Bidirectional effect of vitamin D on brown adipogenesis of C3H10T1/2 fibroblast-like cells.

Background: Brown adipose tissue (BAT) dissipates caloric energy as heat and plays a role in glucose and lipid metabolism. Therefore, augmentation and activation of BAT are the focus of new treatment strategies against obesity, a primary risk factor of metabolic syndrome. The vitamin D system plays a crucial role in mineral homeostasis, bone metabolism, and cell proliferation and differentiation. In this study, we investigated the effects of vitamin D3 [1,25(OH)2D3] on brown adipocyte differentiation. Methods: The mouse fibroblast-like cell line C3H10T1/2 was differentiated into brown adipocytes in the presence of 1,25(OH)2D3. The effect of 1,25(OH)2D3 on brown adipocyte differentiation was assessed by measuring lipid accumulation, the expression of related genes, and cytotoxicity. The viability of C3H10T1/2 cells was measured using the Cell Counting Kit-8 assay. Gene expression was investigated using quantitative reverse transcription-polymerase chain reaction. Protein expression was estimated using western blotting. Results: 1,25(OH)2D3 inhibited adipocyte differentiation and exerted a cytotoxic effect at 1 nM. However, in the physiological concentration range (50-250 pM), 1,25(OH)2D3 promoted uncoupling protein 1 (UCP1) expression in C3H10T1/2 cells. This effect was not observed when 1,25(OH)2D3 was added 48 h after the initiation of differentiation, suggesting that the vitamin D system acts in the early phase of the differentiation program. We showed that 1,25(OH)2D3 increased the expression of two key regulators of brown adipogenesis, PR domain containing 16 (Prdm16) and peroxisome proliferator-activated receptor γ coactivator-1α (Pgc1α ). Furthermore, 1,25(OH)2D3 increased Ucp1 expression in 3T3-L1 beige adipogenesis in a dose-dependent manner. Conclusion: These data indicate the potential of vitamin D and its analogs as therapeutics for the treatment of obesity and related metabolic diseases.

CREG1 improves diet-induced obesity via uncoupling protein 1-dependent manner in mice.

Thermogenic brown and beige adipocytes express uncoupling protein 1 (UCP1) and stimulate energy metabolism, protecting against obesity and metabolic diseases such as type 2 diabetes and hyperlipidemia. Cellular repressor of E1A-stimulated genes 1 (CREG1) can stimulate thermogenic fat formation, induce UCP1, and reduce diet-induced obesity (DIO) in mice at normal room temperature. In this study, we investigated the effect of CREG1 administration and the importance of UCP1 in DIO inhibition under thermoneutral conditions at 30°C, which attenuate thermogenic fat formation. Interestingly, subcutaneous administration of recombinant CREG1 protein via an osmotic pump in C57BL/6J mice for four weeks increased UCP1 expression in interscapular brown adipose tissue (IBAT), inhibited visceral white fat hypertrophy with partial browning, and reduced DIO compared to that in PBS-treated mice. The mRNA expression of energy metabolism-related genes was significantly increased in the IBAT of CREG1-treated mice compared to that in PBS-treated mice. In contrast, adipocyte-specific overexpression of CREG1 failed to improve DIO in UCP1-knockout mice at thermoneutrality. Our results indicate the therapeutic potential of CREG1 administration for obesity under thermogenic fat-attenuating conditions and highlight the indispensable role of UCP1 in the DIO-inhibitory effect of CREG1.

CREG1 administration stimulates BAT thermogenesis and improves diet-induced obesity in mice.

Brown and beige adipocytes, which express thermogenic uncoupling protein-1 (UCP1), stimulate glucose and lipid metabolism, improving obesity and metabolic diseases such as type 2 diabetes and hyperlipidemia. Overexpression of cellular repressor of E1A-stimulated genes 1 (CREG1) promotes adipose tissue browning and inhibits diet-induced obesity (DIO) in mice. In this study, we investigated the effects of CREG1 administration on DIO inhibition and adipose browning. Subcutaneous administration of recombinant CREG1 protein to C57BL/6 mice stimulated UCP1 expression in interscapular brown adipose tissue (IBAT) and improved DIO, glucose tolerance and fatty liver compared with those in phosphate-buffered saline-treated mice. Injection of Creg1-expressing adenovirus into inguinal white adipose tissue (IWAT) significantly increased browning and mRNA expression of beige adipocyte marker genes compared with that in mice injected with control virus. The effect of Creg1 induction on beige adipocyte differentiation was supported in primary culture using preadipocytes isolated from IWAT of Creg1-transgenic mice compared with that of wild-type mice. Our results indicate a therapeutic effect of CREG1 on obesity and its associated pathology and a potential of CREG1 to stimulate brown/beige adipocyte formation.

Glycerol kinase stimulates uncoupling protein 1 expression by regulating fatty acid metabolism in beige adipocytes.

Browning of adipose tissue is induced by specific stimuli such as cold exposure and consists of up-regulation of thermogenesis in white adipose tissue. Recently, it has emerged as an attractive target for managing obesity in humans. Here, we performed a comprehensive analysis to identify genes associated with browning in murine adipose tissue. We focused on glycerol kinase (GYK) because its mRNA expression pattern is highly correlated with that of uncoupling protein 1 (UCP1), which regulates the thermogenic capacity of adipocytes. Cold exposure-induced Ucp1 up-regulation in inguinal white adipose tissue (iWAT) was partially abolished by Gyk knockdown (KD) in vivo Consistently, the Gyk KD inhibited Ucp1 expression induced by treatment with the ß-adrenergic receptors (ßAR) agonist isoproterenol (Iso) in vitro and resulted in impaired uncoupled respiration. Gyk KD also suppressed Iso- and adenylate cyclase activator-induced transcriptional activation and phosphorylation of the cAMP response element-binding protein (CREB). However, we did not observe these effects with a cAMP analog. Therefore Gyk KD related to Iso-induced cAMP products. In Iso-treated Gyk KD adipocytes, stearoyl-CoA desaturase 1 (SCD1) was up-regulated, and monounsaturated fatty acids such as palmitoleic acid (POA) accumulated. Moreover, a SCD1 inhibitor treatment recovered the Gyk KD-induced Ucp1 down-regulation and POA treatment down-regulated Iso-activated Ucp1 Our findings suggest that Gyk stimulates Ucp1 expression via a mechanism that partially depends on the ßAR-cAMP-CREB pathway and Gyk-mediated regulation of fatty acid metabolism.

Lack of UCP1 stimulates fatty liver but mediates UCP1-independent action of beige fat to improve hyperlipidemia in Apoe knockout mice.

Brown adipose tissue (BAT) plays a critical role in lipid metabolism and may protect from hyperlipidemia; however, its beneficial effect appears to depend on the ambient temperature of the environment. In this study, we investigated the effects of uncoupling protein 1 (UCP1) deficiency on lipid metabolism, including the pathophysiology of hyperlipidemia, in apolipoprotein E knockout (APOE-KO) mice at a normal (23 °C) and thermoneutral (30 °C) temperature. Unexpectedly, UCP1 deficiency caused improvements in hyperlipidemia, atherosclerosis, and glucose metabolism, regardless of an increase in hepatic lipid deposition, in Ucp1/Apoe double-knockout (DKO) mice fed a high-fat diet at 23 °C, with BAT hyperplasia and robust browning of inguinal white adipose tissue (IWAT) observed. Proteomics and gene expression analyses revealed significant increases in many proteins involved in energy metabolism and strong upregulation of brown/beige adipocyte-related genes and fatty acid metabolism-related genes in browned IWAT, suggesting an induction of beige fat formation and stimulation of lipid metabolism in DKO mice at 23 °C. Conversely, mRNA levels of fatty acid oxidation-related genes decreased in the liver of DKO mice. The favorable phenotypic changes were lost at 30 °C, with BAT whitening and disappearance of IWAT browning, while fatty liver further deteriorated in DKO mice compared with that in APOE-KO mice. Finally, longevity analysis revealed a significant lifespan extension of DKO mice compared with that of APOE-KO mice at 23 °C. Irrespective of the fundamental role of UCP1 thermogenesis, our results highlight the importance of beige fat for the improvement of hyperlipidemia and longevity under the atherogenic status at normal room temperature.

CREG1 stimulates brown adipocyte formation and ameliorates diet-induced obesity in mice.

Increased formation of brown and beige adipocytes is critical for adaptive thermogenesis to maintain homeothermy in cold or to circumvent diet-induced obesity (DIO). Cellular repressor of adenovirus early region 1A-stimulated genes 1 (CREG1) exhibits the ability to stimulate brown adipogenesis, including the induction of uncoupling protein 1 (UCP1), in vitro. Thus, we aimed to clarify whether CREG1 promotes brown adipocyte formation and inhibits DIO at the whole-animal level. In mouse brown adipose tissue (BAT), CREG1 expression was markedly increased in cold but was decreased under thermoneutrality, suggesting CREG1 involvement in BAT thermogenesis. Moreover, in BAT and white adipose tissue, expression of UCP1 and fibroblast growth factor-21 and browning were both significantly higher in adipocyte P2-Creg1-transgenic (Tg) mice than in wild-type (WT) littermates. Following stimulation with a ß3-adrenergic agonist, energy consumption was elevated in the Tg mice, which showed increased resistance to DIO and improvement of obesity-associated complications including fatty liver relative to WT mice. The CREG1 stimulatory effect on brown adipogenesis was confirmed in Tg-BAT primary cultures. It was also found that CREG1 binds to retinoid X receptor α, which interacts with thyroid hormone receptor for brown adipogenesis. Our findings demonstrate that CREG1 stimulates brown adipocyte formation and browning, ameliorating obesity and its related pathology in vivo.-Hashimoto, M., Kusudo, T., Takeuchi, T., Kataoka, N., Mukai, T., Yamashita, H. CREG1 stimulates brown adipocyte formation and ameliorates diet-induced obesity in mice.

CREG1 promotes uncoupling protein 1 expression and brown adipogenesis in vitro.

Brown adipocytes play a critical role for adaptive thermogenesis to regulate body temperature in cold or to circumvent diet-induced obesity. In this study, we investigated the role of cellular repressor of E1A-stimulated genes 1 (CREG1) on brown adipogenesis and uncoupling protein 1 (UCP1) expression by using in vitro culture models. In murine mesenchymal stem cell line C3H10T1/2, Creg1 mRNA expression significantly increased in a time-dependent manner along with Ucp1 mRNA induction in brown adipogenesis. Creg1 gene overexpression upregulated the expression of brown fat-related genes including Ucp1 but its suppression downregulated these gene expression in C3H10T1/2 cells. Unlike the brown adipogenesis, Creg1 mRNA expression decreased significantly after differentiation stimulation in white adipogenesis of 3T3-L1 cells. Either Creg1 gene overexpression or suppression hardly affected white adipogenesis. In addition, CREG1 protein stimulated brown adipogenesis and rescued the adipogenesis in the absence of thyroid hormone in C3H10T1/2 cells. In reporter assay, CREG1 induction stimulated Ucp1 promoter activity, which was enhanced by co-expression with thyroid hormone receptors. The effect of CREG1 on Ucp1 promoter activity was also stimulated by retinoic acid. These results strongly suggest that CREG1 plays an important role on the regulation of UCP1 expression and brown adipogenesis.

Silencing of FABP1 ameliorates hepatic steatosis, inflammation, and oxidative stress in mice with nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is increasing in prevalence worldwide and has been identified as a risk factor for cirrhosis and hepatocellular carcinoma. However, there is no effective pharmacologic treatment for NAFLD. FABP1 is a liver-specific fatty acid-binding protein (FABP) that plays important roles in intracellular lipid metabolism in the liver. We investigated the effect of repression of FABP1 expression on NAFLD, using adenovirus-mediated silencing of FABP1. FABP1 knockdown in the liver decreased the liver weight and hepatic triglyceride (TG) accumulation. The expression of inflammatory and oxidative stress markers in the liver was also reduced. The level of thiobarbituric acid-reactive substances, a marker of lipid peroxidation, in the liver of FABP1 knockdown mice was significantly decreased. These results suggest that FABP1 reduction in the liver is an effective approach against NAFLD.

Asp3Gly polymorphism affects fatty acid-binding protein 3 intracellular stability and subcellular localization.

Fatty acid-binding proteins (FABP) play a crucial role in intracellular fatty acid transportation and metabolism. In this study, we investigate the effects of the FABP3 Asp3Gly (D3G) polymorphism on protein structure and function. Although the mutation did not alter protein secondary structure or the ability to bind 1-anilinonaphthalene-8-sulfonic acid and palmitate, the intracellular stability of the D3G mutant was significantly decreased. Immunocytochemical analysis reveals that the mutation alters FABP3 subcellular localization. Our results suggest that the D3G polymorphism may impact energy metabolism and physiological functions.

Phenolic compounds from leaves of Casimiroa edulis showed adipogenesis activity.

Casimiroa edulis is known as cochitzapotl, and it belongs to a species of tropical fruiting tree in the family Rutaceae, native to eastern Mexico and Central America south to Costa Rica. In this study, we isolated two furocoumarins and two polymethoxyflavones from leaves of C. edulis and evaluated the functions of glucose and lipid metabolism activity with 3T3-L1 adipocytes. We discovered that the addition of furocoumarins increased glucose uptake and lipid accumulation in 3T3-L1 adipocyte. These results suggest that furocoumarin compounds can be used as functional food-derived compounds, to regulate adipocyte functioning for the management of metabolic syndrome, which is associated with dysfunctions of glucose and lipid metabolism.

A key role for neuropeptide Y in lifespan extension and cancer suppression via dietary restriction.

Knowledge of genes essential for the life-extending effect of dietary restriction (DR) in mammals is incomplete. In this study, we found that neuropeptide Y (Npy), which mediates physiological adaptations to energy deficits, is an essential link between DR and longevity in mice. The lifespan-prolonging effect of lifelong 30% DR was attenuated in Npy-null mice, as was the effect on the occurrence of spontaneous tumors and oxidative stress responses in comparison to wild-type mice. In contrast, the physiological processes activated during adaptation to DR, including inhibition of anabolic signaling molecules (insulin and insulin-like growth factor-1), modulation of adipokine and corticosterone levels, and preferential fatty acid oxidation, were unaffected by the absence of Npy. These results suggest a key role for Npy in mediating the effects of DR. We also provide evidence that most of the physiological adaptations to DR could be achieved in mice without Npy.

Evodiamine inhibits insulin-stimulated mTOR-S6K activation and IRS1 serine phosphorylation in adipocytes and improves glucose tolerance in obese/diabetic mice.

Evodiamine, an alkaloid extracted from the dried unripe fruit of the tree Evodia rutaecarpa Bentham (Rutaceae), reduces obesity and insulin resistance in obese/diabetic mice; however, the mechanism underlying the effect of evodiamine on insulin resistance is unknown. This study investigated the effect of evodiamine on signal transduction relating to insulin resistance using obese/diabetic KK-Ay mice and an in vitro adipocyte culture. There is a significant decrease in the mammalian target of rapamycin (mTOR) and ribosomal S6 protein kinase (S6K) signaling in white adipose tissue (WAT) in KK-Ay mice treated with evodiamine, in which glucose tolerance is improved. In addition, reduction of insulin receptor substrate 1 (IRS1) serine phosphorylation, an indicator of insulin resistance, was detected in their WAT, suggesting suppression of the negative feedback loop from S6K to IRS1. As well as the stimulation of IRS1 and Akt serine phosphorylation, insulin-stimulated phosphorylation of mTOR and S6K is time-dependent in 3T3-L1 adipocytes, whereas evodiamine does not affect their phosphorylation except for an inhibitory effect on mTOR phosphorylation. Moreover, evodiamine inhibits the insulin-stimulated phosphorylation of mTOR and S6K, leading to down-regulation of IRS1 serine phosphorylation in the adipocytes. Evodiamine also stimulates phosphorylation of AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, which may cause down-regulation of mTOR signaling in adipocytes. A similar effect on AMPK, mTOR and IRS1 phosphorylation was found in adipocytes treated with rosiglitazone. These results suggest evodiamine improves glucose tolerance and prevents the progress of insulin resistance associated with obese/diabetic states, at least in part, through inhibition of mTOR-S6K signaling and IRS1 serine phosphorylation in adipocytes.

Bixin activates PPARα and improves obesity-induced abnormalities of carbohydrate and lipid metabolism in mice.

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that regulates the expression of the genes involved in fatty acid oxidation. PPARα activators induce fatty acid oxidation in the liver, thereby improving lipid and carbohydrate metabolism in obese mice. In this study, the dietary cis-carotenoids bixin and norbixin, which are commonly used in the food coloring industry, were found to activate PPARα by luciferase reporter assays using GAL4/PPARα chimeric and full-length PPARα systems. Treatment with bixin and norbixin induced the mRNA expression of PPARα target genes involved in fatty acid oxidation in PPARα-expressing HepG2 hepatocytes. In obese KK-Ay mice, bixin treatment suppressed the development of hyperlipidemia and hepatic lipid accumulation. In the livers of bixin-treated mice, the mRNA levels of PPARα target genes related to fatty acid oxidation were up-regulated. Moreover, bixin treatment also improved obesity-induced dysfunctions of carbohydrate metabolism, such as hyperglycemia, hyperinsulinemia, and hypoadiponectinemia. Glucose tolerance test and insulin tolerance test revealed that glucose intolerance and insulin resistance in KK-Ay obese mice were attenuated by the treatment with bixin. These results indicate that bixin acts as a food-derived agonist of PPARα, and bixin treatment is useful for the management of obesity-induced metabolic dysfunctions in mice.

TRPV4 deficiency increases skeletal muscle metabolic capacity and resistance against diet-induced obesity.

Transient receptor potential channel V4 (TRPV4) functions as a nonselective cation channel in various cells and plays physiological roles in osmotic and thermal sensation. However, the function of TRPV4 in energy metabolism is unknown. Here, we report that TRPV4 deficiency results in increased muscle oxidative capacity and resistance to diet-induced obesity in mice. Although no difference in body weight was observed between wild-type and Trpv4(-/-) mice when fed a standard chow diet, obesity phenotypes induced by a high-fat diet were significantly improved in Trpv4(-/-) mice, without any change in food intake. Quantitative analysis of mRNA revealed the constitutive upregulation of many genes, including those for transcription factors such as peroxisome proliferator-activated receptor α and for metabolic enzymes such as phosphoenolpyruvate carboxykinase. These upregulated genes were especially prominent in oxidative skeletal muscle, in which the activity of Ca(2+)-dependent phosphatase calcineurin was elevated, suggesting that other Ca(2+) channels function in the skeletal muscle of Trpv4(-/-) mice. Indeed, gene expressions for TRPC3 and TRPC6 increased in the muscles of Trpv4(-/-) mice compared with those of wild-type mice. The number of oxidative type I fiber also increased in the mutant muscles following myogenin gene induction. These results strongly suggested that inactivation of Trpv4 induces compensatory increases in TRPC3 and TRPC6 production, and elevation of calcineurin activity, affecting energy metabolism through increased expression of genes involved in fuel oxidation in skeletal muscle and thereby contributing to increased energy expenditure and protection from diet-induced obesity in mice.

Fatty acid-binding protein 3 stimulates glucose uptake by facilitating AS160 phosphorylation in mouse muscle cells.

We studied the relationship between fatty acid-binding protein 3 (FABP3) and obesity in vivo and the effects of FABP3 on signal transduction for glucose uptake in skeletal muscle cells in vitro. In obese mice, the level of FABP3 protein in gastrocnemius muscles increased significantly with an increase in body weight and metabolic phenotypes, suggesting a close relationship between FABP3 expression in the muscle and the development of obesity and/or insulin resistance in mice. In experiments using C2C12 myotubes infected with adenoviruses encoding human FABP3, induction stimulated glucose uptake without insulin stimulation in parallel with increases in the phosphorylation of AMP-activated protein kinase (AMPK) and AS160. Insulin enhanced glucose uptake in an additive fashion with increased phosphorylation of Akt and AS160 in FABP3-induced myotubes compared to control cells. This increased glucose uptake in FABP3-induced myotubes with insulin stimulation was found even in the presence of palmitate, in which a significantly higher Akt phosphorylation was detected compared to controls. These results suggest that FABP3 stimulates glucose uptake by facilitating AMPK-dependent AS160 phosphorylation in skeletal muscle. FABP3 may also contribute to AS160 phosphorylation by maintaining insulin-dependent Akt activation in the cells under a lipotoxic condition.

Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPARgamma activation.

Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPARgamma by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPARgamma target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPARgamma and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

Synthesis of 2alpha-propoxy-1alpha,25-dihydroxyvitamin D3 and comparison of its metabolism by human CYP24A1 and rat CYP24A1.

A new vitamin D(3) analogue, 2alpha-propoxy-1alpha,25-dihydroxyvitamin D(3) (C3O1), was synthesized starting from d-glucose as a chiral template of the A-ring with a CD-ring bromoolefin unit using the Trost coupling method. We studied the metabolism of the new analogue by human CYP24A1 and rat CYP24A1 to learn of species-based differences and found that the former has multiple metabolic pathways, but the latter has only a single pathway.

Induction of fatty acid-binding protein 3 in brown adipose tissue correlates with increased demand for adaptive thermogenesis in rodents.

We investigated the contribution of fatty acid-binding protein 3 (FABP3) to adaptive thermogenesis in brown adipose tissue (BAT) in rodents. The expression of FABP3 mRNA in BAT was regulated discriminatively in response to alteration of the ambient temperature, which regulation was similar and reciprocal to the regulation of uncoupling protein 1 (UCP1) and leptin, respectively. FABP3 expression in the BAT was significantly higher in the UCP1-knockout (KO) mice than in the wild-type ones, and these KO mice showed a higher clearance rate of free fatty acid from the plasma. In addition, FABP3 expression in the BAT was increased greatly with the development of diet-induced obesity in mice. These results indicate that the induction of FABP3 in BAT correlates with an increased demand for adaptive thermogenesis in rodents. FABP3 appears to be essential for accelerating fatty acid flux and its oxidation through UCP1 activity for non-shivering thermogenesis in BAT.

Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes.

Citrus fruit compounds have many health-enhancing effects. In this study, using a luciferase ligand assay system, we showed that citrus auraptene activates peroxisome proliferator-activated receptor (PPAR)-alpha and PPARgamma. Auraptene induced up-regulation of adiponectin expression and increased the ratio of the amount of high-molecular-weight multimers of adiponectin to the total adiponectin. In contrast, auraptene suppressed monocyte chemoattractant protein (MCP)-1 expression in 3T3-L1 adipocytes. Experiments using PPARgamma antagonist demonstrated that these effects on regulation of adiponectin and MCP-1 expression were caused by PPARgamma activations. The results indicate that auraptene activates PPARgamma in adipocytes to control adipocytekines such as adiponectin and MCP-1 and suggest that the consumption of citrus fruits, which contain auraptene can lead to a partial prevention of lipid and glucose metabolism abnormalities.

Sodium cromoglycate inhibits absorption of the major soybean allergen, Gly m Bd 30K, in mice and human intestinal Caco-2 cells.

Our previous data showed that Gly m Bd 30K was absorbed from the gastrointestinal tract and circulated in blood in mice. This study was conducted to determine the mechanism and identify the inhibitor of such absorption. Using sandwich ELISA and immunoblotting, we found that intact Gly m Bd 30K was absorbed from apical to basolateral solutions and intracellularly accumulated by Caco-2 cells in a dose- and time-dependent manner. The absorption and intracellular accumulation of Gly m Bd 30K were significantly suppressed when Caco-2 cells were treated with sodium cromoglycate (SCG) (0-50 mmol/L) in a dose-dependent manner. In 24-d-old mice orally treated with SCG (10-1000 mg/kg body weight), plasma Gly m Bd 30K concentration decreased significantly 30-120 min after Gly m Bd 30K (2000 mg/kg body weight) administration. Moreover, inhibitors that suppress the clathrin-dependent endocytosis dansylcadaverine, the caveolae-dependent endocytosis nystatin and clathrin, and the caveolae-dependent endocytosis methyl-beta-cyclodextrin had inhibitory effects on the absorption and intracellular accumulation of Gly m Bd 30K by Caco-2 cells. These data indicate that Gly m Bd 30K is absorbed and intracellularly accumulated in Caco-2 cells via clathrin- or caveolae-dependent endocytosis. We propose that the absorption and intracellular accumulation of Gly m Bd 30K are inhibited by SCG via clathrin- or caveolae-dependent endocytosis.