Lactoferrin interacts with bile acids and increases fecal cholesterol excretion in rats

Lactoferrin (LF) is a multifunctional cationic protein (pI 8.2 to 8.9) in mammalian milk. We previously reported that enteric-LF prevented hypercholesterolemia and atherosclerosis in a diet-induced atherosclerosis model using MicrominipigTM, although the underlying mechanisms remain unclear. Because LF is assumed to electrostatically interact with bile acids to inhibit intestinal cholesterol absorption, LF could promote cholesterol excretion. In this study, we assessed the interaction between LF and taurocholate in vitro and the effect of LF on cholesterol excretion in rats. The binding rate of taurocholate to LF was significantly higher than that to transferrin (pI 5.2 to 6.3). When rats were administered a high-cholesterol diet (HCD) containing 5% LF, LF was detected using ELISA in the upper small intestine from 7.5 to 60 min after the administration. Rats were fed one of the following diets: Control, HCD, or HCD + 5% LF for 21 days. Fecal neutral steroids and hepatic cholesterol levels in the HCD group were significantly higher than those in the Control group. The addition of LF to a HCD significantly increased fecal neutral steroids levels (22% increase, pThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Effects of Lutein and Astaxanthin Intake on the Improvement of Cognitive Functions among Healthy Adults: A Systematic Review of Randomized Controlled Trials

Background: Fruits and vegetables are generally rich in antioxidants such as carotenoids. Consumption of carotenoids is expected to have benefits on cognitive functions in humans. However, previous randomized controlled trials (RCT) using carotenoids have reported inconsistent results. Therefore, this systematic review (SR) aimed to summarize the effect of carotenoid intake on cognitive functions in humans. Method: PubMed, Cochrane Library, Web of Science, and PsychoINFO were searched for research papers on carotenoid intake with the criteria that 1) oral carotenoid intake was evaluated using RCTs, 2) participants were healthy young, middle-aged, or older, and 3) cognitive functions were measured using RCTs. Results: Five studies using lutein and two studies using astaxanthin met the inclusion criteria. Consumption of lutein and its isomer showed consistent results in selective improvement of visual episodic memory in young and middle-aged adults while inhibition was observed in middle-aged and older adults. One of the two included astaxanthin studies reported a significant improvement of verbal episodic memory performance in middle-aged adults. Conclusion: This SR showed that the 10 mg lutein per day for twelve months can lead to improvement of cognitive functions. Due to the small number of studies, it is difficult to conclude whether astaxanthin would have a positive effect on cognitive functions

Lactoferrin attenuates fatty acid-induced lipotoxicity via Akt signaling in hepatocarcinoma cells

Nonalcoholic fatty liver disease (NAFLD) describes a spectrum of lesions ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). The excess influx of fatty acids (FAs) into the liver is recognized as a main cause of simple steatosis formation and progression to NASH. Recently, administration of lactoferrin (LF), a glycoprotein present in milk, was suggested to prevent NAFLD development. However, the effect of LF on the contribution of FA to NAFLD development remains unclear. In this study, the effects of LF on FA mixture (FAm)-induced lipotoxicity using human hepatocarcinoma G2 cells were assessed. FAm significantly decreased cell viability and increased intracellular lipid accumulation, whereas LF significantly recovered cell viability without affecting lipid accumulation. FAm-induced lactic dehydrogenase (LDH) and caspase-3/7 activities were significantly decreased by LF and SP600125, a c-Jun N-terminal kinase (JNK) specific inhibitor. We also found that LF added to FAm-treated cells induced Akt phosphorylation which contributed to inhibition of JNK signaling pathway-dependent apoptosis. Akt inhibitor VIII, an allosteric Akt inhibitor, significantly attenuated the effect of LF on LDH activity and abrogated the ones on cell viability and caspase-3/7 activity. In summary, the present study has revealed that LF has a protective effect on FAm-induced lipotoxicity in a HepG2 model of NAFLD and identified the activation of the Akt signaling pathway as a possibly major mechanism.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Role of LRP1 and ERK and cAMP Signaling Pathways in Lactoferrin-Induced Lipolysis in Mature Rat Adipocytes

<div><p>Lactoferrin (LF) is a multifunctional glycoprotein present in milk. A clinical study showed that enteric-coated bovine LF tablets decrease visceral fat accumulation. Furthermore, animal studies revealed that ingested LF is partially delivered to mesenteric fat, and <i>in vitro</i> studies showed that LF promotes lipolysis in mature adipocytes. The aim of the present study was to determine the mechanism underlying the induction of lipolysis in mature adipocytes that is induced by LF. To address this question, we used proteomics techniques to analyze protein expression profiles. Mature adipocytes from primary cultures of rat mesenteric fat were collected at various times after exposure to LF. Proteomic analysis revealed that the expression levels of hormone-sensitive lipase (HSL), which catalyzes the rate-limiting step of lipolysis, were upregulated and that HSL was activated by protein kinase A within 15 min after the cells were treated with LF. We previously reported that LF increases the intracellular concentration of cyclic adenosine monophosphate (cAMP), suggesting that LF activates the cAMP signaling pathway. In this study, we show that the expression level and the activity of the components of the extracellular signal-regulated kinase (ERK) signaling pathway were upregulated. Moreover, LF increased the activity of the transcription factor cAMP response element binding protein (CREB), which acts downstream in the cAMP and ERK signaling pathways and regulates the expression levels of adenylyl cyclase and HSL. Moreover, silencing of the putative LF receptor low-density lipoprotein receptor-related protein 1 (LRP1) attenuated lipolysis in LF-treated adipocytes. These results suggest that LF promoted lipolysis in mature adipocytes by regulating the expression levels of proteins involved in lipolysis through controlling the activity of cAMP/ERK signaling pathways via LRP1.</p></div

Enteric lactoferrin attenuates the development of high-fat and high-cholesterol diet-induced hypercholesterolemia and atherosclerosis in Microminipigs

<div><p>Previously, we found that enteric lactoferrin (eLF) could reduce the visceral fat accumulation known to associate strongly with metabolic syndrome symptoms and consequently with an increased risk of atherosclerosis. In this study, the atherosclerosis-preventive potential of LF was assessed in a high-fat and high-cholesterol diet (HFCD)-induced hypercholesterolemia and atherosclerosis model using Microminipig™. Eight-week orally administered eLF remarkably reduced the HFCD-induced serum total and low-density lipoprotein cholesterol levels but not high-density lipoprotein cholesterol levels. A histological analysis of 15 arteries revealed that eLF systemically inhibited the development of atherosclerotic lesions. Pathway analysis using identified genes that characterized eLF administration in liver revealed significant changes in the steroid biosynthesis pathway (ssc00100) and all affected genes in this pathway were upregulated, suggesting that cholesterol synthesis inhibited by HFCD was recovered by eLF. In summary, eLF could potentially prevent the hypercholesterolemia and atherosclerosis through protecting homeostasis from HFCD-induced dysfunction of cholesterol metabolism.</p></div

Analysis of the effect of LF on CREB activation.

<p><b>(A)</b> Phosphorylation of CREB-Ser133 in adipocytes treated with LF. Phosphorylated CREB was detected in the presence or absence (0 min) of 1 mg/ml of LF. Phosphorylation levels normalized to the protein expression level of CREB. Changes in protein expression levels of <b>(B)</b> HSL and <b>(C)</b> AC isomers (AC1, 2, and 6) in the presence or absence (0 min) of 1 mg/ml LF normalized to the protein expression level of β-actin. The statistical significance of the data at each sampling time compared with the 0-min sample was evaluated using Dunnett’s multiple comparison test, and the data represent the mean ± SD values of triplicate determinations of one of three identical experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. AC, adenylyl cyclase; CREB, cAMP response element binding protein; HSL, hormone-sensitive lipase; LF, lactoferrin; SD, standard deviation.</p

LF-induced lipolysis in LRP1-silenced adipocytes.

<p><b>(A)</b> Activation of lipolysis by LF. To quantitate lipolysis, the amount of glycerol in the medium was analyzed 24 h after adding 1 mg/ml of LF. The statistical significance of the differences between LF treated and untreated cells was evaluated using the Student <i>t</i> test. **<i>p</i> < 0.01. The data represent the mean ± SD values of triplicate determinations of one of three identical experiments. <b>(B)</b> Activation of HSL by LF treatment. Phosphorylation of HSL was detected in the presence or absence of 1 mg/ml LF 15 min after the addition of LF. Phosphorylation levels normalized to protein expression levels are shown. The statistical significance was evaluated using the Student <i>t</i> test vs LF untreated control. **<i>p</i> < 0.01; n.s., no significant difference. The data represent the mean ± SD values of triplicate determinations of one of three identical experiments. <b>(C)</b> LRP1 silencing by siRNA. Adipocytes were transiently transfected with negative control siRNA (siNC) or LRP1 siRNA (siLRP1) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141378#sec002" target="_blank">Materials and methods</a>). LRP1 protein expression was monitored by immunoblotting during each assay. Distinctive data is shown. β-actin was used as a loading control. HSL, hormone-sensitive lipase; LF, lactoferrin; LRP1, lipoprotein receptor-related protein 1; SD, standard deviation.</p