Genome-wide association meta-analysis identifies two novel loci associated with dental caries.

BACKGROUND: Tooth loss significantly impacts oral function and overall health deterioration. Dental caries and periodontal disease are major contributors to tooth loss, emphasizing the critical need to prevent these conditions. Genetic studies have played a crucial role in deepening our understanding of the underlying mechanisms of these diseases. While large-scale genome-wide association studies (GWAS) on dental caries and periodontal disease have been conducted extensively, research focusing on Asian populations remains limited. Given substantial genetic and lifestyle variations across ethnicities, conducting studies across diverse populations is imperative. This study aimed to uncover new insights into the genetic mechanisms of these diseases, contributing to broader knowledge and potential targeted interventions. METHODS: We conducted a GWAS using genome data from 45,525 Japanese individuals, assessing their self-reported history of dental caries and periodontal disease. Additionally, we performed a meta-analysis by integrating our results with those from a previous large-scale GWAS predominantly involving European populations. RESULTS: While no new loci associated with periodontal disease were identified, we discovered two novel loci associated with dental caries. The lead variants of these loci were intron variant rs10974056 in GLIS3 and intron variant rs4801882 in SIGLEC5. CONCLUSION: Our study findings are anticipated to advance understanding of the underlying mechanisms of dental caries and periodontal disease. Thes insights may inform better management strategies for patients affected by these conditions.

Transcriptome and Methylome Profiling in Rat Skeletal Muscle: Impact of Post-Weaning Protein Restriction.

Skeletal muscle is programmable, and early-life nutritional stimuli may form epigenetic memory in the skeletal muscle, thus impacting adult muscle function, aging, and longevity. In the present study, we designed a one-month protein restriction model using post-weaning rats, followed by a two-month rebound feeding, to investigate how early-life protein restriction affects overall body growth and muscle development and whether these influences could be corrected by rebound feeding. We observed comprehensive alterations immediately after protein restriction, including retarded growth, altered biochemical indices, and disturbed hormone secretion. Transcriptome profiling of the gastrocnemius muscle followed by gene ontology analyses revealed that "myogenic differentiation functions" were upregulated, while "protein catabolism" was downregulated as a compensatory mechanism, with enhanced endoplasmic reticulum stress and undesired apoptosis. Furthermore, methylome profiling of the gastrocnemius muscle showed that protein restriction altered the methylation of apoptotic and hormone secretion-related genes. Although most of the alterations were reversed after rebound feeding, 17 genes, most of which play roles during muscle development, remained altered at the transcriptional level. In summary, early-life protein restriction may undermine muscle function in the long term and affect skeletal muscle development at the both transcriptional and methylation levels, which may hazard future muscle health.

Disruption of insulin receptor substrate-2 impairs growth but not insulin function in rats.

Insulin receptor substrate (IRS)-2, along with IRS-1, is a key signaling molecule that mediates the action of insulin and insulin-like growth factor (IGF)-I. The activated insulin and IGF-I receptors phosphorylate IRSs on tyrosine residues, leading to the activation of downstream signaling pathways and the induction of various physiological functions of insulin and IGF-I. Studies using IRS-2 knockout (KO) mice showed that the deletion of IRS-2 causes type 2 diabetes due to peripheral insulin resistance and impaired ß-cell function. However, little is known about the roles of IRS-2 in other animal models. Here, we created IRS-2 KO rats to elucidate the physiological functions of IRS-2 in rats. The body weights of IRS-2 KO rats at birth were lower compared with those of their WT littermates. The postnatal growth of both male and female IRS-2 KO rats was also suppressed. Compared with male WT rats, the glucose and insulin tolerance of male IRS-2 KO rats were slightly enhanced, whereas a similar difference was not observed between female WT and IRS-2 KO rats. Besides the modestly increased insulin sensitivity, male IRS-2 KO rats displayed the enhanced insulin-induced activation of the mTOR complex 1 pathway in the liver compared with WT rats. Taken together, these results indicate that in rats, IRS-2 plays important roles in the regulation of growth but is not essential for the glucose-lowering effects of insulin.

A translation repressor, 4E-BP1, regulates the triglyceride level in rat liver during protein deprivation.

Protein deprivation has been shown to induce fatty liver in humans and animals, but the molecular mechanisms underlying such induction are largely unknown. Our previous studies have shown that a low-protein diet increases eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein and triglyceride (TG) levels in rat liver. 4E-BP1 is known to repress translation by binding to eIF4E. There is also evidence indicating that 4E-BP1 regulates lipid metabolism. Here, we examined the role of 4E-BP1 on TG accumulation in the livers of rats under protein deprivation. The low-protein diet rapidly increased the hepatic 4E-BP1 mRNA level within 1 day, followed by the induction of hepatic TG accumulation. The knockdown of hepatic 4E-BP1 attenuated the TG accumulation in rat liver induced by the low-protein diet. 4E-BP1 knockdown also increased the protein level of carnitine palmitoyltransferase 1A (CPT1A), a regulator of fatty acid oxidation, in the liver of rats fed a low-protein diet. These results indicate that a low-protein diet increases the amount of 4E-BP1, leading to TG accumulation in rat liver. We thus conclude that 4E-BP1 plays an important role in inducing hepatic steatosis under protein deprivation.

Strong association between the 12q24 locus and sweet taste preference in the Japanese population revealed by genome-wide meta-analysis.

The sweet taste preference of humans is an important adaptation to ensure the acquisition of carbohydrate nutrition; however, overconsumption of sweet foods can potentially lead to diseases such as obesity and diabetes. Although previous studies have suggested that interindividual variation of human sweet taste preference is heritable, genetic loci associated with the trait have yet to be fully elucidated. Here, we genotyped 12,312 Japanese participants using the HumanCore-12+ Custom BeadChip or the HumanCore-24 Custom BeadChip microarrays. The sweet taste preference of the participants was surveyed via an internet-based questionnaire, resulting in a five-point scale of sweet taste preference. The genome-wide meta-analysis of the Japanese participants revealed a strong association between the 12q24 locus and sweet taste preference scale (P = 2.8 × 10-70). The lead variant rs671 is monoallelic in non-East Asian populations and is located in the aldehyde dehydrogenase (ALDH2) gene, encoding an enzyme involved in alcohol metabolism. The association between the minor allele of rs671 and sweet taste preference was attenuated by adjusting for alcohol drinking. The subgroup analysis showed that the effect of rs671 on sweet taste preference was greater in males than in females. In conclusion, we found an association between the 12q24 locus and sweet taste preference in the Japanese population, and showed that the adjustment for drinking habits attenuated the association. This novel genetic association may provide new clues to elucidate mechanisms determining sweet taste preferences.

GWAS of habitual coffee consumption reveals a sex difference in the genetic effect of the 12q24 locus in the Japanese population.

BACKGROUND: Studies on genetic effects of coffee consumption are scarce for Asian populations. We conducted a genome-wide association study (GWAS) of habitual coffee consumption in Japan using a self-reporting online survey. RESULTS: Candidate genetic loci associated with habitual coffee consumption were searched within a discovery cohort (N = 6,264) and confirmed in a replication cohort (N = 5,975). Two loci achieved genome-wide significance (P < 5 × 10- 8) in a meta-analysis of the discovery and replication cohorts: an Asian population-specific 12q24 (rs79105258; P = 9.5 × 10- 15), which harbors CUX2, and 7p21 (rs10252701; P = 1.0 × 10- 14), in the upstream region of the aryl hydrocarbon receptor (AHR) gene, involved in caffeine metabolism. Subgroup analysis revealed a stronger genetic effect of the 12q24 locus in males (P for interaction = 8.2 × 10- 5). Further, rs79105258 at the 12q24 locus exerted pleiotropic effects on body mass index (P = 3.5 × 10- 4) and serum triglyceride levels (P = 8.7 × 10- 3). CONCLUSIONS: Our results consolidate the association of habitual coffee consumption with the 12q24 and 7p21 loci. The different effects of the 12q24 locus between males and females are a novel finding that improves our understanding of genetic influences on habitual coffee consumption.

Low-protein diet enhances adiponectin secretion in rats.

Previous studies including ours have shown that a low-protein diet up-regulates insulin signaling in the liver and muscle and induces fatty liver in rats. Adiponectin is known as an insulin-sensitizing adipocytokine. We, therefore, examined the effect of a low-protein diet on the adiponectin levels in rats. The low-protein diet significantly increased serum adiponectin level. However, mRNA and protein levels of adiponectin in white adipose tissue (WAT) were not changed by the low-protein diet. Since it is known that oligomerization is important to control serum adiponectin level, we examined the population of adiponectin oligomeric forms in WAT and found that low-protein diet did not change it. Despite these events, the amount of its secretion was significantly increased in the adipocytes isolated from WAT of low-protein diet-fed rats. These results indicate that a low-protein diet enhances adiponectin secretion, which is not due to the increased intracellular amount and oligomerization of adiponectin.

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.

Triglyceride synthesis in hepatocytes isolated from rats fed a low-protein diet is enhanced independently of upregulation of insulin signaling.

It is known that protein malnutrition develops fatty liver in rats. However, the mechanisms by which protein malnutrition enhances lipid accumulation in the liver are not fully understood. Our previous studies have demonstrated that protein malnutrition upregulates insulin signaling with an increase in TG levels in rat livers. Here, we examined whether the upregulated insulin signaling contributes to an enhancement of TG accumulation under protein malnutrition. As it is difficult to analyze insulin-induced hepatic TG synthesis in vivo, the isolated hepatocytes derived from rats fed a low-protein diet were used. The hepatocytes were isolated from rats fed a 15% casein diet (15C) as a control diet or a 5% casein diet (5C) as a low-protein diet and then treated with insulin. As shown in vivo, insulin signaling was upregulated in isolated hepatocytes from 5C-fed rats (5C hepatocytes). However, the insulin-induced increase in the mRNA levels of lipogenic enzymes, including acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS), was similar in both groups. The amounts of TG synthesized from both glucose and palmitate, as well as ACC1 and FAS protein levels, were increased at the basal state in 5C hepatocytes, but were not further increased by insulin. These results indicate that TG synthesis via both de novo fatty acid synthesis and esterification is enhanced in 5C hepatocytes, which is independent of the upregulation of insulin signaling.

Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α.

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein B (apoB)-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α.

Dietary Genistein Prevents Denervation-Induced Muscle Atrophy in Male Rodents via Effects on Estrogen Receptor-α.

BACKGROUND: Genistein has high estrogenic activity. Previous studies have shown beneficial effects of estrogen or hormone replacement therapy on muscle mass and muscle atrophy. OBJECTIVE: We investigated the preventive effects and underlying mechanisms of genistein on muscle atrophy. METHODS: In Expt. 1, male Wistar rats were fed a diet containing no genistein [control (CON)] or 0.05% genistein (GEN; wt:wt diet) for 24 d. On day 14, the sciatic nerve in the left hind leg was severed, and the right hind leg was sham-treated. In Expt. 2, male C57BL6J mice were subcutaneously administered a vehicle (Veh group) or the estrogen receptor (ER) antagonist ICI 182,780 (ICI group) via an osmotic pump for 27 d, and each group was subsequently fed CON or GEN diets from day 3 to day 27. Muscle atrophy was induced on day 17 as in Expt. 1. In Expt. 3, male C57BL6J mice were subcutaneously administered vehicle or a selective ER agonist-ER-α [4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT)] or ER-ß [2,3-bis(4-hydroxyphenyl)-propionitrile (DPN)]-or genistein (GEN-sc-i) via an osmotic pump for 13 d, and muscle atrophy was induced on day 3 as in Expt. 1. The ratio of denervated soleus muscle weight to sham-operated soleus muscle weight (d/s ratio) was used as the index of muscle atrophy. RESULTS: Expt. 1: The d/s ratio in the GEN group was 20% higher than that in the CON group (P < 0.05). Expt. 2: The d/s ratio in the Veh-GEN group was 14% higher than that in the Veh-CON group (P < 0.05), although there was no significant difference between ICI-CON and ICI-GEN groups (P = 0.69). Expt. 3: The d/s ratio in the PPT-treated group was 20% greater than that in the Veh group (P < 0.05), but DPN and GEN-sc-i had no effect on the d/s ratio (P ≥ 0.05 compared with vehicle). CONCLUSION: Genistein intake mitigated denervation-induced soleus muscle atrophy. ER-α was related to the preventive effect of genistein on muscle atrophy.

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.

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.

Rapid increase in fibroblast growth factor 21 in protein malnutrition and its impact on growth and lipid metabolism.

Protein malnutrition promotes hepatic steatosis, decreases insulin-like growth factor (IGF)-I production and retards growth. To identify new molecules involved in such changes, we conducted DNA microarray analysis on liver samples from rats fed an isoenergetic low-protein diet for 8 h. We identified the fibroblast growth factor 21 gene (Fgf21) as one of the most strongly up-regulated genes under conditions of acute protein malnutrition (P<0·05, false-discovery rate<0·001). In addition, amino acid deprivation increased Fgf21 mRNA levels in rat liver-derived RL-34 cells (P<0·01). These results suggested that amino acid limitation directly increases Fgf21 expression. FGF21 is a polypeptide hormone that regulates glucose and lipid metabolism. FGF21 also promotes a growth hormone-resistance state and suppresses IGF-I in transgenic mice. Therefore, to determine further whether Fgf21 up-regulation causes hepatic steatosis and growth retardation after IGF-I decrease in protein malnutrition, we fed an isoenergetic low-protein diet to Fgf21-knockout (KO) mice. Fgf21-KO did not rescue growth retardation and reduced plasma IGF-I concentration in these mice. Fgf21-KO mice showed greater epididymal white adipose tissue weight and increased hepatic TAG and cholesterol levels under protein malnutrition conditions (P<0·05). Overall, the results showed that protein deprivation directly increased Fgf21 expression. However, growth retardation and decreased IGF-I were not mediated by increased FGF21 expression in protein malnutrition. Furthermore, FGF21 up-regulation rather appears to have a protective effect against obesity and hepatic steatosis in protein-malnourished animals.

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.

Beneficial effects of soluble dietary Jerusalem artichoke (Helianthus tuberosus) in the prevention of the onset of type 2 diabetes and non-alcoholic fatty liver disease in high-fructose diet-fed rats.

Jerusalem artichoke (JA) has the potential to attenuate lipid disturbances and insulin resistance (IR), but the underlying mechanisms are not well understood. In the present study, we elucidated the physiological responses and mechanisms of JA intervention with a comprehensive transcriptome analysis. Wistar rats were fed a control diet, a 60 % fructose-enriched diet (FRU), or a FRU with 10 % JA (n 6-7) for 4 weeks. An oral glucose tolerance test was carried out on day 21. Liver samples were collected for biochemical and global gene expression analyses (GeneChip® Rat Genome 230 2.0 Array, Affymetrix). Fructose feeding resulted in IR and hepatic TAG accumulation; dietary JA supplementation significantly improved these changes. Transcriptomic profiling revealed that the expression of malic enzyme 1 (Me1), associated with fatty acid synthesis; decorin (Dcn), related to fibrosis; and cytochrome P450, family 1, subfamily a, polypeptide 2 (Cyp1a2) and nicotinamide phosphoribosyltransferase (Nampt), associated with inflammation, was differentially altered by the FRU, whereas dietary JA supplementation significantly improved the expression of these genes. We established for the first time the molecular mechanisms driving the beneficial effects of JA in the prevention of type 2 diabetes and non-alcoholic fatty liver disease. We propose that 10 % JA supplementation may be beneficial for the prevention of the onset of these diseases.

Tissue-specific effects of protein malnutrition on insulin signaling pathway and lipid accumulation in growing rats.

Our previous studies have revealed that protein malnutrition enhances insulin signaling in rat liver and muscle in response to a bolus insulin injection. However, it has not been established whether protein malnutrition up-regulates insulin signaling under physiological conditions, such as feeding. Here, we studied the effects of protein malnutrition on insulin signaling after feeding in rat liver, muscle and white adipose tissue (WAT). Six-week-old rats were fed a 15% casein diet (15C) or a calorie-matched 5% casein diet (5C) for 8 h/day during 14 days. On the 15th day, blood and tissues were collected at various time points after feeding. Feeding-induced insulin secretion was reduced in 5C-fed rats compared to 15C-fed rats. The 5C-feeding suppressed immediate activation of insulin receptor after feeding in the liver, muscle, and WAT. However, 5C-feeding constantly increased tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and threonine phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in the liver during the examined periods, corresponding to the changes of their amounts. In skeletal muscle, 5C-feeding did not appreciably alter insulin signaling. In WAT, 5C-feeding decreased tyrosine phosphorylation of IRS-1 compared to 15C-feeding. Furthermore, hepatic triglyceride content was increased and feeding-induced acetyl-CoA carboxylase 1 gene expression was enhanced in 5C-fed rats. The 5C-feeding decreased insulin-dependent glucose uptake in adipocytes. These results suggest that enhanced insulin signaling through increased IRS-2 and 4E-BP1 levels in the liver and repressed insulin signaling through decreased IRS-1 levels in WAT contribute to the preferential hepatic lipid accumulation under protein malnutrition.

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.

Effects of Thyme (Thymus vulgaris L.) Essential Oil on Aging-Induced Brain Inflammation and Blood Telomere Attrition in Chronologically Aged C57BL/6J Mice.

Chronological aging is commonly accompanied by chronic low-grade inflammation (or "inflammaging"), a contributor to the development of age-related chronic diseases. Aging increases oxidative stress that accelerates telomere shortening, leading to cell senescence and the generation of senescence-associated secretory phenotype (SASP) that exacerbates inflammation. Dietary antioxidants may help protect telomeres and attenuate inflammation. Thyme essential oil (TEO), reported for its potency against neuroinflammation, was fed to chronologically aged C57BL/6J mice for 24 weeks. The TEO diet showed notable impacts on the hippocampus, indicated by lower expression of the aging-related gene p16INK4A (p = 0.0783) and significantly lower expression of cyclin D kinase Cdk4 and Cdk6 (p < 0.05) compared to the age-matched control mice. The TEO group also showed significantly lower gene expression of the pro-inflammatory cytokine Il6 (p < 0.05) in the hippocampus and lower Il1b expression in the liver and cerebellum (p < 0.05). In vitro experiments conducted on NIH-3T3 cells expressing SASP revealed the dose-dependent anti-inflammatory activity of TEO. Remarkably, TEO diet-fed mice showed higher survival rates and significantly longer blood telomere lengths than the control mice. Monoterpene antioxidants in TEO, particularly thymol and p-cymene, may primarily contribute to the anti-inflammatory and telomere-protecting activities of TEO.