in the goodness of prediction (Q2), of 0.64 using data from the positive ionization mode. For negative ionization mode data, better separation appears with a Q2of 0.73.

Cloud plot is a new multidimensional data visualization method for global metabolomic data (Patti et al., 2013). Data characteristics, such as the p-value, fold change, retention time, mass-to-charge ratio and signal intensity of features, can be presented simultaneously using the cloud plot. In this study, the cloud plot was used to illustrate the ion features causing the group separation. In Fig. 2 and 82 features with p < 0.05 and fold change >2, including visualisation of the p-value, the directional fold change, the retention time and the mass to charge ratio of features, are shown. Also, the total ion chromato-grams for each sample were shown. The upper panel in (2A) shows the chromatograms of plasma samples from pigs fed the high fat diet, while the lower panel shows the chromatograms of samples from pigs fed the regular diet. Features whose intensity is increased are shown in green, whereas features whose intensity is decreased are shown in pink (2A). The size of each bubble corresponds to the log fold change of the feature: the larger the bubble, the larger the fold changes. The statistical significance of the fold change, as calculated by a Welch t-test with unequal variances, is represented by the intensity of the feature’s color where features with low p-values are brighter compared to features with high p-values. The Y coordinate for each feature corresponds to the mass-to-charge ratio of the compound, as determined by mass spectrometry. Each feature is also color coded, such as features that are shown with a black outline have database hits in METLIN, whereas features shown without a black outline do not have any database hits.

From the cloud plot (Fig. 2A), 82 discriminating ion features from positive data and 48 discriminating ions features from negative data were considered as of great importance for class separation. After filtering out the fragment ions, isotope annotations, and adduct ions, thirty-one metabolites were tentatively assigned using a Metlin library search (Table S4).

Among the assigned metabolites detected, five of the highest abundant metabolites were identified as bile acid and bile acid conjugates (Fig. 2B). This series of compounds shared the following characteristics; the unconjugated bile acids showed [M-H] ion as base peak in the negative mode.

The characteristic consistent with bile acid hyodeoxycholic acid (HDCA) was confirmed with a reference standard. For the conjugated bile acids (usually with glycine and taurine), the [M-H] and [M+H]+ are always observed as the base peaks. For example, the ion feature m/z 448.3065 at 21.18 min was identified as chenodeoxycholic acid glycine conjugate. The neutral loss of 62 amu (H2O + CO2) was considered as a characteristic fragmentation pathway for bile acid glycine conjugates. This above mentioned characteristic can easily identify a series of bile acids compounds. The five metabolite ions detected in plasma were significantly different between pigs fed the high fat diet (Fig. 2B, red bars) and regular diet (Fig. 2B, blue bars) for 11 weeks, and were identified as chenodeoxycholic acid glycine conjugate, tauroursodeoxycholic acid, hyodeoxycholic acid, deoxycholic acid glycine conjugate and glycocholic acid; chenodeoxycholic acid glycine and hyodeoxycholic acid.

Figures 1-4 , not shown.
Fig 1. The PCA score plot of plasma (A) (+)ESI data with all the ion features; (B) (+)ESI data with selected ion features; (C) (-)ESI data with all ion features; (D) (-)ESI data with selected ion features. Samples were taken from pigs fed diet A (BS, blue) and diet B (HF, red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig 2. Cloud plot showing 82 discriminatory ion features (negative ion data) in plasma, and (B) box-plot of data set of the five most abundant bile acids identified in plasma (negative ion data) samples.

Fig. 3. PCA score plot of fecal samples from pigs fed diet A (BS, blue) and diet B (HF, red) (A) week 0, (B) week 2, (C) week 4 (D) week 6, (E) week 11 for distal samples (F) week 11 for proximal colon samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. PCA and PLS-DA score plot of urine samples from (+)ESI-data (A and C) and (-)ESI-data (B and D) taken at the end of the study (week 11) from pigs fed diet A (BS, blue) and diet B (HF, red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Plasma, fecal and urine metabolites from pigs fed either a high fat or regular diet were investigated using a UHPLC–HRMS based metabolomic approach. Their metabolic profiles were compared by multivariate statistical analysis.
Diet is logically believed to have a close relationship with metabolic profiles. Feeding a high fat and high cholesterol diet to pigs for 11 weeks resulted in
an increase in bile acids and their derivatives in plasma, fecal and urine samples, though at this stage, there was no significant weight gain observed.

In a previous study, a significantly higher level of muricholic acid, but not cholic acid, was found in pigs fed a high fat diet. The gut microbiota of these pigs were altered by diet and considered to regulate bile acid metabolism by reducing the levels of tauro-beta-muricholic acid. In our study, the unconjugated bile acids, hyodeoxycholic acid and deoxycholic acid were found to be significantly higher in the fecal samples of pigs fed a high-fat diet.

Chenodeoxycholic acid glycine was 8.6 times higher in pigs fed a high fat and high cholesterol diet compared to those fed a regular diet. These results confirm that feeding a high fat and high cholesterol diet leads to a changing metabolomic pattern over time, represented by excretion of certain bile acids in the feces. We also found that several metabolites associated with lipid metabolism were increased in the feces of pigs fed the high-fat diet. Feeding the high fat diet to pigs for 11 weeks did not induce any overt expression of disease, except for significantly higher levels of circulating cholesterol and triglycerides in the blood. It is likely, however, that longer periods of feeding would increase expression of metabolic syndrome disorders and features of cardiovascular disease in pigs, as have been previously demonstrated. Products of lipid metabolism that changed early in the dietary treatment could be useful as biomarkers. This may be important because the composition of the fats in the diet, used in this study, was complex and from multiple sources including lard, soybean oil and coconut oil.

In summary, a number of metabolite differences were detected in the plasma, urine and feces of pigs fed a high fat and high cholesterol diet versus a regular diet that significantly increased over time. PCA showed a clear separation of metabolites in all biological samples tested from pigs fed the different diets. This methodology could be used to associate metabolic profiles with early markers of disease expression or the responsiveness of metabolic profiles to alterations in the diet. The ability to identify metabolites from bio-fluids, feces, and tissues that change with alterations in the diet has the potential to identify new biomarkers and to better understand mechanisms related to diet and health.

Amino acid, mineral, and polyphenolic profiles of black vinegar, and its lipid lowering and antioxidant effects in vivo

Chung-Hsi Chou, Cheng-Wei Liu, Deng-Jye Yang, Yi-Hsieng S Wuf, Yi-Chen Chen
Food Chemistry 168 (2015) 63–69
http://dx.doi.org/10.1016/j.foodchem.2014.07.035

Black vinegar (BV) contains abundant essential and hydrophobic amino acids, and polyphenolic contents, especially catechin and chlorogenic acid via chemical analyses. K and Mg are the major minerals in BV, and Ca, Fe, Mn, and Se are also measured. After a 9-week experiment, high-fat/cholesterol-diet (HFCD) fed hamsters had higher (p < 0.05) weight gains, relative visceral-fat sizes, serum/liver lipids, and serum cardiac indices than low-fat/cholesterol diet (LFCD) fed ones, but BV supplementation decreased (p < 0.05) them which may resulted from the higher (p < 0.05) fecal TAG and TC contents. Serum ALT value, and hepatic thiobarbituric acid reactive substances (TBARS), and hepatic TNF-α and IL-1β contents in HFCD-fed hamsters were reduced (p < 0.05) by supplementing BV due to increased (p < 0.05) hepatic glutathione (GSH) and trolox equivalent antioxidant capacity (TEAC) levels, and catalase (CAT) and glutathione peroxidase (GPx) activities. Taken together, the component profiles of BV contributed the lipid lowering and antioxidant effects on HFCD fed hamsters.

World Health Organization (WHO) reported that more than 1.4 billion adults were overweight (WHO, 2013). As we know, imbalanced fat or excess energy intake is one of the most important environmental factors resulted in not only increased serum/liver lipids but also oxidative stress, further leading cardiovascular disorders and inflammatory responses. Food scientists strive to improve serum lipid profile and increase serum antioxidant capacity via  medical foods or functional supplementation.

Vinegar is not only used as an acidic seasoning but also is shown to have some beneficial effects, such as digestive, appetite stimulation, antioxidant, exhaustion recovering effects, lipid lowering effects, and regulations of blood pressure. Polyphenols exist in several food categories, such as vegetable, fruits, tea, wine, juice, and vinegar that have effects against lipid peroxidation, hypertension, hyperlipidemia, inflammation, DNA damage, and. Black vinegar (BV) (Kurosu) is produced from unpolished rice with rice germ and bran through a stationary surface fermentation and contains higher amounts of amino acids and organic acids than other vinegars. Black vinegar is also characterised as a health food rather than only an acidic seasoning because it was reported to own a DPPH radical scavenging ability and decrease the adipocyte size in rat models. Moreover, the extract of BV shows the highest radical scavenging activity in a DPPH radical system than rice, grain, apple, and wine vinegars. The extract suppresses increased lipid peroxidation in mouse skin treated with 12-o-tetradecanoylphorbol-13-acetate.

This study focused on the nutritional compositions in BV, and the in-vivo lipid lowering and antioxidant effects. First, the amino acid, mineral, and polyphenolic profile of BV were identified. Hypolipidemic hamsters induced by a high-fat/cholesterol diet (HFCD) were orally administered with different doses of BV. Serum lipid profile and liver damage indices liver and fecal lipid contents, as well as hepatic antioxidant capacities [thiobarbituric acid reactive substances (TBARS), glutathione (GSH), trolox equivalent antioxidant capacity (TEAC), and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)] and hepatic cytokine levels were assayed to demonstrated physiological functions of BV.

Higher serum AST, ALT, and free fatty acids, as well as hepatic cholesterol, triacylglycerol, MDA, hydroperoxide, and cytokine (IL-1β and TNF-α) levels were easily observed in a high-fat-consumption rodent. Several reports indicated some amino acids antioxidant activities in vitro and in vivo. Acidic amino acids, such as Asp and Glu and hydrophobic amino acids, such as Ile, Leu, and Val display high antioxidant properties. Recently, an in vivo study indicated that a pepsin hydrolyzation significantly enhanced Asp, Glu, Leu, and Val contents in chicken livers; meanwhile, chicken-liver hydrolysates showed an antioxidant capacity in brain and liver of D-galactose treated mice. In addition, it was also reported that Mg and Se play important roles in SOD and GPx activities, respectively. Uzun and Kalender (2013) used chlorpyrifos, an organophosphorus insecticide, to induce hepatotoxic and hematologic changes in rats, but they observed that catechin can attenuate the chlorpyrifos-induced hepatotoxicity by increasing GPx and glutathione-S-transferase activities and decreasing MDA contents. Meanwhile, chlorogenic acid elevated SOD, CAT, and GPx activities with concomitantly decreased lipid peroxidation of liver and kidney in streptozotocin-nicotinamide induced type-2 diabetic rats. Hence, it is reasonable to assume that increased antioxidant capacities and decreased damage in livers of HFCD fed hamsters supplemented with BV should be highly related to the components, i.e. amino acid profile, mineral profile, and polyphenol contents, as well as the lowered liver lipid accumulations.

In analyses of amino acids, minerals and polyphenols, BV contained abundant essential amino acids and hydrophobic amino acids. Mg, K, Ca, Fe, Mn, and Se were measured in BV where K and Mg were major. Gallic acid, catechin, chlorogenic acid, p-hydroxybezoic acid, p-cumeric acid, ferulic acid, and sinapic acid were also identified in BV where catechin and chlorogenic acid were the majorities. Meanwhile, the lipid-lowering and antioxidant effects of BV were also investigated via a hamster model. BV supplementation apparently decreased weight gain (g and %), relative size of visceral fat, serum/liver TC levels, serum cardiac index, and hepatic TBARS values and damage indices (serum ALT and hepatic TNF-α and IL-1β) but increased fecal lipid contents and hepatic antioxidant capacities (GSH level, TEAC level, CAT activity, and GPx activity) in HFCD fed hamsters. To sum up, those benefits could be attributed to a synergetic effect of compounds in BV.

Analysis of pecan nut (Carya illinoinensis) unsaponifiable fraction – Effect of ripening stage on phytosterols and phytostanols composition

Intidhar Bouali, Hajer Trabelsi, Wahid Herchi, Lucy Martine, et al.
Food Chemistry 164 (2014) 309–316
http://dx.doi.org/10.1016/j.foodchem.2014.05.029

Changes in 4-desmethylsterol, 4-monomethylsterol, 4,4-dimethylsterol and phytostanol composition were quantitatively and qualitatively investigated during the ripening of three varieties of Tunisian grown pecan nuts. These components have many health benefits, especially in lowering LDL-cholesterol and preventing heart disease. The phytosterol composition of whole pecan kernel was quantified by Gas Chromatography–Flame Ionization Detection (GC–FID) and identified by Gas Chromatography–Mass Spectrometry (GC–MS). Fifteen phytosterols and one phytostanol were quantified. The greatest amount of phytosterols (2852.5 mg/100 g of oil) was detected in Mahan variety at 20 weeks after the flowering date (WAFD). Moore had the highest level of phytostanols (7.3 mg/100 g of oil) at 20 WAFD. Phytosterol and phytostanol contents showed a steep decrease during pecan nut development. Results from the quantitative characterization of pecan nut oils revealed that β-sitosterol, D5-avenasterol, and campesterol were the most abundant phytosterol compounds at all ripening stages.

Association between HMW adiponectin, HMW-total adiponectin ratio and early-onset coronary artery disease in Chinese population

Ying Wang, Aihua Zheng, Yunsheng Yan, Fei Song, et al.
Atherosclerosis 235 (2014) 392-397
http://dx.doi.org/10.1016/j.atherosclerosis.2014.05.910

Objective: Adiponectin is an adipose-secreting protein that shows atheroprotective property and has inverse relation with coronary artery disease (CAD). High-molecular weight (HMW) adiponectin is reported as the active form of adiponectin. In the present study, we aimed to investigate the association between total adiponectin, HMW adiponectin, HMW-total adiponectin ratio and the severity of coronary atherosclerosis, and to compare their evaluative power for the risk of CAD. Methods: Serum levels of total and HMW adiponectin were measured in 382 early-onset CAD (EOCAD) patients and 305 matched controls undergoing coronary angiography by enzyme-linked immunosorbent assay (ELISA). Gensini score was used to evaluate the severity of coronary atherosclerosis. Results: CAD onset age was positively correlated with HMW adiponectin (r = 0.383, P < 0.001) and HMW-total adiponectin ratio (r = 0.429, P < 0.001) in EOCAD patients. Total and HMW adiponectin and HMW-total adiponectin ratio were all inversely correlated with Gensini score (r=0.417, r=0.637, r=0.578, respectively; all P < 0.001). Multivariate binary logistic regression analysis demonstrated that HMW adiponectin and HMW-total adiponectin ratio were both inversely correlated with the risk of CAD (P < 0.05). ROC analysis indicated that areas under the ROC curves of HMW adiponectin and HMW-total adiponectin ratio were larger than that of total adiponectin (P < 0.05). Conclusions: Adiponectin is cardioprotective against coronary atherosclerosis onset in EOCAD patients. HMW adiponectin and HMW-total adiponectin ratio show stronger negative associations with the severity of coronary atherosclerosis than total adiponectin does. HMW adiponectin and HMW-total adiponectin ratio are effective biomarkers for the risk of CAD in Chinese population.

Gender and age were well matched between patients and controls. EOCAD patients were tended to have a history of diabetes or hypertension, more current smoking, and more use of lipid lowering drugs. Levels of total cholesterol, LDL-c, FPG, HbA1c and triglycerides were significantly higher in the patients than in controls, while HDL-cholesterol, total adiponectin, HMW adiponectin, and HMW-total adiponectin ratio were significantly lower in the patients. EOCAD patients developed different degrees of coronary atherosclerosis, and had significantly higher levels of high-sensitivity CRP and larger circumferences of waist and hip than controls.

Spearman correlation coefficients between selected cardiovascular risk factors, Gensini score and adiponectin were significant. Total and HMW adiponectin and HMW-total adiponectin ratio were all inversely correlated with Gensini score, BMI and pack years of cigarette smoking. Total and HMW adiponectin were negatively associated with triglycerides and circumference of waist and hip. LDL-cholesterol and high-sensitivity CRP were inversely correlated with HMW adiponectin and HMW-total adiponectin ratio, while HDL-cholesterol and age were positively correlated with them. FPG was only inversely associated with HMW-total adiponectin ratio.

All participants were divided into four groups according to their Gensini score, group A (control, n = 305), group B (<20, n = 154), group C (20-40, n = 121) and group D (>40, n = 105). With the increasing of Gensini score, a stepwise downward trend was observed in levels of total and HMW adiponectin and HMW-total adiponectin ratio (P < 0.001). Specifically, total adiponectin of four groups were 1.58 (0.61-4.36) mg/ml, 1.21 (0.70-2.83) mg/ml, 1.00 (0.73-1.88) mg/ml, and 0.76 (0.37-1.19) mg/ml, respectively. Except group A with B and group B with C, the differences of pairwise comparisons among all the other groups were statistically significant (all P < 0.05). HMW adiponectin of four groups were 0.91 (0.39-3.26) mg/ml, 0.55 (0.32-1.49) mg/ml, 0.46 (0.21-0.876) mg/ml, and 0.23 (0.14-0.39) mg/ml, respectively. The differences of pairwise comparisons among all the other groups were statistically significant (all P < 0.05) except group B with C. HMW-total adiponectin ratio of four groups were 0.58 (0.31-0.81), 0.47 (0.26-0.69), 0.41 (0.24-0.57), and 0.36 (0.21-0.42), respectively. The differences of pairwise comparisons among all the other groups were statistically significant (all P < 0.05) except group B with C. In the model of multivariate binary logistic regression analysis, after adjustment for conventional cardiovascular risk factors, HMW adiponectin (OR = 0.234, P < 0.011) and HMW-total adiponectin ratio (OR = 0.138, P < 0.005) remained inversely correlated with the risk of CAD, while no significant association was observed between total adiponectin and CAD

Areas under the ROC curves were compared pairwise to identify the diagnostic power for CAD among total adiponectin, HMW adiponectin, and HMW-total adiponectin ratio. HMW adiponectin and HMW-total adiponectin ratio showed greater capability for identifying CAD than total adiponectin did (0.797 vs. 0.674, 0.806 vs. 0.674; respectively, all P < 0.05); however, no significant difference was observed between HMW and HMW-total ratio (P > 0.05).

Associations between total adiponectin, HMW adiponectin, HMW-total adiponectin ratio and the severity of coronary atherosclerosis

Associations between total adiponectin, HMW adiponectin, HMW-total adiponectin ratio and the severity of coronary atherosclerosis in EOCAD patients (evaluated by Gensini score). *P < 0.05; **P < 0.001; ***P < 0.005 by Mann-Whitney U test.

Compares diagnostic power

Compares diagnostic power

Fig. Compares diagnostic power among total adiponectin, HMW adiponectin and HMW-total adiponectin ratio for CAD by ROC curves. Diagnostic power for CAD was based on discriminating patients with or without coronary atherosclerosis. The area under the curve for HMW-total adiponectin ratio (dotted black line) was larger than that for total adiponectin (fine black line) (0.806 [95%CI 0.708-0.903] vs. 0.674 [95%CI 0.552-0.797], P < 0.05) and HMW adiponectin (bold black line) (0.806 [95%CI 0.708-0.903] vs. 0.797 [95%CI 0.706-0.888], no statistically difference). Sensitivity, specificity and optimal cut off value for them were total adiponectin (57.38%, 75.86%, 1.11 mg/ml), HMW (55.74%, 93.1%, 0.49 mg/ml) and H/T (78.69%, 75.86%, 0.52), respectively.

There are two strengths in our study. One is the precise Gensini scoring system to carefully evaluate stenosis of coronary artery or branches > 0% diameter as coronary lesion, another is the specific study subjects of EOCAD in a Chinese Han population that is particularly genetically determined and not influenced by racial/ethnic disparities. The limitations of our study lie in the interference of medications such as the effect of lipid lowering drugs on the levels of adiponectin, and cardiovascular risk factors. Smoking is a conventional cardiovascular risk factor, whose interaction with HMW adiponectin level is rarely investigated, but it has been revealed to be associated with HMW adiponectin level in men according to the study from Kawamoto R et al. We did not adjust the result for the pack/year variable in the multivariate logistic regression analysis for the limitation of small sample size of male subjects in our study. The relatively small study sample also restrained our conclusion generalizable to all populations. Future researches in larger study samples and different populations are in need to validate our findings, and to explore the association of smoking with adiponectin in male subgroup analysis, and to investigate the potential mechanisms by which adiponectin affects the progression of coronary atherosclerosis.

In summary, the present study has demonstrated that adiponectin is protective against coronary atherosclerosis onset in EOCAD patients. HMW adiponectin and HMW-total adiponectin ratio show stronger negative associations with the severity of coronary atherosclerosis than total adiponectin does. HMW adiponectin and HMW-total adiponectin ratio are more effective biomarkers for the risk of CAD than total adiponectin.

Berberis aristata combined with Silybum marianum on lipid profile in patients not tolerating statins at high doses

Giuseppe Derosa, Davide Romano, Angela D’Angelo, Pamela Maffioli
Atherosclerosis 239 (2015) 87-92
http://dx.doi.org/10.1016/j.atherosclerosis.2014.12.043

Aim: To evaluate the effects of Berberis aristata combined with Silybum marianum in dyslipidemic patients intolerant to statins at high doses.
Methods: 137 euglycemic, dyslipidemic subjects, with previous adverse events to statins at high doses, were enrolled. Statins were stopped for 1 month (run-in), then they were re-introduced at the half of the previously taken dose. At randomization, patients tolerating the half dose of statin, were assigned to
add placebo or B. aristata/S. marianum 588/105 mg, 1 tablet during the lunch and 1 tablet during the dinner, for six months. We evaluated lipid profile and safety parameters variation at randomization, and after 3, and 6 months.
Results: B. aristata/S. marianum reduced fasting plasma glucose (-9 mg/dl), insulin (-0.7 mU/ml), and HOMA-index (-0.35) levels compared to baseline and also to placebo. Lipid profile did not significantly change after 6 months since the reduction of statin dosage and the introduction of B. aristata/S. marianum, while it worsened in the placebo group both compared to placebo and with active treatment (+23.4 mg/dl for total cholesterol, +19.6 mg/dl for LDL-cholesterol, +23.1 mg/dl for triglycerides with placebo compared to B. aristata/S. marianum). We did not record any variations of safety parameters
in either group. Conclusions: B. aristata/S. marianum can be considered as addition to statins in patients not tolerating high dose of these drugs.

Statins, also known as 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are effective medications for reducing the risk of death and future cardiovascular disease. In the latest years, however, statin intolerance (including adverse effects related to quality of life, leading to decisions to decrease or stop the use of an otherwise-beneficial drug) has come to the forefront of clinical concern, whereas the safety of statins has come to be regarded as largely favorable. Statin intolerance is defined as any adverse symptoms, signs, or laboratory abnormalities attributed by the patient or physician to the statin and in most cases perceived by the patient to interfere unacceptably with activities of daily living, leading to a decision to stop or reduce statin therapy. The physician might also decide to stop or reduce statin therapy on the basis of clinical/laboratory assessment [abnormal liver function tests, creatine phosphokinase values (CPK)] suggesting undue risk. Adverse events are more common at higher doses of statins, and often contribute to patients low adherence to treatment. For this reason, researchers are testing alternative strategies for lipid treatment when statin intolerance is recognized. One strategy to reduce the risk of statin-induced adverse events includes using a low-dose of statin combined with nonstatin drugs in order to achieve the goals of therapy. Nonstatin drugs include nutraceuticals; in the latest years relatively large number of dietary supplements and nutraceuticals have been studied for their supposed or demonstrated ability to reduce cholesterolemia in humans, in particular Berberis Aristata, has been studied in randomized clinical trials and proved to be effective in improving lipid profile. In particular, B. aristata acts up-regulating LDL-receptor (LDL-R) expression independent of sterol regulatory element binding proteins, but dependent on extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) activation leading to total cholesterol (TC) and LDL-C reduction of about 30 and 25%, respectively. Hwever, B. aristata is a problem in terms of oral bioavailability, affected by a P-glycoprotein (P-gp) mediated gut extrusion process. P-gp seems to reduce by about 90% the amount of B. aristata able to cross the enterocytes, but the use of a potential P-gp inhibitor could ameliorate its oral poor bioavailability improving its effectiveness. Among the potential Pgp inhibitors, silymarin from S. marianum, an herbal drug used as liver protectant, could be considered a good candidate due to its high safety profile.

Analyzing the results of our study, it can appear, at a first glance, that B. aristata/S. marianum has a neutral effect of lipid profile that did not change during the study after the addition of the nutraceutical combination. This lack of effect, however, is only apparent, because, when we analyzed what happens in placebo group, we observed a worsening of lipid profile after statin dose reduction. In other words, the addition of B. aristata/S. marianum neutralized the worsening of lipid profile observed with placebo after statins dose reduction. These results are in line with what was reported by Kong et al., who evaluated the effects of a combination of berberine and simvastatin in sixty-three outpatients diagnosed with hypercholesterolemia. As compared with monotherapies, the combination showed an improved lipid lowering effect with 31.8% reduction of serum LDL-C, and similar efficacies were observed in the reduction of TC as well as Tg in patients. Considering the results of this study, B. aristata/S. marianum can be considered as addition to statins in patients not tolerating high dose of these drugs.

CETP inhibitors downregulate hepatic LDL receptor and PCSK9 expression in vitro and in vivo through a SREBP2 dependent mechanism

Bin Dong, Amar Bahadur Singh, Chin Fung, Kelvin Kan, Jingwen Liu
Atherosclerosis 235 (2014) 449-462
http://dx.doi.org/10.1016/j.atherosclerosis.2014.05.931

Background: CETP inhibitors block the transfer of cholesteryl ester from HDL-C to VLDL-C and LDL-C, thereby raising HDL-C and lowering LDL-C. In this study, we explored the effect of CETP inhibitors on hepatic LDL receptor (LDLR) and PCSK9 expression and further elucidated the underlying regulatory mechanism. Results: We first examined the effect of anacetrapib (ANA) and dalcetrapib (DAL) on LDLR and PCSK9 expression in hepatic cells in vitro. ANA exhibited a dose-dependent inhibition on both LDLR and PCSK9 expression in CETP-positive HepG2 cells and human primary hepatocytes as well as CETP-negative mouse primary hepatocytes (MPH). Moreover, the induction of LDLR protein expression by rosuvastatin in MPH was blunted by cotreatment with ANA. In both HepG2 and MPH ANA treatment reduced the amount of mature form of SREBP2 (SREBP2-M). In vivo, oral administration of ANA to dyslipidemic C57BL/6J mice at a daily dose of 50 mg/kg for 1 week elevated serum total cholesterol by approximately 24.5% (p < 0.05%) and VLDL-C by 70% (p < 0.05%) with concomitant reductions of serum PCSK9 and liver LDLR/SREBP2-M protein. Finally, we examined the in vitro effect of two other strong CETP inhibitors evacetrapib and torcetrapib on LDLR/PCSK9 expression and observed a similar inhibitory effect as ANA in a concentration range of 1-10 µM. Conclusion: Our study revealed an unexpected off-target effect of CETP inhibitors that reduce the mature form of SREBP2, leading to attenuated transcription of hepatic LDLR and PCSK9. This negative regulation of SREBP pathway by ANA manifested in mice where CETP activity was absent and affected serum cholesterol metabolism.

Effect of Eclipta prostrata on lipid metabolism in hyperlipidemic animals

Yun Zhao, Lu Peng, Wei Lu, Yiqing Wang, Xuefeng Huang, et al.
Experimental Gerontology 62 (2015) 37–44
http://dx.doi.org/10.1016/j.exger.2014.12.017

Eclipta prostrata (Linn.) Linn. is a traditional Chinese medicine and has previously been reported to have hypolipidemic effects. However, its mechanism of action is not well understood. This study was conducted to identify the active fraction of Eclipta, its toxicity, its effect on hyperlipidemia, and its mechanism of action. The ethanol extract (EP) of Eclipta and fractions EPF1–EPF4, obtained by eluting with different concentrations of ethanol from a HPD-450 macroporous resin column chromatography of the EP, were screened in hyperlipidemic mice for lipid lowering activity, and EPF3 was the most active fraction. The LD50 of EPF3 was undetectable because no mice died with administration of EPF3 at 10.4 g/kg. Then, 48 male hamsters were used and randomly assigned to normal chow diet, high-fat diet, high-fat diet with Xuezhikang (positive control) or EPF3 (75, 150 and 250 mg/kg) groups. We evaluated the effects of EPF3 on body weight gain, liver weight gain, serum lipid concentration, antioxidant enzyme activity, and the expression of genes involved in lipid metabolism in hyperlipidemic hamsters. The results showed that EPF3 significantly decreased body-weight gain and liver-weight gain and reduced the serum lipid levels in hyperlipidemic hamsters. EPF3 also increased the activities of antioxidant enzymes; upregulated the mRNA expression of peroxisome proliferator-activated receptor α (PPARα), low density lipoprotein receptor (LDLR), lecithin-cholesterol transferase (LCAT) and scavenger receptor class B type Ι receptor (SR-BI); and down-regulated the mRNA expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) in the liver. These results indicate that EPF3 ameliorates hyperlipidemia, in part, by reducing oxidative stress and modulating the transcription of genes involved in lipid metabolism.

Although Eclipta has long been used as a food additive, no studies or reports have clearly shown any liver or kidney toxicity from its use. Therefore, E. prostrata is safe and beneficial for preventing hyperlipidemia in experimental animals and can be used as an alternative medicine for the regulation of dyslipidemia.

Effect of high fiber products on blood lipids and lipoproteins in hamsters

HE Martinez-Floresa, Y Kil Chang, F Martinez-Bustosc, V Sgarbieri
Nutrition Research 24 (2004) 85–93
http://dx.doi.org:/10.1016/S0271-5317(03)00206-9

Serum and liver lipidemic responses in hamsters fed diets containing 2% cholesterol and different dietary fiber sources were studied. The following diets were made from: a) the control diet made from extruded cassava starch (CSH) contained 9.3% cellulose, b) cassava starch extruded with 9.7% resistant starch (CS-RS), c) cassava starch extruded with 9.9% oat fiber (CS-OF), d) the reference diet contained 9.5% cellulose, and no cholesterol was added. Total cholesterol, LDLVLDL-cholesterol and triglycerides were significantly lower (P < 0.05) in serum of hamsters fed on the CS-RS (17.87%, 62.92% and 9.17%, respectively) and CS-OF (15.12%, 67.41% and 18.35%, respectively) diets, as compared to hamster fed with the CSH diet. Similar results were found in the livers of hamsters fed on the CS-RS and CS-OF diets, as compared to hamsters fed with the CSH diet. The diets containing these fibers could be used as active ingredients in human diets to improve the human health.

A new piece in the puzzling effect of n-3 fatty acids on atherosclerosis?

Wilfried Le Goff
Atherosclerosis 235 (2014) 358-362
http://dx.doi.org/10.1016/j.atherosclerosis.2014.03.038

Omega-3 fatty acids (ω-3) FA are reported to be protective against cardiovascular disease (CVD), notably through their beneficial action on atherosclerosis development. In this context dietary intake of long chain marine eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is recommended and randomised trials largely support that EPA and DHA intake is associated with a reduction of CVD. However, mechanisms governing the atheroprotective action of ω-3 FA are still unclear and numerous studies using mouse models conducted so far do not allow to reach a precise view of the cellular and molecular effects of ω-3 FA on atherosclerosis. In the current issue of Atherosclerosis, Chang et al. provide important new information on the anti-atherogenic properties of ω-3 FA by analyzing the incremental replacement of saturated FA by pure fish oil as a source of EPA and DHA in Ldlr -/- mice fed a high fat/high cholesterol diet.

Cardiovascular disease (CVD) is the leading causes of death in the world and is frequently associated with atherosclerosis, a pathology characterized by the accumulation of lipids, mainly cholesterol in the arterial wall. Among major risk factors for CVD, circulating levels of lipids and more especially those originating from diets are closely linked to development of atherosclerosis. In this context, not only cholesterol, but also dietary fatty acids (FA) may appear particularly deleterious in regards to atherosclerosis and associated CVD. However, although saturated fats are proatherogenic, omega-3 fatty acids (ω-3 FA), and more especially long-chain marine eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exert atheroprotective properties through several potential underlying mechanisms. Therefore, the intake of EPA and DHA is recommended around the world and randomised trials with ω-3 FA confirmed that EPA and DHA intake reduced risk for CVD events. However benefits of ω-3 FA intake were challenged by recent clinical trials that failed to replicate protective effects of EPA + DHA on CVD, raising the controversy on the healthy side of marine ω-3 FA.

Animal models are commonly employed in order to decipher mechanisms by which ω-3 FA exert their beneficial actions regarding lipid metabolism and atherosclerosis. Since the last past 20 years, mouse models, and more especially genetically modified mouse models, became the reference model to evaluate the effects of dietary fatty acids, especially ω-3 FA, on atherosclerosis development [7-20]. However, the use of different mouse models of atherosclerosis (Apoe-/-, Ldlr-/-, double Apoe-/- x Ldlr-/- , Ldlr-/- x hApoB mice), as well as diet composition (chow, high cholesterol, high fat, high cholesterol/high fat), source of ω-3 FA supplementation (fish oil, perilla seed oil, flaxseed, pure ALA, EPA or DHA), duration of the diet (from 4 to 32 weeks), size of atherosclerotic lesions in control animals (from 51 to 700.103 mm2) in

those studies led to heterogeneous results and therefore to a partial understanding of the effects of ω-3 FA on atherosclerosis.

Contrary to what observed in Apoe-/- mice, dietary supplementation of Ldlr-/- mice with ω-3 FA led to a reproducible reduction of aortic atherosclerosis, although to various degrees, confirming that Ldlr-/- mice constitute the most appropriate model for studying the atheroprotective effects of ω-3 FA. When evaluated, the decrease of atherosclerosis upon ω-3 FA-rich diet was accompanied by a reduction in the macrophage content as well as inflammation in aortic lesions highlighting the major impact of ω-3 FA on monocyte recruitment and subsequent macrophage accumulation in the arterial wall. However, although supplementation with ω-3 FA allows an efficacious lowering of plasma lipid levels in humans, studies in mouse models suggest that the antiatherogenic action of ω-3 FA is independent of any effects on plasma cholesterol or triglyceride levels. However, that must be asserted with caution as lipid metabolism is quite different in mouse in comparison to humans, highlighting the need to study in the future the effects of ω-3 FA on atherosclerosis in a mouse model exhibiting a more “humanized” lipid metabolism as achieved in hApoB/CETP mice.

In a previous issue of Atherosclerosis, Chang et al. reevaluate the impact of fish oil ω-3 FA on atherosclerosis development by operating an incremental replacement of saturated fats (SAT) by ω-3 FA (pure fish oil, EPA- and DHA-rich) in Ldlr-/- mice fed a high-fat (21%, w/w)/high-cholesterol (0.2%, w/w) diet for a 12-week period. This experimental approach is quite pertinent as dietary fat intake in developed countries, as in United States, derived mostly from saturated FA and is poor in ω-3 FA. Then, using this strategy the authors were able to evaluate the potential beneficial effects of a supplementation with fish oil ω-3 FA in a dietary context for which ω-3 FA intake is relevant.

Here, Chang et al. demonstrated that the progressive increase of dietary intake of fish oil ω-3 FA (EPA and DHA) abrogated the deleterious effects of a SAT diet, thereby suggesting that a dietary ω-3 FA intake on a SAT background is potentially efficient to decrease CVD in humans. Indeed, replacement of SAT by fish oil ω-3 FA markedly reduced plasma cholesterol and triglycerides levels and abolished diet-induced atherosclerosis mediated by SAT in Ldlr-/-mice. To note that in the present study, Ldlr-/- mice only developed small atherosclerosic lesions (~100.103 mm2) after 12 weeks of diet with SAT.

As previously reported, decreased atherosclerotic lesions were accompanied by a reduced content of aortic macrophages and inflammation. Based on their previous works, the authors proposed that the reduction of atherosclerosis upon ω-3 FA resulted from an impairment of cholesterol uptake by arterial macrophages consecutive to the decrease of Lipoprotein Lipase (LPL) expression in those cells. Indeed, beyond its lipolysis action on triglycerides, LPL was reported to promote lipid accumulation, in particular in macrophages, by binding to lipoproteins and cell surface proteoglycans and then acting as a bridging molecule that facilitates cellular lipid uptake. Coherent with this mechanism, macrophage LPL expression was reported to promote foam cell formation and atherosclerosis. In the present study, replacement of SAT by ω-3 FA both decreased expression and altered distribution of arterial LPL. Such a mechanism for ω-3 FA (EPA and DHA) was proposed by this group in earlier studies to favor reduction of arterial LDL-cholesterol. It is noteworthy that lipid rafts alter distribution of LPL at the cell surface and subsequently the LPL dependent accumulation of lipids in macrophages and foam cell formation. As incorporation of ω-3 FA, such as DHA, into cell membrane phospholipids disrupts lipid rafts organization, it cannot be exclude that reduction of lipid accumulation in arterial macrophages upon addition of ω-3 FA results in part from an impairment of the localization and of the anchoring function of LPL at the cell surface of macrophages. Indeed Chang et al. observed that progressive replacement of SAT by ω-3 FA affected aortic FA composition leading to a pronounced increase of arterial EPA and DHA, then suggesting that content of ω-3 FA in macrophage membrane may be equally altered. However, the implication of LPL in the atheroprotective effects of ω-3 FA need to be validated using an appropriate mouse model for which LPL expression may be controlled.

Among the various mechanisms by which ω-3 FA exert anti-inflammatory properties, EPA and DHA repressed inflammation by shutting down NF-kB activation in macrophages. Since expression of TLR-4 and NF-kB target genes, IL-6 and TNFα, in aorta from mice fed diets containing ω-3 FA were decreased when compared to SAT, those results strongly support the contention that ω-3 FA repress inflammation by inhibiting the TLR4/NF-kB signaling cascade likely through the macrophage ω-3 FA receptor GPR120.

Although further studies are needed to explore the complete spectrum of actions of ω-3 FA on atherosclerosis development and CVD, this study provides important information that supports that ω-3 FA intake is a pertinent strategy to reduce risk of CVD.

Effects of dietary hull-less barley β-glucan on the cholesterol metabolism of hypercholesterolemic hamsters

Li-Tao Tong, Kui Zhong, Liya Liu, Xianrong Zhou, Ju Qiu, Sumei Zhou
Food Chemistry 169 (2015) 344–349
http://dx.doi.org/10.1016/j.foodchem.2014.07.157

The aim of the present study is to investigate the hypocholesterolemic effects of dietary hull-less barley β-glucan (HBG) on cholesterol metabolism in hamsters which were fed a hypercholesterolemic diet. The hamsters were divided into 3 groups and fed experimental diets, containing 5‰ HBG or 5‰ oat β-glucan (OG), for 30 days. The HBG, as well as OG, lowered the concentration of plasma LDL-cholesterol significantly. The excretion of total lipids and cholesterol in feces were increased in HBG and OG groups compared with the control group. The activity of 3-hydroxy-3-methyl glutaryl-coenzyme A (HMG-CoA) reductase in liver was reduced significantly in the HBG group compared with the control and OG groups. The activity of cholesterol 7-α hydroxylase (CYP7A1) in the liver, in the HBG and OG groups, was significantly increased compared with the control group. The concentrations of acetate, propionate and total short chain fatty acids (SCFAs) were not significantly different between the HBG and control groups. These results indicate that dietary HBG reduces the concentration of plasma LDL cholesterol by promoting the excretion of fecal lipids, and regulating the activities of HMG-CoA reductase and CYP7A1 in hypercholesterolemic hamsters.

Effects of dietary wheat bran arabinoxylans on cholesterolmetabolism of hypercholesterolemic hamsters

Li-Tao Tong, Kui Zhong, Liya Liu, Ju Qiu, Lina Guo, et al.
Carbohydrate Polymers 112 (2014) 1–5
http://dx.doi.org/10.1016/j.carbpol.2014.05.061

The aim of the present study is to investigate the effects of dietary wheat bran arabinoxylans (AXs) on cholesterol metabolism in hypercholesterolemic hamsters. The hamsters were divided into 3 groups and fed the experimental diets containing AXs or oat β-glucan at a dose of 5 g/kg for 30 days. As the results,the AXs lowered plasma total cholesterol and LDL-cholesterol concentrations, and increased excretions of total lipids, cholesterol and bile acids, as well as oat β-glucan. The AXs reduced the activity of 3-hydroxy-3-methyl glutaryl-coenzyme A (HMG-CoA) reductase, and increased the activity of cholesterol 7-α hydroxylase (CYP7A1) in liver. Moreover, the AXs increased propionate and the total short-chain fatty acids (SCFAs) concentrations. These results indicated that dietary AXs reduced the plasma total cholesterol and LDL-cholesterol concentrations by promoting the excretion of fecal lipids, regulating the activities of HMG-CoA reductase and CYP7A1, and increasing colonic SCFAs in hamsters.

High-fructose feeding promotes accelerated degradation of hepatic LDL receptor and hypercholesterolemia in hamsters via elevated circulating PCSK9 levels

Bin Dong, Amar Bahadur Singh, Salman Azhar, Nabil G. Seidah, Jingwen Liu
Atherosclerosis 239 (2015) 364-374
http://dx.doi.org/10.1016/j.atherosclerosis.2015.01.013

Background: High fructose diet (HFD) induces dyslipidemia and insulin resistance in experimental animals and humans with incomplete mechanistic understanding. By utilizing mice and hamsters as in vivo models, we investigated whether high fructose consumption affects serum PCSK9 and liver LDL receptor (LDLR) protein levels. Results: Feeding mice with an HFD increased serum cholesterol and reduced serum PCSK9 levels as compared with the mice fed a normal chow diet (NCD). In contrast to the inverse relationship in mice, serum PCSK9 and cholesterol levels were co-elevated in HFD-fed hamsters. Liver tissue analysis revealed that PCSK9 mRNA and protein levels were both reduced in mice and hamsters by HFD feeding, however, liver LDLR protein levels were markedly reduced by HFD in hamsters but not in mice. We further showed that circulating PCSK9 clearance rates were significantly lower in hamsters fed an HFD as compared with the hamsters fed NCD, providing additional evidence for the reduced hepatic LDLR function by HFD consumption. The majority of PCSK9 in hamster serum was detected as a 53 kDa N-terminus cleaved protein. By conducting in vitro studies, we demonstrate that this 53 kDa truncated hamster PCSK9 is functionally active in promoting hepatic LDLR degradation. Conclusion: Our studies for the first time demonstrate that high fructose consumption increases serum PCSK9 concentrations and reduces liver LDLR protein levels in hyper-lipidemic hamsters. The positive correlation between circulating cholesterol and PCSK9 and the reduction of liver LDLR protein in HFD-fed hamsters suggest that hamster is a better animal model than mouse to study the modulation of PCSK9/LDLR pathway by atherogenic diets.

High-oleic canola oil consumption enriches LDL particle cholesteryl oleate content and reduces LDL proteoglycan binding in humans

Peter J.H. Jones, Dylan S. MacKay, Vijitha K. Senanayake, Shuaihua Pu, et al.
Atherosclerosis 238 (2015) 231-238
http://dx.doi.org/10.1016/j.atherosclerosis.2014.12.010

Oleic acid consumption is considered cardio-protective according to studies conducted examining effects of the Mediterranean diet. However, animal models have shown that oleic acid consumption increases LDL particle cholesteryl oleate content which is associated with increased LDL-proteoglycan binding and atherosclerosis. The objective was to examine effects of varying oleic, linoleic and docosahexaenoic acid consumption on human LDL-proteoglycan binding in a non-random subset of the Canola Oil Multi-center Intervention Trial (COMIT) participants. COMIT employed a randomized, double-blind, five-period, crossover trial design. Three of the treatment oil diets: 1) a blend of corn/safflower oil (25:75); 2) high oleic canola oil; and 3) DHA-enriched high oleic canola oil were selected for analysis of LDL-proteoglycan binding in 50 participants exhibiting good compliance. LDL particles were isolated from frozen plasma by gel filtration chromatography and LDL cholesteryl esters quantified by mass-spectrometry. LDL-proteoglycan binding was assessed using surface plasmon resonance. LDL particle cholesterol ester fatty acid composition was sensitive to the treatment fatty acid compositions, with the main fatty acids in the treatments increasing in the LDL cholesterol esters. The corn/safflower oil and high-oleic canola oil diets lowered LDL-proteoglycan binding relative to their baseline values (p < 0.0005 and p < 0.0012, respectively). At endpoint, high-oleic canola oil feeding resulted in lower LDL-proteoglycan binding than corn/safflower oil (p < 0.0243) and DHA-enriched high oleic canola oil (p < 0.0249), although high-oleic canola oil had the lowest binding at baseline (p < 0.0344). Our findings suggest that high-oleic canola oil consumption in humans increases cholesteryl oleate percentage in LDL, but in a manner not associated with a rise in LDL-proteoglycan binding.

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Mechanisms of Drug Resistance

Curator: Larry H. Bernstein, MD, FCAP

Leaders in Pharmaceutical Intelligence, CSO

 

Mechanisms of Drug Resistance

This discussion is a continuing discussion of matters of metabolomics and the
essential role of genomic or epigenetic mechanisms to guide the development of
proteomic driven effectors of resistance to drug therapy.
We start with the elucidation of efflux pumps in bacteria, and we conclude with
consideration of cancer cells.

Part 1. Antimicrobial Resistance

Antimicrobial resistance is the ability of microbes, such as bacteria, viruses,
parasites, or
fungi, to grow in the presence of a chemical (drug) that would normally kill it
or limit its growth.

difference between non-resistant bacteria and drug resistant bacteria

difference between non-resistant bacteria and drug resistant bacteria

http://www.niaid.nih.gov/SiteCollectionImages/topics/antimicrobialresistance/1whatIs
DrugResistance.gif

Non-resistant bacteria multiply, and upon drug treatment, the bacteria die. Drug
resistant bacteria multiply as well, but upon drug treatment, the bacteria continue
to spread.

Many infectious diseases are increasingly difficult to treat because of antimicrobial-resistant organisms, including HIV infection, staphylococcal infection, tuberculosis,
influenza, gonorrhea, candida infection, and malaria.

Between 5 and 10 percent of all hospital patients develop an infection. About 90,000
of these patients die each year as a result of their infection, up from 13,300 patient
deaths in 1992.

According to the Centers for Disease Control and Prevention (April 2011), antibiotic
resistance in the United States costs an estimated $20 billion a year in excess health
care costs. In addition, a cost of $35 million in other societal costs and more than 8
million additional days that people spend in the hospital. This is because people
infected with antimicrobial-resistant organisms are more likely to have longer hospital stays and may require more complicated treatment.

Diagnostic tests designed to determine which microbe is causing infection and to
which antimicrobials the microbe might be resistant take a few days or weeks to give
results because of a requirement for the microbe to grow for it to be identified.

Part 2. Antibiotic Tolerance   
Reported By Jef Akst | June 25, 2014

Optimization of lag time underlies antibiotic tolerance in evolved bacterial
populations

O. Fridma et al.    Nature, 2014 
http://dx.doi.org://10.1038/nature13469

Populations of Escherichia coli grown in the lab develop tolerance when exposed to
repeated treatments with the antibiotic ampicillin. The bacteria evolved to stay in a
dormant “lag” phase for just longer than three-, five-, or eight-hour-long treatment
courses. Antibiotic tolerance, which allows bacteria to survive even high levels of
antibiotics by remaining dormant. Tolerance may lead to an inaccurate assumption
that an unsuccessful antibiotic treatment failed as a result of resistance, in which
the microbe has evolved to grow in the presence of the drug. Resistance is very well
known; but the issue of tolerance is much less known,” according to Tom Coenye of
the Laboratory of Pharmaceutical Microbiology (LPM) at Gent University in Belgium,
who was not involved in the research.  This is a new phenomenon, extended lag,
where mutants have a longer lag time, and that extended lag allows them to survive
an attack by antibiotics.

To gain a better understanding of how bacterial populations might evolve to tolerate
antibiotic exposure, Nathalie Q. Balaban, a microbiologist and physicist at The Hebrew
University of Jerusalem in Israel and her colleagues exposed cultures of E. coli to high
concentrations of ampicillin for three, five, or eight hours, then washed the drug away
and suspended the bacteria in fresh media to be grown overnight. The next day, the
team repeated these treatments. In 10 cycles we could see that tolerance had evolved,
” Balaban said. Indeed, while the ampicillin treatments killed more than 99.9 percent of
the E. coli, by day 10, bacterial survival had increased 100-fold.

Moreover, the bacteria were also tolerant to norfloxacin, an antibiotic with a different mechanism of action than ampicillin but also ineffective during the dormant stage,
further supporting the idea that the E. coli populations had evolved to tolerate certain
durations of antibiotic exposure. “This is characteristic of tolerance,” said Balaban.
“The bacteria that have evolved tolerance under ampicillin are also more tolerant to
this completely different class of antibiotics.” Resistance, on the other hand, is usually
class-specific, she noted.

The researchers identified three genes that seemed to play a functional role in antibiotic
tolerance. While the exact mechanism of how mutations in these genes may have
lengthened the bacteria’s lag time is not yet known, two of the genes are part of pathways
that were previously implicated in bacterial persistence, including an antitoxin in a
common toxin-antitoxin module
 that may help regulate that bacteria’s growth.

Part 3. Multidrug Resistance Perspective

Mechanisms of antibiotic resistance in salmonella: efflux pumps, genetics,
quorum sensing and biofilm formation.

Perspectives in Drug Discovery and Design 02/2011; 8:114-123.
Martins M, McCusker, Amaral, Fanning S

Multidrug resistance (MDR) to antibiotics presents a serious therapeutic problem
in the treatment of bacterial infections. The importance of this mechanism of resistance
in clinical settings is reflected in the increasing number of reports of multidrug resistant
isolates. In Salmonella enterica, the most common etiological agent of food borne
salmonellosis worldwide, MDR is becoming a major concern.

In Salmonella the main mechanisms of antibiotic resistance are mutations in target
genes (such as DNA gyrase and topoisomerase IV) and the over-expression of efflux pumps. However, other mechanisms such as

  1. changes in the cell envelope;
  2. down regulation of membrane porins;
  3. increased lipopolysaccharide (LPS) component of the outer cell membrane;
  4. quorum sensing and
  5. biofilm formation

can also contribute to the resistance seen in this microorganism. To overcome
this problem new therapeutic approaches are urgently needed.

In the case of efflux-mediated multidrug resistant isolates, one of the treatment
options could be

  • the use of efflux pump inhibitors (EPIs)
  • in combination with the antibiotics to which the bacteria is resistant.

By blocking the efflux pumps

  • resistance is partly or wholly reversed,
  • allowing antibiotics showing no activity against the MDR strains
  • to be used to treat these infections.

Compounds that show potential as an EPI are therefore of interest, as well as new
strategies to target the efflux systems. Quorum sensing (QS) and biofilm formation
are systems also known to be involved in antibiotic resistance. Consequently,
compounds that

  • can disrupt or inhibit these bacterial “communication systems” will be of use in
    the treatment of these infections.

Part 5. Effux pumps and S. Aureus

Multidrug Efflux Pumps in Staphylococcus aureus: an Update

SS Costa, M Viveiros, L Amaral and I Couto
1Grupo de Micobactérias, Unidade de Microbiologia Médica, Instituto de Higiene e
Medicina Tropical, Universidade Nova de Lisboa (IHMT, UNL), 2Centro de Recursos
Microbiológicos (CREM), UNL, Portugal,3COST ACTION BM0701 (ATENS), Brussels,
Belgium
The Open Microbiology Journal 2013;(Suppl 1-M5): 59-71

The emergence of infections caused by multi- or pan-resistant bacteria in the hospital
or in the community settings is an increasing health concern. Albeit there is no single
resistance mechanism behind multi-resistance, multidrug efflux pumps,

  • proteins that cells use to detoxify from noxious compounds,

seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria.
During the last decades, experimental data has established their contribution to low
level resistance to antimicrobials in bacteria and their

  • potential role in the appearance of MDR phenotypes, by the extrusion of multiple,
    unrelated compounds.

Recent studies suggest that

  • efflux pumps may be used by the cell as a first-line defense mechanism,

avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration
occurs, that allows survival in the presence of that agent.

In this paper we review the current knowledge on

  • MDR efflux pumps and their
  • intricate regulatory network in Staphylococcus aureus,

a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that

  • aureus MDR efflux pumps,
  • either chromosomal or plasmid-encoded, have
  • on resistance towards different antimicrobial agents and
  • on the selection of drug – resistant strains.

We will also discuss the many questions that still remain on the role of each specific
efflux pump and the need to establish appropriate methodological approaches to
address all these questions.

        Table 1. Multidrug Efflux Pumps Described for Staphylococcus aureus

Efflux Pump  Family Regulator(s) Substrate Specificity  References 
Chromosomally-encoded Efflux Systems 
NorA MFS MgrA,
NorG(?)
Hydrophilic fluoroquinolones (ciprofloxacin,
norfloxacin) QACs (tetraphenylphosphonium,
benzalkonium chloride) Dyes (e.g. ethidium
bromide, rhodamine)
[16,18,19]
NorB MFS MgrA,
NorG
Fluoroquinolones (e.g. hydrophilic: ciprofloxacin,
norfloxacin and hydrophobic: moxifloxacin,
sparfloxacin) Tetracycline QACs (e.g.
tetraphenylphosphonium, cetrimide) Dyes (e.g. ethidium bromide)
[31]
NorC MFS MgrA(?),
NorG
Fluoroquinolones (e.g. hydrophilic: ciprofloxacin
and hydrophobic: moxifloxacin) Dyes
(e.g. rhodamine)
[35,36]
MepA MATE MepR Fluoroquinolones (e.g. hydrophilic: ciprofloxacin,
norfloxacin and hydrophobic: moxifloxacin,
sparfloxacin) Glycylcyclines (e.g. tigecycline) QACs (e.g. tetraphenylphosphonium, cetrimide, benzalkonium chloride) Dyes
(e.g. ethidium bromide)
[37,38]
MdeA MFS n.i. Hydrophilic fluoroquinolones (e.g. ciprofloxacin,
norfloxacin) Virginiamycin, novobiocin, mupirocin,
fusidic acid QACs (e.g. tetraphenylphosphonium,
benzalkonium chloride, dequalinium) Dyes (e.g. ethidium bromide)
[39,40]
SepA n.d. n.i. QACs (e.g. benzalkonium chloride) Biguanidines
(e.g. chlorhexidine) Dyes (e.g. acriflavine)
[41]
SdrM MFS n.i. Hydrophilic fluoroquinolones (e.g. norfloxacin) Dyes (e.g. ethidium bromide, acriflavine) [42]
LmrS MFS n.i. Oxazolidinone (linezolid) Phenicols
(e.g. choramphenicol, florfenicol) Trimethoprim, erythromycin, kanamycin,
fusidic acid QACs (e.g. tetrapheny-
lphosphonium) Detergents (e.g. sodium
docecyl sulphate) Dyes (e.g. ethidium
bromide)
[43]
Plasmid-encoded Efflux Systems

QacA MFS QacR QACs (e.g. tetraphenylphosphonium,
benzalkonium chloride, dequalinium)
Biguanidines (e.g. chlorhexidine)
Diamidines (e.g. pentamidine) Dyes
(e.g. ethidium bromide,
rhodamine, acriflavine)
[45,49]
QacB MFS QacR QACs (e.g. tetraphenylphosphonium,
benzalkonium chloride)Dyes (e.g. ethidium bromide, rhodamine,
acriflavine)
[53]
Smr SMR n.i. QACs (e.g. benzalkonium chloride,
cetrimide) Dyes (e.g. ethidium bromide)
[58,61]
QacG SMR n.i. QACs (e.g. benzalkonium chloride,
cetyltrymethylammonium) Dyes
(e.g. ethidium bromide)
[67]
QacH SMR n.i. QACs (e.g. benzalkonium chloride,
cetyltrymethylammonium) Dyes
(e.g. ethidium bromide)
[68]
QacJ SMR n.i. QACs (e.g. benzalkonium chloride,
cetyltrymethylammonium) Dyes
(e.g. ethidium bromide)
[69]

a n.d.: The family of transporters to which SepA belongs is not elucidated to date.
b n.i.: The transporter has no regulator identified to date.
QACs: quaternary ammonium compounds

Identification of the plasmid-encoded qacA efflux pump gene
in meticillin-resistant Staphylococcus aureus (MRSA)
strain HPV107, a representative of the MRSA Iberian clone

S.S. Costaa,b, E. Ntokouc, A. Martinsa,d, M. Viveirosa,e, S. Pournarasc,
I. Coutoa,b, L. Amarala,d,e,∗
a Unidade de Micobactérias, Instituto de Higiene e Medicina Tropical,
Universidade Nova de Lisboa (IHMT, UNL), b Centro de Recursos Microbiológicos,
Universidade Nova de Lisboa (CREM, UNL), d Unidade de Parasitologia e
Microbiologia Médica (UPMM), Instituto de Higiene e Medicina Tropical, Universidade
Nova de Lisboa (IHMT, UNL), Lisbon, Portugal; e COST ACTION BM0701 (ATENS)
c Department of Microbiology, Medical School, University of Thessaly, Larissa, Greece;
Int J Antimicrobial Agents  2010; 36: 557–561
http://www.elsevier.com/locate/ijantimicag

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial
bacterium for which prevention and control measures consist mainly of

  • the application of biocides with antiseptic and disinfectant activity.

In this study, we demonstrated the presence of

  • the plasmid-located efflux pump gene qacA in MRSA strain HPV107,

a clinical isolate representative of the MRSA Iberian clone. The existence
of efflux activity in strain HPV107 due to the QacA pump was found and

  • this QacA efflux activity was linked with a phenotype of
  • reduced susceptibility towards several biocide compounds.

No association could be made with antibiotic resistance. This work
emphasises the potential of QacA pump activity in

  • the maintenance and dissemination of important MRSA strains in
    the hospital setting and, increasingly, in the community.

Efflux-mediated response of Staphylococcus aureus exposed to
ethidium bromide

I Couto1,2, S S Costa1, M Viveiros1, M Martins1,3 and L Amaral1,3*
1Unidade de Micobacterias, Instituto de Higiene e Medicina Tropical,
Universidade Nova de Lisboa (UNL), 2Centro de Recursos Microbiolo´gicos (CREM), Faculdade de Cieˆncias e Tecnologia, UNL,3UPMM,
Instituto de Higiene e Medicina Tropical, UNL, Portugal
J Antimicrob Chemother  (2008) 62, 504–513
http://dx.doi.org:/10.1093/jac/dkn217

By adapting an antibiotic-susceptible Staphylococcus aureus strain to
increasing concentrations of ethidium bromide, a known substrate
of efflux pumps (EPs), and

  • by phenotypically and genotypically analysing the resulting progeny,
  • we characterized the molecular mechanisms of S. aureus
    adaptation to ethidium bromide.

ATCC 25923 was grown in increasing concentrations of ethidium bromide.
The MICs of representatives of eight classes of antibiotics, eight biocides
and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny
ATCC 25923EtBr were determined

  • with or without six efflux pump inhibitors (EPIs).

Efflux activity in the presence/absence of EPIs was evaluated by realtime
fluorometry. The presence and expression of eight EP genes were assayed
by PCR and quantitative RT–PCR (qRT–PCR), respectively. Mutations in
grlA, gyrA and norA promoter regions were screened by DNA sequencing.

Compared with its parental strain, ATCC 25923EtBr was

  • 32-fold more resistant to ethidium bromide and
  • also more resistant to biocides and hydrophilic fluoroquinolones.
  • Resistance to these could be reduced by the EPIs chlorpromazine,
    thioridazine and reserpine.

Increased efflux of ethidium bromide by ATCC 25923EtBr could be
inhibited by the same EPIs. qRT–PCR showed that

  • norA was 35-fold over-expressed in ATCC 25923EtBr,

whereas the remaining EP genes showed no significant increase in their

expression. Sequencing of the norA promoter region revealed

  • a 70 bp deletion in ATCC 25923EtBr.

Exposure of S. aureus to quaternary compounds such as ethidium bromide
results in decreased susceptibility of the organism to a wide variety of
compounds, including quinolones and biocides

  • through an efflux-mediated response, which
  • for strain ATCC 25923 is mainly NorA-mediated.

This altered expression may result from alterations in the norA
promoter region
.

Ethnic consumption of plant leaf extracts and appraisal of
their nutraceutical efficacy against multidrug resistant
staphylococcus aureus

Kaushik S1, 2*, Tomar Rs1, Shrivastav V1, Shrivastav A2 And Jain Sk3
Amity Institute of Biotechnology, Amity University Madhya Pradesh,
Gwalior (M.P.);  2: College of Life Sciences, Cancer Hospital and
Research Institute, Gwalior (M.P.); 3: Department of Microbiology,
Vikram University, Ujjain (M.P.), INDIA
IJBPAS, Feb, 2014, 3(2): 204-209

Nutraceuticals are natural bioactive chemical compounds that have
health promoting, disease preventing or medicinal properties.
Emergence of Multi Drug Resistant Staphylococci is increasing at
alarming rates and diseases caused by these strains leave patients
against multiple resistant Staphylococcus aureus.

The test bacteria were isolated and characterized by standard and
NCCLS recommended microbiological techniques. A total of eighteen
plant extracts were analysed for their antimicrobial activity. The
selection of medicinal plants was based on their traditional uses in
India. However most of these plants were not previously screened.
Antibacterial activity of these components was performed by standard
Kirby Bauer Disk Diffusion method approved by NCCLS and the
inhibitory effect was analysed by calculating Zone of inhibition.

Among the eighteen plant extracts analysed we found highest
activity in the effect of chemotherapy and as promising bio control agents

  • Guava,
  • Mango,
  • Jamun and
  • Pomengrate plant extracts,

while most of the other plants were either showing very moderate/
least activity against test bacteria. Our recent experiment indicated
that phytochemicals extracted with methanol can be utilized as
nutraceutical to lower the side.

Part 6. Efflux pumps and gram-negative organisms

Efflux Pumps that Bestow Multi-Drug Resistance of Pathogenic Gram-
negative 
Bacteria 

Amaral L1,2*, Spengler G2, Martins A2,3 and Molnar J2
1Travel Medicine of the Centre for Malaria and Other Tropical Diseases (CMDT),
Institute of Hygiene and Tropical Medicine, Lisbon, Portugal 2Department of
Medical Microbiology and Immunobiology, Faculty of Medicine, University of
Szeged, Szeged, Hungary 3Unit of Parasitology and Medical Microbiology
(UPMM), Institute of Hygiene and Tropical Medicine, Lisbon, Portugal
Amaral et al., Biochem Pharmacol 2013; 2:3
http://dx.doi.org/10.4172/2167-0501.1000119

The efflux pump

The efflux pump

Efflux pumps are integral plasma membrane protein systems that recognize and bind
noxious compounds present in the cytoplasm (toxic products produced by metabolism;
compounds that have penetrated the cell), or periplasm of the bacterial cell and extrude
it into the environment in which the bacterium resides [1].

The efflux pump machinery gives the cell additional protection to the one provided by

  • the constituents of its cell wall (example: lipopolysaccharides), and
  • provides an initial protection to noxious agents present in its
    natural environment that have penetrated into the cell (example: bile
    salts in the colon) [1].

The efflux pump machinery is divided into five superfamily classes;

  • the major facilitator (MF),
  • the ATP-binding cassette (ABC),
  • the resistance-nodulation-division (RND),
  • the small multi-drug resistance (SMR) and
  • the multi-drug and toxic compound extrusion (MATE).

With respect to Gram-negative bacteria, although they all play
important roles in the protection of the bacterium from noxious
agents present in the environment, the

  • main efflux pump of the Gram negative bacterium is a
    member of the RND superfamily, and
  • because multi-drug resistance of clinical isolates have
    been associated with the over-expression of this pump,

it has received a great deal of attention [2].

The first in vitro response of bacteria to a given noxious agent,
such as an antibiotic, is to over-express its main efflux pump [2].
If the bacterium is serially exposed in vitro to increasing
concentrations of that compound, it responds by increasing
the effective number of its main efflux pump, as well as others
that provide redundant protection [2].

However, if that “adapted” bacterium is now maintained at a
constant level of a noxious agent, the level of efflux pump
activity increases up to a maximum, followed by a gradual
return of efflux pump activity to its basal level. Concomitant
to this process, an accumulation of mutations of essential
proteins located in the plasma membrane (example penicillin
binding proteins), mutations 30 S component of the ribosome
and gyrase take place [3]. These events suggest that when
the organism is faced with an environment that contains a
constant toxic level of a compound, and the cost for
maintaining an energy consuming system, such as that
needed for the energy dependent efflux pump, is too
great a price to pay.

Therefore, in order to survive in this unchanging environment,
other mechanisms are activated. For example, activation of a
mutator master gene is thought to be an important step at this
level, which results in the mutation of genes that code for
essential proteins, reversing the over-expression of efflux-
pumps, but still conferring the bacterial resistant to the
environmental pressure via other mechanism(s), yet
to be understood [4,5].

During therapy, the level of resistance increases many fold
higher than that of the initial infecting strain. Hence, clinical
isolates from treated patients often show much higher levels
of antibiotic resistance than that of their wild type counterpart
(sometimes it can even present a 1000 fold increase) [6].
At this stage, resistance is usually related to the presence
of mutations, which reduces the survival of the resistant
bacteria,

  • once it is transferred to a noxious agent-free environment

that contains the competing wild type counterpart [3,4].

Depending upon when during therapy a clinical strain is isolated,
its resistance to two or more antibiotic classes (multi-drug
resistance (MDR)), may be due entirely to over-expressed
efflux pumps; to a mixture of over-expressed efflux pumps
and increasing accumulation of mutations; and only to mutations [3,4].

The degree of resistance can readily be determined with
methods that employ compounds known for their modulation
of efflux pump activity, such as

  • phenothiazines [7] or phenyl-arginine-betanaphthylamide
    (PAβN),
  • the latter which competes with the antibiotic as
    substrate of the efflux pump [8].

If in presence of such compounds,

  • the MDR bacterium is rendered fully susceptible
    to the antibiotic(s) to which it was initially resistant,
  • resistance is most likely due to its overexpressed
    efflux pump systems.
  • Contributions made by accumulated mutations
    render the organism less and less affected by the EPI.

This type of information is of great value to clinicians faced
with long-term therapy of a bacterial infection that
progresses to an MDR phenotype. It should be understood
that although the Gram-negative bacterium has essentially
one main efflux pump, such as

  • the AcrAB (Escherichia coli) or
  • the MexAB (Pseudomonas aeruginosa),

the deletion of the main efflux pump results in the over-
expression of one or more other RND efflux pumps,
such as is the case for deletion of the AcrAB, followed by

  • the over-expression of the AcrEF pump [2].

Redundancy of as many as nine RND efflux pumps [2],
provides additional protection to the organism.

The pumps belonging to the RND family form

  • a tripartite complex together with
  • the periplasmic proteins belonging to the
    membrane fusion-protein (MFP) family and
  • the outer membrane channels.

RND transporters consist of

  • a transporter protein that recognises and
    binds the noxious agent
    in the cytoplasm or periplasm and
  • transports it to the contiguous channel (TolC),
  • ending at the surface of the outer membrane.

The transporter is attached to the plasma membrane
by two or three fusion proteins, which are believed to assist the

  • extrusion of the substrate by peristaltic actions [9].

Although the actual structure of RND efflux pumps
in the cell envelop is not completely understood,

  • the structure of the transporter, TolC and fusion
    proteins are well established for major Gram-negative
    bacteria [10].

The PMF energy dependent efflux pump most likely needs the
passage of hydronium ions through its internal cavity,

  • for the release of the substrate that is
  • in turn ejected into the TolC channel via the
  • peristaltic action of the fusion proteins [11].

A low pH,

  • the concentration of hydronium ions at the surface of the cell
  • results in a pH difference of 2 or 3 pH units compared
    to that of the milieu,

the surface concentration of hydronium ions

  • provides the force for the mobility of hydronium ions
  • through porins leading to the acidification of the periplasm,
  • providing the low pH needed by the transporter
  • for the release of the substrate.

At high pH, these hydronium ions come from

  • hydrolysis of ATP by ATP synthase, and
  • are passed into the transporter, thereby
  • reducing its internal pH, so that
  • the release of the substrates can take place [11,12].

EPIs, such as the phenothiazines chlorpromazine or thioridazine,

  • exert their inhibition at pH above 6, and
  • are thought to affect hydrolysis of ATP
  • denying the efflux pump transporter hydronium ions needed

for release of the bound substrate [11,12].

The search for EPIs that are clinically useful continues, although

with respect to thioridazine, this old neuroleptic has been shown

  • to inhibit efflux pumps of pathogenic mycobacteria [13], and
  • has been successfully used to treat extensively drug resistant
    tuberculosis infections [14].

The regulation of the main efflux pump of Escherichia coli may
take place via   distinct pathways. The induced synthesis of the
transporter component of the AcrAB efflux pump, when the
organism is exposed in vitro to a noxious agent,

  1. involves the activation of the stress gene soxS,
  2. followed by the activation of the local regulator marA,
  3. then by the activation of the transporter gene acrB [8].

In the case of Salmonella spp. two component resistance
mechanisms, such as the PmrA/PmrB system, directly
activate the master efflux pump regulator ram A gene [15].
The activation of the PmrA/PmrB system takes place
readily when Salmonella spp. is phagocytosed due to
the acidic nature of the phagolysosome [15], as follows:

  1. PmrB is a sensor that self-phosphorylates, and
  2. then transfers the phosphate to PmrA.
  3. PmrA activates a nine gene operon, which
  4. codes for Lipid A introduced into the nascent
    lipopolysaccharide layer of the outer membrane.
  5. The increased presence of Lipid A renders the
    phagocytosed bacterium practically immune to
    everything, including the hydrolases of the
    phagolysososome [15].

Although some EPIs are in clinical trials, none have yet to
reach the marketplace,    mainly due to their common
toxicity against healthy mammalian cells, affecting
intrinsic mammalian efflux pumps, as for example
those of the blood brain barrier. Lastly, it should be
noted that compounds that inhibit the efflux pump
of bacteria also have the capacity to promote the
removal of plasmids that carry antibiotic resistant
genes [16,17].

  1. Nikaido H, Pages JM (2012) Broad-specificity efflux
    pumps and their role in multidrug resistance of Gram-
    negative bacteria. FEMSMicrobiol Rev 36: 340-363.
  2. Viveiros M, Jesus A, Brito M, Leandro C, Martins M,
    et al. (2005) Inducement and reversal of tetracycline
    resistance in Escherichia coli K-12 and expression of
    proton gradient-dependent multidrug efflux pump
    genes. Antimicrob Agents Chemother 49: 3578-3582.
  3. Martins A, Couto I, Aagaard L, Martins M, Viveiros M
    (2007) Prolonged exposure of methicillin-resistant
    Staphylococcus aureus (MRSA) COL strain to
    increasing concentrations of oxacillin results in a
    multidrug-resistant phenotype. Int J Antimicrob
    Agent 29: 302-305.
  4. Martins A, Spengler G, Molnar J, Amaral L (2012)
    Sequential responses of bacteria to noxious agents
    (antibiotics) leading to accumulation of mutations
    and permanent resistance. Biochem Pharmacol J
    Open Access 1: 7.

Inhibitors of efflux pumps of Gram-negative
bacteria inhibit Quorum Sensing

Leonard Amaral, Joseph Molnar
1 Grupo de Micobacterias, Unidade de Microbacterilogia,
Centro de Malaria e Doenças Tropicais (CMDT), Instituto de
Higiene e Medicina Tropical, Universidade Nova de Lisboa,
Lisbon, Portugal; 2 Cost Action BM0701 (ATENS) of the
European Commission/European Science Foundation;
3 Department of Medical Microbiology and Immunobiology,
University of Szeged, Szeged, Hungary
Open Journal of Pharmacology, 2012, 2-2

Quorum Sensing (QS) systems of bacteria consist of

  • a producer of the QS signal and the responder.

The generation of a QS signal provides the means by which
a population can behave in a concerted manner such as

  • swarming, swimming and secretion of biofilm, etc.

Because concerted bahaviour bestows protection to the bacterial
species, and hence factors involved in the severity of an infection
such as virulence are products of QS systems, compounds that
inhibit the QS system have significant clinical relevance. Recent
evidence suggests that

  • the secretion of QS signals takes place via
  • the efflux pump system of the producer of the signal.

Interestingly, compounds such as phenothiazines and
trifluoromethyl ketones (TFs)

  • that inhibit proton motive force (PMF) activities such
    as swarming and swimming also
  • inhibit the PMF dependent efflux pump systems of
    bacteria and their QS   systems.

This review discusses the relationship between the efflux
pump, the QS system and the compounds that affect both.
Lastly, suggestions are made regarding classes of compounds
that have been shown

  • to inhibit PMF dependent efflux pumps and the need
  • to evaluate them for QS inhibitory properties.

Keywords: Quorum Sensing, QS signal, acylated hydroxyl
lactone (AHL), efflux pumps, Proton Motive Force (PMF),
inhibitors of efflux pumps, inhibitors of QS systems,
phenothiazines, Trifluormethyl Ketones (TFs), plants
sources for QS inhibitors

Efflux pumps of bacteria provide protection from noxious
agents that are present in the environment in which they
exist. Noxious agents may be naturally occurring compounds
present in environments outside and within the human.

Because over-expressed efflux pumps render antibiotic
therapy problematic, an intense search for agents that
inhibit specific efflux pumps of specific bacteria has
been conducted during the past decade [9].

Communication between bacteria of the same strain
or species and between species contributes to their
survival [11-13]. Communication involves the secretion
of signals that invoke a specific response from the responder
[11-13]. This  communication process is termed Quorum
sensing (QS). When it takes place between strains of the
same species,

  • communication is directed towards the reduction
    of population growth and
  • reducing the possibility of exceeding the nutritional
    support of the environment

Other signals may involve a population response that involves

  • the secretion of bioactive molecules that inhibit the
    replication of a competing population species [14-16]
    or even kill [biocidins) [17-21] or
  • promote a swarming effect that recruits members
    of the same species to migrate  to a specific location [22-24]
    similar to swarming by insects subsequent to signals
    indicating site of food [example bees).
  • biofilm, encase the bacteria at distances from each other
    [25-29] and within the matrix of this biofilm are
    channels used for further communication [30].

Biofilms are produced in the wild, at sites such as surfaces
of rocks which maintain the bacterial population in situ [31]
and are also produced at sites of the human colonized by
infecting bacteria [32, 33].

Agents that inhibit the QS response of the infecting bacterium
are obviously important and hence, the search for such agents
that inhibit the QS system and biofilm formation has been in
effect for the past two decades [11-13].

There is a relationship between efflux pumps (EP), QS and
biofilm (BF) secretion which has come to the forefront only
recently [13]. Control of this relationship is critical for
successful therapy of MDR bacterial infections which have
become rather commonplace. It is the intent of this review
to identify agents which may serve to interfere with the
complex system of EP-QS-BF interaction.

Proton motive force (PMF) dependent transporters obtain
their energy for function from the proton motive force. The
proton motive force is the result of cellular metabolism which
yields protons that are not used for coupling with molecular
oxygen and which are exported to the surface of the cell [43-45]
where they are distributed and bound to components of
the protective lipopolysaccharide layer that covers the cell
and constitutes a part of the outer cell wall of Gram-negative
[46] and the cell wall of Gram positive bacteria [47].

The larger the concentration of protons (hydronium ions)
on the surface of the cell with respect to their lower
concentration on the medial side of the cytoplasmic
membrane creates an electrochemical gradient that
is termed the proton motive force (PMF) [48].

Because hydronium ions cannot penetrate the cell wall
or the membrane, they may re-enter the cell only
through channels such as porins in general [49, 50].
The movement of these hydromium ions from the
surface of the cell to the periplasm or cytoplasm is
predicated upon systems that use the PMF as source
of energy-namely the resistance nodulation division
(RND) family of transporters.

E. coli has a multiplicity of efflux pumps that may
exceed 30 in number [51]. However, the main
efflux pump of this organism is the AcrAB-TolC
efflux pump [52, 53] which when deleted, its
function is replaced by the AcrEF-TolC efflux
pump [51]. Both efflux pumps are members
of the resistance nodulation division family of
transporters [51] and consist of three proteins:

  1. The transporter AcrB coded by the gene acrB and
    is intimately attached to the  plasma membrane;
  2. Two fusion proteins AcrA coded by the gene acrA
    that flank the AcrB transporter and are thought
    to assist the movement of a substrate through
    the AcrB transporter [35]; and,
  3. TolC which is also part of other tri-unit efflux pumps
    of the organism [35], is contiguous with the AcrB
    transporter and provides a conduit for the extrusion
    of the substrate [38].

Although the means for the recognition of the substrate to
be extruded appears to involve a pocket within the transporter,
it appears to be

  • defined by a phenyalanine residue [54].

Nevertheless, studies employing fluorochromes recognised by
the AcrB transporter indicate that the binding and release of
the substrate are pH dependent [55].

  • At low pH the dissociation of the substrate is high and
  • at high pH it is very slow.

In a physiological environment of ca. pH 7, if the dissociation
of the substrate is slow or not at all, then the effectiveness of
the pump to extrude a noxious agent would be nullified.
However, since the pump functions at this pH, conditions that
result in the dissociation of the substrate needed for continuous
pump action must involve a

  • decrease of the pH of the internal cavity of the pump
    to which the substrate is bound.

It has been postulated that the lowering of the pH takes place
by the generation of hydronium ions from metabolism [6] which

  • pass from the cytoplasmic side of the plasma membrane
    through the transporter.

At lower pH, there is no need for the generation of metabolically
derived  hydronium ions since these ions can be

  • diverted by the PMF from the surface of the cell
    to the periplasm via porins.

Whether hydronium ions are to be generated from the
hydrolysis of ATP at high pH or used for the synthesis
of ATP at low pH is a special

  • function of ATP synthase [56-58].

Model of the AcrAB-TolC efflux pump of a Gram-
negative bacterium

AcrAB-TolC efflux pump of a Gram-negative bacterium

AcrAB-TolC efflux pump of a Gram-negative bacterium

Hypothesis. At near neutral pH, Hydronium ions from hydrolysis of ATP
by ATP synthase pass through the AcrB

transporter, reduce the pH to a point that causes the release of the
substrate. When the hydronium ions reach the surface of the cell they
are distributed over that surface and bind to lipopolysaccharides
and basic amino acids. When there is a need for hydronium ions for
activity of the efflux pump and the pH is lower than neutral, and
the hydrolysis of ATP is not favoured, hydronium ions from the
surface of cell via the PMF mobilize through the Aqua porins
and reach the transporter where they are pushed through
the transporter by the peristaltic action caused by the fusion
proteins. Substrates bound to the transporter dissociate
when the pH is reduced by the flow of hydronium ions and
are carried out by the flow of water.

Inhibitors of bacterial efflux pumps
Inhibitors of the QS of bacteria

Because phenothiazines inhibit many energy dependent systems
of bacteria such as motility [89, 90, 95], and these phenothiazines
also inhibit efflux pumps of bacteria [6, 7, 9, 41, 51, 73, 74, 76-83],
there seems to be a correlation between an active efflux pump
system and a functional QS system. That this assumption is correct,
recent evidence has been provided showing that the efflux pumps of
the AHL responding environmental Chromobacterium violaceum
(CV026) bacterium and that of E. coli are inhibited by the phenothiazine
thioridazine (TZ) [12]. Because TZ is known to inhibit genes that
regulate and code for efflux pumps of bacteria [41, 119, 120], it is
possible that the inhibition of the responding CV0126 bacterium to
AHLs [12] involves the inhibition of genes that code and regulate
the efflux pump of the responder which is assumed to recognise the
AHL signal as an noxious agent and hence would extrude it to the
environment [12]. The inhibition of an efflux pump should manifest
itself as an inhibitor of the QS component responsible for biofilm
formation.

Since the discovery of berberine a powerful inhibitor of bacterial
efflux pumps [159], plants have become sources of inhibitors of
efflux pumps [160-164]. Given that efflux pumps and the  QS of
bacteria have an intimate relationship as described in this review,
attention has been focused on plants for potential sources of inhibitors
of efflux pumps and QS systems. Essential oils from Columbian
plants have yielded a large number of compounds that inhibit the
QS system of responding bacteria such as

  1. limonene-carvone , the
  2. citral (geranial-neral) (isolated from Lippia alba),
  3. α-pinene (from Ocotea sp.),
  4. β-pinene (from Swinglea glutinosa),
  5. cineol (from Elettaria cardamomun),
  6. α-zingiberene (from Zingiber officinale) and
  7.  pulegone (from Minthostachys mollis) [165].

Several other essential oils, in particular were shown to present
promising inhibitory properties for the short chain AHL quorum
sensing (QS) system in Escherichia coli containing the biosensor

  •  plasmid pJBA132, in  particular Lippia alba.

Citral was the only  essential oil that presented some activity for
the long chain AHL QS system in Pseudomonas putida containing

  •  the plasmid pRK-C12 [165].

The essence of this review is to correlate the relationship of the
efflux pump system to the QS system of bacteria via the use of
compounds that inhibit both systems. Simply put, inhibitors of
the efflux pump system also, when studied, inhibit the QS system
as well. Because the PMF dependent efflux pump system of Gram-
negatives that is overexpressed is responsible for the multi-drug
phenotype of the bacterium, compounds that affect the PMF of
the bacterium are candidates that will inhibit the activity of the
pump. Consequently, this inhibition will inhibit the secretion of
biofilm, and because biofilm is a deterrent to the action of antibiotics,
compounds that affect the efflux pump system are promising
candidates for clinical evaluation.

Limiting and controlling carbapenem-resistant
Klebsiella pneumonia

L Saidel-Odes, A Borer.
1Infection Control and Hospital Epidemiology Unit, 2Infectious
Diseases Institute, Soroka University Medical Center and the
Faculty of Health Sciences, Ben-Gurion University of the Negev,
Beer-Sheva, Israel
Infection and Drug Resistance 2014:7 9–14

Carbapenem-resistant Klebsiella pneumoniae (CRKP)

  • is resistant to almost all antimicrobial agents,
  • is associated with substantial morbidity and mortality, and
  • poses a serious threat to public health.

The ongoing worldwide spread of this pathogen emphasizes the
need for immediate intervention. This article reviews the global
spread and risk factors for CRKP colonization/infection, and
provides an overview of the strategy to combat CRKP dissemination

Outbreaks of CRKP that have occurred around the world have
been associated with the plasmid-encoded carbapenemase
K. pneumoniae carbapenemase (KPC),

  • a carbapenem-hydrolyzing β-lactamase.19

CRKP isolates are resistant to almost all available antimicrobials
and are susceptible

  • only to polymyxins and tigecycline;
  • a minority to the few remaining aminoglycosides,
    though resistance to these agents is increasingly reported.20,21

Several investigators have evaluated predictors for CRKP colonization.
The following summarizes various studies.

  1. In a multivariate analysis, prior use of macrolides and
    any antibiotic exposure $14 days remained the only
    independent factors associated with CRKP bacteremia
  2. Nosocomial isolation of CRKP was strongly favored by the
    selection pressure of carbapenem. In this study, prior
    treatment with fluoroquinolones was associated with
    decreased risk for the emergence of CRKP.
  3. Previous use of carbapenem and cephalosporin
  4. Nursing home residency before hospital admission, bedridden
    status, and previous antibiotic therapy
  5. exposure to fluoroquinolones
  6. the recipient of antibiotics
  7. intensive care unit (ICU) stay, and
  8. Poor functional status,
  9. Independent predictors of subsequent carbapenem-
    resistant Enterobacteriaceae (CRE) infection were
  • admission to the ICU,
  • having a central venous  catheter,
  • receipt of antibiotics, and
  • diabetes mellitus

Schwaber et al and the Israeli CRE Working Group enforced the
Israel Ministry of Health guidelines mandating physical separation
of hospitalized carriers of CRE and dedicated staffing and appointed
a professional task force charged with containment.19 The monthly
incidence of nosocomial CRE was reduced from 55.5 to 11.7 cases
per 100,000 patient days within 15 months.

Part 7.  Tuberculosis

The Mechanism by which the Phenothiazine Thioridazine
Contributes to Cure Problematic Drug-Resistant Forms
of Pulmonary Tuberculosis: Recent Patents for “New Use”

L Amaral1*, A Martins2,3, G Spengler2, A Hunyadi4 and J Molnar2
Recent Patents on Anti-Infective Drug Discovery 2013; 8(3):000-000

At this moment, over half million patients suffer from multi-drug
resistant tuberculosis (MDR-TB) according to the data from the WHO.
A large majority is terminally ill with essentially incurable pulmonary
tuberculosis. This herein mini-review provides the experimental and
observational evidence that a specific phenothiazine,

  • thioridazine,

will contribute to cure any form of drug-resistant tuberculosis. This
antipsychotic agent is no longer under patent  protection for its
initial use. The reader is informed on the recent developments

  • in patenting this compound for “new use” with a special
  • emphasis on the aspects of drug-resistance.

Given that economic motivation can stimulate the use of this drug
as an antitubercular agent, future prospects are also discussed.

Thioridazine is not the only phenothiazine that has been recommended
for therapy of pulmonary tuberculosis. In general, many phenothiazines
have been implicated for antitubercular activity [62, 80-86]. Among
these are

  • trifluoperazine [87-94],
  • methdilazine [95, 96],
  • promazine [97, 98],
  • promethazine [97, 98],
  • fluphenazin [99],
  • propiomazine [100], and
  • the methylene blue related toluidine blue [101].

There are phenothiazine compounds derived from the parental
methylene blue for therapy of pathologies unrelated to tuberculosis
that also possess

  •  antitubercular [44, 48] and/or antimalarial properties [44].

Moreover, derivatives made from any of the phenothiazines that
have in vitro activity against Mycobacterium tuberculosis are also
active [61, 67, 102, 103], suggesting ample opportunities for
patenting of new analogs developed from known, active phenothiazines
with even less side effects than those of TZ, as recently suggested by
Musuka and co-authors [104]. It is important to mention, that the
commercially available phenothiazines such as for example

  •  trifluoperazine, methdilazine, promazine, promethazine,
    fluphenazin and propiomazine

are beyond patent protection as initially intended. Nevertheless,
these compounds have been patented as adjuvants for the treatment
of MDR cancer (patent expired in 2011 [105]; and, right afterwards,
a new patent has been filed with a priority date of 28th March, 2012,
claiming combination therapy of cancer with a chemotherapeutic
agent and a dopamine receptor antagonist against Cancer stem cells (CSC).

Taking into account that intrinsic MDR is considered as one of the key
properties of CSCs [107], the subject to be covered is indeed related.
According to the MDR, XDR and TDR Mycobacterium tuberculosis,
subjects of this herein paper, the initial step for actually reaching those
in need has been made: a patent has been published in December, 2007,
for the use of TZ and its derivatives for reversing anti-microbial drug
resistance [108]. We must note, however, that, despite the six years
passed since, we were unable to find any related clinical trials, which
would certainly be of outmost importance and urgency in order to
proceed towards an effective therapy of highly resistant mycobacterial
infections.

Mechanism Of Action Of Tz: Why It Cures Multi-Drug,
Extensively Drug Resistant And Probably Totally Drug
Resistant Tuberculosis

Over-expressed efflux pumps of Mycobacterium tuberculosis render
the organism multi-drug resistant [13]. Special attention has been
given to those coded by the

  • mmpL7, p55, efpA, mmr, Rv1258c and Rv2459 genes [109].

The activity of these efflux pumps can be suppressed by

  • concentrations of TZ that have no effect on the viability of
    Mycobacterium tuberculosis
  • rendering the organism susceptible to the antibiotic to
    which it was initially resistant
  • as a consequence of the over-expression of its
    efflux pumps [109].

TZ has also been shown to inhibit the activity of the main

  • efflux pumps of bacteria belonging to other species.

TZ has strong inhibitory activity against the genes that code for
essential proteins of M. tuberculosis [122-124].  Consequently, we
may conclude that the in vitro activity of TZ involves

  • the inhibition of the efflux pumps of M. tuberculosis and that
  • the in vitro exposure of this organism to TZ renders the organism
  • susceptible to antibiotics to which it was initially resistant
  • as a consequence of over-expressed efflux pumps [21].

Phenothiazines such as CPZ, TZ, trifluoperazine, etc., also inhibit

  • the binding of calcium to calcium binding proteins such as

calmodulin in eukaryotes [125], and

  • interfere with other proteins involved in
  • the regulation of cellular activity [126].

They inhibit the transport of calcium and potassium systems

  • in eukaryotic cells [127-129] as well as in
  • mycobacteria [89, 130] and
  • E. coli [113].

In fact, in the latter case, calcium was shown essential to

  • the continuous activity of the thioridiazine sensitive
    efflux system [113].

The killing activity of the human macrophage as well as that
of the neutrophil

  • is dependent upon the retention of calcium and potassium
  • within the phagolysosome of the cell [131].

Considering this, several alternative choices are available for
patenting under “new use”, which would allow a “fresh start”
for the compound to be developed. However, the needed
experimental proof that these phenothiazine agents have
activity at the pulmonary macrophage of the alveolar unit
(the site where the causative organism of pulmonary tuberculosis
resides) is still absent.

Targeting the Human Macrophage with Combinations
of Drugs and Inhibitors of Ca2+ and K+ Transport to
Enhance the Killing of Intracellular Multi-Drug Resistant
M. tuberculosis (MDR-TB) – a Novel, Patentable Approach
to Limit the Emergence of XDR-TB

Marta Martins
UCD Centre for Food Safety, School of Agriculture, Food Science and
Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
& Unit of Mycobacteriology and UPMM; Instituto de Higiene e Medicina
Tropical, Universidade Nova de Lisboa (IHMT/UNL),  Lisbon, Portugal
Recent Patents on Anti-Infective Drug Discovery, 2011, 6, 000-000

The emergence of resistance in Tuberculosis has become a serious
problem for the control of this disease. For that reason, new therapeutic
strategies that can be implemented in the clinical setting are urgently
needed. The design of new compounds active against mycobacteria
must take into account that Tuberculosis is mainly an intracellular
infection of the alveolar macrophage and therefore must maintain
activity within the host cells.

An alternative therapeutic approach will be described in this review,
focusing on the activation of the phagocytic cell and the subsequent
killing of the internalized bacteria. This approach explores the combined
use of antibiotics and phenothiazines, or Ca2+ and K+ flux inhibitors,
in the infected macrophage.

Targeting the infected macrophage and not the internalized bacteria
could overcome the problem of bacterial multi-drug resistance. This
will potentially eliminate the appearance of new multi-drug resistant
tuberculosis (MDR-TB) cases and subsequently prevent the emergence
of extensively-drug resistant tuberculosis (XDR-TB).

Patents resulting from this novel and innovative approach could be
extremely valuable if they can be implemented in the clinical setting.
Other patents will also be discussed such as the treatment of TB
using immunomodulator compounds (for example: betaglycans).

Role of Phenothiazines and Structurally Similar
Compounds of Plant Origin in the Fight against
Infections by Drug Resistant Bacteria


SG. Dastidar 1, JE. Kristiansen 2, J Molnar 3 and L Amaral
Antibiotics 2013; 2: 58-71;
http://dx.doi.org:/10.3390/antibiotics2010058

Phenothiazines have their primary effects on the plasma membranes
of prokaryotes and eukaryotes. Among the components of the
prokaryotic plasma membrane affected are

  • efflux pumps,
  • their energy sources
  • and energy providing enzymes, such as ATPase,
  • and genes that regulate and code for the permeability
    aspect of a bacterium.

The response of multidrug and extensively drug resistant
tuberculosis to phenothiazines shows an alternative therapy for the
treatment of these dreaded diseases, which are claiming more and
more lives every year throughout the world.

Many phenothiazines have shown

  • synergistic activity with several antibiotics thereby
  • lowering the doses of antibiotics administered to patients
    suffering from specific bacterial infections.

Trimeprazine is synergistic with trimethoprim. Flupenthixol (Fp)
has been found to be synergistic with penicillin and chlorpromazine
(CPZ); in addition, some antibiotics are also synergistic. Along with
the antibacterial action described in this review,

  • many phenothiazines possess plasmid curing activities, which
  • render the bacterial carrier of the plasmid sensitive to antibiotics.

Thus, simultaneous applications of a phenothiazine like TZ would not
only act as an additional antibacterial agent but also would help

  • to eliminate drug resistant plasmid from
    the infectious bacterial cells.

Part 8.  Cancer Cytotherapy

Synthesis and Structure-Activity Relationships of Novel
Dioxolanes as MDR Modulators in Cancer

A Martins 1,2,†,*, J Csábi 3,†, A Balázs 4, DKitka 1, L Amaral 5,
J Molnár 1, A Simon 6, G Tóth 6 and A Hunyadi 3,
Molecules 2013, 18, 15255-15275;
http://dx.doi.org:/10.3390/molecules181215255

Ecdysteroids, molting hormones of insects, can exert several mild,
non-hormonal bioactivities in mammals, including humans. In a
previous study, we have found a significant effect of certain derivatives

  • on the ABCB1 transporter mediated multi-drug resistance of a
  • transfected murine leukemia cell line.

In this paper, we present a structure-activity relationship study
focused on

  • the apolar dioxolane derivatives of 20-hydroxyecdysone.

Semi-synthesis and bioactivity of a total of 32 ecdysteroids, including
20 new compounds, is presented, supplemented with their

  • complete 1H- and 13C-NMR signal assignment

As published before [9], the 20,22-diol moiety of 20E is more reactive
than the 2,3-diol, probably due to the free rotation of the 20,22-bond
of 20E that allows the 20,22-dioxolane ring to form with less strain.

This allowed us to selectively obtain the 20,22-mono-dioxolane
derivatives 2–14, or, depending on the amount of reagent and the
reaction time, the 2,3;20,22-bis-homo-dioxolanes 17 and 21–25.

By utilizing the 20,22-monodioxolane ecdysteroids, another aldehyde
or ketone could be coupled to position 2,3, resulting in several bis-hetero-
dioxolane derivatives 26–33. For this, however, gradually decreasing
reactivity with the increase of the size of the reagent was a limiting factor:

  • larger aldehydes or ketones (mainly those containing a
    substituted aromatic ring) could not be coupled at the 2,3-position.
  • The 2,3-monodioxolane derivatives also appeared to be present as
    minor side-products of the reactions, and as a consequence of their
    low amount, only one such compound (compound 15) was isolated and studied.

To selectively obtain this kind of a compound (16) in a more reasonable
yield, another, three-step approach was successfully applied:

  • after protecting the 20,22-diol with phenylboronic acid, the
    2,3-acetonide could be prepared, and
  • removal of the 20,22 protecting group afforded the desired
    2,3-monoacetonide in a one-pot procedure.

In the case of the reactions with aldehydes or asymmetric ketones,
the new C-28 and C-29 central atoms of the dioxolane rings are
stereogenic centers and thus two possible diastereomers can be
formed at both diols. Their configuration was elucidated by two-
dimensional ROESY or selective one-dimensional ROESY experiments,
e.g., in the doubly substituted

  • dioxolane derivative 22 (R1 = R4 = n-Bu, R2 = R3 = H)
  • the unambiguous differentiation of the 1H and 13C signals of
    the two n-butyl groups was achieved in the following way
    (see Figure 2).

Assignment of the H-C(28) atoms (δ = 4.93/105.9 ppm) was supported by

  • the H-2/C-28 and H-3/C-28 HMBC correlations, and
  • that of H-C(29) (δ = 4.91/105.6 ppm) by the H-22/C-29
    cross peak, respectively.

The selective ROESY experiment irradiating at 4.93 ppm showed

  • contacts with the Hα-2 and Hα-3 atoms proving the
    α position of the R2 = H atom.

The ROESY response obtained irradiating H = R3 signal (δ = 4.91)
on H-22 (δ = 3.64 ppm) revealed their

  • cis arrangement and the R configuration around C-29.

The unambiguous assignments of the signals

  • of the two n-butyl groups R1 and R4 were achieved by
  • selective TOCSY experiments (irradiation at
  • δ = 4.93 and 4.91, respectively).

Figure 2

Stereostructure of 22. Red-ROESY proximitries. Blue- 1H. Black-1 001

Stereostructure of 22. Red-ROESY proximitries. Blue- 1H. Black-1 001

Stereostructure of 22. Red arrows indicate the detected ROESY
steric proximities, the blue numbers give the characteristic 1H,
and the black numbers the 13C chemical shifts.

 

Related Material

Identification of Efflux Pump-mediated Multidrug-resistant
Bacteria by the Ethidium Bromide-agar Cartwheel Method

M Martins, M Viveiros, I Couto, SS. Costa, T Pacheco, S Fanning,
Jean-Marie Pagès, and L Amaral
in vivo 2011; 25: 171-178  

Index for efflux activity of the MDR strains. The capacity to efflux EtBr
of each bacterial strain was ranked relative to the reference strain
according to the following formula:

 

Index for efflux activity of the MDR strains

Index for efflux activity of the MDR strains

A Simple Method for Assessment of MDR Bacteria for
Over-Expressed Efflux Pumps

M Martinsa,b*, MP. McCuskera,b, M Viveirosa,c, I Coutoc,d,
S Fanninga,b, Jean-Marie Pagès b,e, L Amaral,b,
The Open Microbiol J 2013; 7: 1-5  1874-2858/13 Bentham

Flowchart followed to test bacterial strains using the EtBr-agar
Cartwheel method.

Flowchart followed to test bacterial strains using the EtBr-agar Cartwheel method.

Flowchart followed to test bacterial strains using the EtBr-agar Cartwheel method.

EtBr-agar cartwheel method applied to different bacterial species

EtBr-agar cartwheel method applied to different bacterial species

EtBr-agar cartwheel method applied to different bacterial species

The effect of selected EPIs on the resistance of the induced and
MDR Gram-positive bacteria.

TET
Enterococcus EFC
ATCC29212
HSEFM-D
1.5
>2.5
w/o
EPI
+
TZ
+
CPZ
+
RES
4
16
4
4
4
4
4
8
(4×) (4×) (2×)
                                MCEtBr NOR  (mg/l) MIC NOR (mg/l)
HSEFM-E >2.5 0.125 0.125 0.125 0.125

EPI: Efflux pump inhibitor; w/o: without; TZ: thioridazine; CPZ:
chlorpromazine; PAN: phenyl arginine β-naphthylamide. Values
in bold-type correspond to a decrease of 4-fold or higher on
the MIC values in comparison to those in the absence of inhibitor.
Values in parenthesis indicate the MIC decrease relative to that
of the original culture. The concentration of each EPI used is
defined in the Materials and Methods section.

Macrocyclic diterpenes resensitizing multidrug
resistant phenotypes 

MA. Reis a, A Paterna a, RJ. Ferreira a, H Lage b,
Maria-José U. Ferreira a,⇑
a Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade
de Farmácia, Universidade de Lisboa, Lisboa, Portugal
b Charité Campus Mitte, Institute of Pathology, Berlin, Germany
Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx

Herein, collateral sensitivity effect was exploited as a strategy to
select effective compounds to overcome multidrug resistance in
cancer. Thus, eleven macrocyclic diterpenes, namely jolkinol D (1),
isolated from Euphorbia piscatoria, and its derivatives (2–11) were
evaluated for their activity on three different Human cancer entities:

  • gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29)

each with a variant selected for resistance to mitoxantrone

  1. EPG85-257RN;
  2. EPP85-181RN;
  3. HT-29RN and
  • one to daunorubicin (EPG85-257RD; EPP85-181RD; HT-29RD).

Jolkinol D (1) and most of its derivatives (2–11) exhibited significant
collateral sensitivity effect towards the cell lines

  • EPG85-257RN (associated with P-glycoprotein overexpression) and
  • HT-29RD (altered topoisomerase II expression).

The benzoyl derivative, jolkinoate L (8) demonstrated ability to

  • target different cellular contexts with
  • concomitant high antiproliferative activity.

These compounds were previously assessed as
P-glycoprotein modulators,

  • at non-cytotoxic doses, on MDR1-mouse lymphoma cells.

A regression analysis between

  1. the antiproliferative activity presented herein and
  2. the previously assessed P-glycoprotein modulatory effect

showed a strong relation between the compounds that presented

  • both high P-glycoprotein modulation and cytotoxicity.

Molecular Docking Characterizes Substrate-Binding Sites
and Efflux Modulation Mechanisms within P
Glycoprotein.

Ferreira,† Maria-José U. Ferreira,† and DJVA dos Santos*,†,‡
†Research Institute for Medicines and Pharmaceutical Sciences
(iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
‡REQUIMTE, Department of Chemistry & Biochemistry, Faculty of
Sciences, University of Porto, Porto, Portugal
J. Chem. Inf. Model. XXXX, XXX, XXX−XXX
http://dx.doi.org:/10.1021/ci400195v

P-Glycoprotein (Pgp) is one of the best characterized ABC
transporters
, often involved

  • in the multidrug-resistance phenotype
  • overexpressed by several cancer cell lines.

Experimental studies contributed to important knowledge concerning
substrate polyspecificity, efflux mechanism, and drug binding sites.
This information is, however, scattered through different perspectives,
not existing a unifying model for the knowledge available for this transporter.
Using a previously refined structure of murine Pgp,

  • three putative drug-binding sites were hereby characterized
  • by means of molecular docking.

The modulator site (M-site) is characterized by

  • cross interactions between both Pgp halves

herein defined for the first time, having an important role in

  • impairing conformational changes leading to substrate efflux.

Two other binding sites, located next to the inner leaflet of the lipid bilayer,

  • were identified as the substrate binding H and R sites
  • by matching docking and experimental results.

A new classification model

  • with the ability to discriminate substrates from modulators

is also proposed, integrating a vast number of theoretical and experimental data.

conformational changes leading to substrate efflux

conformational changes leading to substrate efflux

conformational changes leading to substrate efflux

http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jcisd8/
2013/jcisd8.2013.53.issue-7/ci400195v/production/pdfimages_v02/normal.img-000.jpg

 

 

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