Clinical and phototrichogrammatic evaluation of estradiol replacement therapy on hair growth in postmenopausal Japanese women with female pattern hair loss: a pilot study.

Background: Female pattern hair loss (FPHL) is known to present with characteristic pathological conditions, including reduced overall hair density. Female hormones affect hair condition; however, the detailed mechanism is unknown. Furthermore, research on the topic is complicated by the fact that senescent alopecia often occurs concurrently with FPHL. Therefore, we investigated the effect of estradiol, a female hormone, on hair growth by eliminating aging factors and objectively evaluating hair changes caused by female hormone replacement therapy (HRT). Objective: This study was conducted to elucidate the mechanism through which female hormones exert their effects on hair. Methods: The study included 11 female patients undergoing HRT who were evaluated before initiating HRT, 3 months after initiating HRT, and 6 months after initiating HRT. The thinning hair score, hair density, telogen hair rate, telogen plucking strength, hair growth rate, and hair thickness were measured and evaluated. Furthermore, hematological tests were performed to assess the general physical condition of the participants. Results: HRT increased the telogen hair rate (P = .010, paired t test) at 3 months, improved frontal hairline thinning score (P = .008, Wilcoxon test), and increased the plucking strength (P = .013, paired t test) at 6 months. Limitations: The limitation of this study included the relatively small sample size, inability to conduct further long-term tests because of participant burden, and lack of a control group. Conclusion: The results suggested that HRT improved the appearance of the frontal hairline. As few studies have analyzed the effects of female hormones on human hair, a novel finding of this study was the effects of estradiol on the plucking strength after excluding age as a factor. We believe that these findings will contribute to understanding FPHL and developing female hormone-related treatments.

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.

Reversal of the hair loss phenotype by modulating the estradiol-ANGPT2 axis in the mouse model of female pattern hair loss.

BACKGROUND: Despite high demand for a remedy, the treatment options for female pattern hair loss (FPHL) are limited. FPHL is frequent in postmenopausal women. In ovariectomized (OVX) mice, which lack ß-estradiol (E2) and manifest hair loss mimicking FPHL, E2 supplementation has been shown to increase hair density. However, the mechanism by which E2 exhibits its biological activity remains elusive. OBJECTIVE: To identify the downstream targets of E2 in the context of FPHL pathophysiology and discover a potential therapeutic agent for the E2-dependent subtype of FPHL. METHODS: Human dermal papilla cells (hDPCs) were cultured with E2, and a microarray analysis was performed to identify the genes regulated by E2. Using OVX mice, the identified gene product was intradermally administered and then quantitative image analysis of hair density was conducted. In silico analysis to link E2 and the identified gene was performed. RESULTS: Global gene expression and bioinformatics analyses revealed that the genes associated with the angiopoietin-2 (ANGPT2) pathway were upregulated by E2 in hDPCs. ANGPT2 was significantly downregulated in OVX mice than in sham-operated mice (P < 0.01). Importantly, hair density was higher in OVX mice treated with ANGPT2 than in control mice (P < 0.05). In silico analysis showed DNA sequences with high possibility of estrogen receptor binding in the promoter region of ANGPT2. CONCLUSION: The E2-ANGPT2 axis is present in hair follicles. ANGPT2 provides a strategy for the management of E2-dependent and postmenopausal subsets of FPHL.

Bovine lactoferrin promotes energy expenditure via the cAMP-PKA signaling pathway in human reprogrammed brown adipocytes.

Lactoferrin (LF) is a multifunctional protein in mammalian milk. We previously reported that enteric-coated bovine LF reduced the visceral fat in a double-blind clinical study. We further demonstrated that bovine LF (bLF) inhibited adipogenesis and promoted lipolysis in white adipocytes, but the effect of bLF on brown adipocytes has not been clarified. In this study, we investigated the effects of bLF on energy expenditure and cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway using human reprogrammed brown adipocytes generated by gene transduction. bLF at concentrations of ≥ 100 µg/mL significantly increased uncoupling protein 1 (UCP1) mRNA levels, with the maximum value observed 4 h after bLF addition. At the same time point, bLF stimulation also significantly increased oxygen consumption. Signaling pathway analysis revealed rapid increases of intracellular cAMP and cAMP response element-binding protein (CREB) phosphorylation levels beginning 5 min after bLF addition. The mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were also significantly increased after 1 h of bLF stimulation. H-89, a specific PKA inhibitor, abrogated bLF-induced UCP1 gene expression. Moreover, receptor-associated protein (Rap), an antagonist of low-density lipoprotein receptor-related protein 1 (LRP1), significantly reduced bLF-induced UCP1 gene expression in a dose-dependent manner. These results suggest that bLF promotes UCP1 gene expression in brown adipocytes through the cAMP-PKA signaling pathway via the LRP1 receptor, leading to increased energy expenditure.

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

Lactoferrin (LF) is a multifunctional cationic protein (pI 8.2-8.9) in mammalian milk. We previously reported that enteric-LF prevented hypercholesterolemia and atherosclerosis in a diet-induced atherosclerosis model using Microminipig, 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 5.2-6.3). 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, p < 0.05) and reduced hepatic cholesterol levels (17% decrease, p < 0.05). These parameters were inversely correlated (R = -0.63, p < 0.05). These results suggest that LF promotes cholesterol excretion via interactions with bile acids.

Dammarane-type triterpene extracts of Panax notoginseng root ameliorates hyperglycemia and insulin sensitivity by enhancing glucose uptake in skeletal muscle.

Skeletal muscle is an important organ for controlling the development of type 2 diabetes. We discovered Panax notoginseng roots as a candidate to improve hyperglycemia through in vitro muscle cells screening test. Saponins are considered as the active ingredients of ginseng. However, in the body, saponins are converted to dammarane-type triterpenes, which may account for the anti-hyperglycemic activity. We developed a method for producing a dammarane-type triterpene extract (DTE) from Panax notoginseng roots and investigated the extract's potential anti-hyperglycemic activity. We found that DTE had stronger suppressive activity on blood glucose levels than the saponin extract (SE) did in KK-Ay mice. Additionally, DTE improved oral glucose tolerance, insulin sensitivity, glucose uptake, and Akt phosphorylation in skeletal muscle. These results suggest that DTE is a promising agent for controlling hyperglycemia by enhancing glucose uptake in skeletal muscle.

Proinflammatory M1 Macrophages Inhibit RANKL-Induced Osteoclastogenesis.

In response to a defined panel of stimuli, immature macrophages can be classified into two major phenotypes: proinflammatory (M1) and anti-inflammatory (M2). Although both phenotypes have been implicated in several chronic inflammatory diseases, their direct role in bone resorption remains unclear. The present study investigated the possible effects of M1 and M2 macrophages on RANKL-induced osteoclastogenesis. In osteoclastogenesis assays using RAW264.7 cells or bone marrow cells as osteoclast precursors, addition of M1 macrophages significantly suppressed RANKL-induced osteoclastogenesis compared to nonstimulated conditions (M0), addition of M2 macrophages, or no macrophage addition (P < 0.05), suggesting that M1 macrophages can downregulate osteoclastogenesis. This effect was maintained when direct contact between M1 and osteoclast precursors was interrupted by cell culture insertion, indicating engagement of soluble factors released from M1. M1 macrophages developed from interferon gamma (IFN-γ) knockout (IFN-γ-KO) mice lost the ability to downregulate osteoclastogenesis. Antibody-based neutralization of interleukin-12 (IL-12), but not IL-10, produced by M1 macrophages also abrogated M1-mediated downregulation of osteoclastogenesis. Real-time PCR analyses showed that IFN-γ suppressed gene expression of NFATc1, a master regulator of osteoclastogenesis, whereas IL-12 increased the apoptosis of osteoclasts, suggesting molecular mechanisms underlying the possible roles of IFN-γ or IL-12 in M1-mediated inhibition of osteoclastogenesis. These findings were confirmed in an in vivo ligature-induced mouse periodontitis model in which adoptive transfer of M1 macrophages showed a significantly lower level of bone loss and less tartrate-resistant acid phosphatase (TRAP)-positive cell induction than M0 or M2 macrophage transfer. In conclusion, by its secretion of IFN-γ and IL-12, M1, but not M0 or M2, was demonstrated to inhibit osteoclastogenesis.

Oral administration of Japanese sake yeast (Saccharomyces cerevisiae sake) promotes non-rapid eye movement sleep in mice via adenosine A2A receptors.

We have demonstrated previously that Japanese sake yeast improves sleep quality in humans. In the present study, we examined the molecular mechanisms of sake yeast to induce sleep by monitoring locomotor activity, electromyogram and electroencephalogram in mice. Oral administration of Japanese sake yeast (100, 200, and 300 mg kg-1 ) decreased the locomotor activity by 18, 46 and 59% and increased the amount of non-rapid eye movement (NREM) sleep by 1.5-, 2.3- and 2.4-fold (to 37 ± 6, 57 ± 8, and 60 ± 4 min from 25 ± 6 min in the vehicle-administered group, respectively) in a dose-dependent manner for 4 h after oral administration. However, Japanese sake yeast did not change the amount of rapid eye movement (REM) sleep, the electroencephalogram power density during NREM sleep or show any adverse effects, such as rebound of insomnia, during 24 h postadministration and on the next day. An intraperitoneal pretreatment with an adenosine A2A receptor-selective antagonist, ZM241385 (15 mg kg-1 ), reduced the amount of NREM sleep of sake yeast-administered mice to the basal level, without changing basal amount of sleep. Conversely, an A1 receptor-selective antagonist, 8-cyclopentyltheophylline (10 mg kg-1 ), did not affect the sleep-promoting effect of Japanese sake yeast. Thus, Japanese sake yeast promotes NREM sleep via activation of adenosine A2A but not A1 receptors.

Japanese sake yeast supplementation improves the quality of sleep: a double-blind randomised controlled clinical trial.

Activation of adenosine A2a receptors in cerebral neurons induces sleep in various mammals. It was previously found that Japanese sake yeast enriched in adenosine analogues activates A2a receptors in vitro and induces sleep in mice. Here it is reported that sake yeast activated A2a receptors in a cultured human cell line and improved human sleep quality in a clinical trial. Sake yeast activated A2a receptors in HEK cells in a dose-dependent manner with an EC50 of 40 µg mL(-1), and the activation was attenuated almost completely by the A2a receptor antagonist ZM241385 with an IC50 of 73 nm. In a double-blind placebo-controlled crossover clinical study, 68 healthy participants ingested tablets containing either 500 mg of sake yeast powder or a placebo (cellulose) 1 h before sleep for 4 days. Electroencephalograms were recorded during sleep at home with a portable device for 4 week days. Electroencephalogram analyses revealed that sake yeast supplementation significantly (P = 0.03) increased delta power during the first cycle of slow-wave sleep by 110%, without changing other sleep parameters. Sake yeast supplementation also significantly increased growth hormone secretion in the urine on awakening by 137% from 3.17 ± 0.41 (placebo) to 4.33 ± 0.62 (sake yeast) pg mg(-1) creatinine (P = 0.03). Subjective sleepiness (P = 0.02) and fatigue (P = 0.06) in the morning were improved by sake yeast. Given these benefits and the absence of adverse effects during the study period, it was concluded that sake yeast supplementation is an effective and safe way to support daily high-quality, deep sleep.

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

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.

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

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 in vitro 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.

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.

Antipyretic analgesic drugs have different mechanisms for regulation of the expression of inducible nitric oxide synthase in hepatocytes and macrophages.

Antipyretic analgesic drugs (including non-steroidal anti-inflammatory drugs) inhibit cyclooxygenase-2 and inducible nitric oxide synthase (iNOS), resulting in decreases of the proinflammatory mediators prostaglandin E2 and nitric oxide (NO), respectively. Both mediators are regulated by nuclear factor-kappa B (NF-κB), a key transcription factor in inflammation. Few reports have compared the efficacy and potency of anti-inflammatory drugs as NO inhibitors. In our study, we examined the effects of four popular antipyretic analgesic drugs on NO production induced in hepatocytes and macrophages. Mouse RAW264.7 macrophages treated with bacterial lipopolysaccharide showed the highest efficacy with regard to NO production; aspirin, loxoprofen, ibuprofen, and acetaminophen dose-dependently suppressed NO induction. Ibuprofen showed the highest potency in suppressing the induced production of NO. In rat hepatocytes, all the drugs inhibited interleukin 1ß-induced NO production and ibuprofen and loxoprofen inhibited NO induction effectively. Unexpectedly, the potency of NO suppression of each drug in hepatocytes did not always correlate with that observed in RAW264.7 cells. Microarray analyses of mRNA expression in hepatocytes revealed that the effects of the four antipyretic analgesic drugs modulated the NF-κB signaling pathway in a similar manner to the regulation of the expression of genes associated with inflammation, including the iNOS gene. However, the affected signal-transducing molecules in the NF-κB pathway were different for each drug. Therefore, antipyretic analgesic drugs may decrease NO production by modulating the NF-κB pathway in different ways, which could confer different efficacies and potencies with regard to their anti-inflammatory effects.

Protective effects of acetaminophen on ibuprofen-induced gastric mucosal damage in rats with associated suppression of matrix metalloproteinase.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to cause gastric mucosal damage as a side effect. Acetaminophen, widely used as an analgesic and antipyretic drug, has gastroprotective effects against gastric lesions induced by absolute ethanol and certain NSAIDs. However, the mechanisms that underlie the gastroprotective effects of acetaminophen have not yet been clarified. In the present study, we examined the role and protective mechanism of acetaminophen on ibuprofen-induced gastric damage in rats. Ibuprofen and acetaminophen were administered orally, and the gastric mucosa was macroscopically examined 4 hours later. Acetaminophen decreased ibuprofen-induced gastric damage in a dose-dependent manner. To investigate the mechanisms involved, transcriptome analyses of the ibuprofen-damaged gastric mucosa were performed in the presence and absence of acetaminophen. Ingenuity pathway analysis (IPA) software revealed that acetaminophen suppressed the pathways related to cellular assembly and inflammation, whereas they were highly activated by ibuprofen. On the basis of gene classifications from the IPA Knowledge Base, we identified the following five genes that were related to gastric damage and showed significant changes in gene expression: interleukin-1ß (IL-1ß), chemokine (C-C motif) ligand 2 (CCL2), matrix metalloproteinase-10 (MMP-10), MMP-13, and FBJ osteosarcoma oncogene (FOS). Expression of these salient genes was confirmed using real-time polymerase chain reaction. The expression of MMP-13 was the most reactive to the treatments, showing strong induction by ibuprofen and suppression by acetaminophen. Moreover, MMP-13 inhibitors decreased ibuprofen-induced gastric damage. In conclusion, these results suggest that acetaminophen decreases ibuprofen-induced gastric mucosal damage and that the suppression of MMP-13 may play an important role in the gastroprotective effects of acetaminophen.

Potent lipolytic activity of lactoferrin in mature adipocytes.

Lactoferrin (LF) is a multifunctional glycoprotein found in mammalian milk. We have shown in a previous clinical study that enteric-coated bovine LF tablets decreased visceral fat accumulation. To address the underlying mechanism, we conducted in vitro studies and revealed the anti-adipogenic action of LF in pre-adipocytes. The aim of this study was to assess whether LF could increase the lipolytic activity in mature adipocytes. Pre-adipocytes were prepared from rat mesenteric fat and differentiated into mature adipocytes for assays of lipolysis. The addition of LF significantly increased the glycerol concentration in the medium in a dose-dependent manner, whereas pepsin-degraded LF did not. A DNA microarray analysis demonstrated that LF decreased the expression of perilipin and affected the cAMP pathway. These findings are supported by the results of quantitative RT-PCR of perilipin and assays of cAMP. These data collectively indicate that visceral fat reduction by LF may result from the promotion of lipolysis and the additional anti-adipogenic activity of LF.

Bovine lactoferrin reduces visceral fat and liver triglycerides in ICR mice.

Lactoferrin (LF) is a multi-functional glycoprotein found in milk. In a previous clinical trial, we showed that enteric-coated bovine LF (bLF) tablets could reduce visceral fat accumulation. We also showed that bLF had anti-adipogenic activity in vitro. However, the mechanisms responsible for these phenomena remain unclear. In this study, we established an animal model of visceral fat reduction via oral bLF administration. We used gastric intubation to ensure that LF was absorbed in the small intestine. bLF administration for 4 weeks significantly reduced mesenteric fat tissue (P < 0.05) and hepatic triglyceride levels (P < 0.01). Furthermore, these two outcomes were positively correlated (R = 0.581, P < 0.05). Overall, these findings suggest that bLF affects mesenteric adipocytes and fatty acid metabolism in the liver.

Effects of Enteric-coated Lactoferrin Tablets Containing Lactobacillus brevis subsp. coagulans on Fecal Properties, Defecation Frequency and Intestinal Microbiota of Japanese Women with a Tendency for Constipation: a Randomized Placebo-controlled Crossover Study.

The effects of oral administration of enteric-coated tablets containing lactoferrin (LF; 100 mg/tablet) and heat-killed Lactobacillus brevis subsp. coagulans FREM BP-4693 (LB; 6×10(9) bacteria/tablet) on fecal properties were examined in 32 Japanese women (20-60 years of age) with a tendency for constipation (defecation frequency at equal to or less than 10 times/2 weeks) by a double-blind placebo-controlled crossover design. A significant increase in defecation days per week was obserbed in the subjects who ingested the tablets containing LF and LB compared with the placebo group. The number of bifidobacteria in feces also significantly increased compared with the placebo group. In an in vitro study, LF and tryptic hydrolysate of LF, but not peptic hydrolysate of LF, upregulated the growth of Bifidobacterium longum ATCC15707 when added to the culture. These results demonstrate the capability of the enteric-coated tablets containing LF and LB in improving intestinal function and suggest that they have a growth promoting function for bifidobacteria.

Effects of pepsin and trypsin on the anti-adipogenic action of lactoferrin against pre-adipocytes derived from rat mesenteric fat.

Lactoferrin (LF) is a multifunctional glycoprotein in mammalian milk. In a previous report, we showed that enteric-coated bovine LF tablets can decrease visceral fat accumulation, hypothesising that the enteric coating is critical to the functional peptides reaching the visceral fat tissue and exerting their anti-adipogenic activity. The aim of the present study was to assess whether ingested LF can retain its anti-adipogenic activity. We therefore investigated the effects of LF and LF treated with digestive enzymes (the stomach enzyme pepsin and the small intestine enzyme trypsin) on lipid accumulation in pre-adipocytes derived from the mesenteric fat tissue of male Sprague-Dawley rats. Lipid accumulation in pre-adipocytes was significantly reduced by LF in a dose-dependent manner and was associated with reduction in gene expression of CCAAT/enhancer binding protein delta, CCAAT/enhancer binding protein alpha and PPARγ as revealed by DNA microarray analysis. Trypsin-treated LF continued to show anti-adipogenic action, whereas pepsin-treated LF abrogated the activity. When an LF solution (1000 mg bovine LF) was administered by gastric intubation to Sprague-Dawley rats, immunoreactive LF determined by ELISA could be detected in mesenteric fat tissue at a concentration of 14·4 µg/g fat after 15 min. The overall results point to the importance of enteric coating for action of LF as a visceral fat-reducing agent when administered in oral form.