Syntaxin 17 promotes lipid droplet formation by regulating the distribution of acyl-CoA synthetase 3.

Lipid droplets (LDs) are ubiquitous organelles that contain neutral lipids and are surrounded by a phospholipid monolayer. How proteins specifically localize to the phospholipid monolayer of the LD surface has been a matter of extensive investigations. In the present study, we show that syntaxin 17 (Stx17), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein whose expression in the liver is regulated by diet, participates in LD biogenesis by regulating the distribution of acyl-CoA synthetase (ACSL)3, a key enzyme for LD biogenesis that redistributes from the endoplasmic reticulum (ER) to LDs during LD formation. Stx17 interacts with ACSL3, but not with LD formation-unrelated ACSL1 or ACSL4, through its SNARE domain. The interaction occurs at the ER-mitochondria interface and depends on the active site occupancy of ACSL3. Depletion of Stx17 impairs ACSL3 redistribution to nascent LDs. The defect in LD maturation due to Stx17 knockdown can be compensated for by ACSL3 overexpression, suggesting that Stx17 increases the efficiency of ACSL3 redistribution to LDs. Moreover, we show that the interaction between Stx17 and ACSL3 during LD maturation may be regulated by synaptosomal-associated protein of 23 kDa.

Expression and distribution of acyl-CoA thioesterases in the white adipose tissue of rats.

Acyl-CoA thioesterases (Acots) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and coenzyme A, and have the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, Acot1, is expressed and distributed in immature adipocytes located in the perivascular region of the white adipose tissue (WAT) of rats. Immunoblot analyses detected Acot1 in all of the WATs examined, while immunohistochemistry revealed positively stained layered structures surrounding the adventitia of blood vessels in the subcutaneous WAT. When the subcutaneous WAT was digested with collagenase and centrifuged, Acot1 was recovered in the stromal vascular fraction (SVF), and not in the large mature adipocytes. In the SVF, undigested cells attached to short tubular fragments of blood vessels showed positive immunostaining, as well as a proportion of the dispersed cells. These fibroblast-like cells contained fine particulate lipid droplets, stained by oil-red O dye, in their cytoplasm, or expressed fatty acid-binding protein 4, an adipocyte marker. After induction of adipocyte differentiation following a 15-day preculture without insulin, the dedifferentiated cells showed increased Acot1 expression with a diffuse distribution throughout the cytosol. These findings suggest that Acot1 expression is transiently upregulated at an early stage of adipocyte maturation, possibly to maintain cytosolic acyl-CoAs below a certain level until the cells acquire their full capability for fat storage.

Identification of acyl-CoA thioesterase in mouse mesenteric lymph nodes.

Acyl-CoA thioesterases (ACOTs) are a group of enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and CoA, with the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, ACOT7, is expressed at a significant level in the mesenteric lymph nodes (MLNs) of mice. While crude preparations of the mesenteric visceral fat contained significant levels of palmitoyl-CoA thioesterase activity, enzyme activity was concentrated 6.9-fold in MLNs compared with the residual adipose portion after excision of MLNs. When MLN homogenates were centrifuged, 82% of the enzyme activity was recovered in the cytosolic fraction, concomitant with almost exclusive recovery of ACOT7. Immunoprecipitation using anti-ACOT7 antibody estimated that 87% of enzyme activity in the homogenates was accounted for by ACOT7. On MLN sections, the germinal centers of secondary lymphoid follicles were immunostained with the antibody. In MLNs of mice fasted for 16 h, ACOT7 levels were induced 1.8-fold, which reflected a 1.5-fold increase in enzyme activity. These findings suggest that ACOT7 may be involved in dietary intake-associated responses in fatty acid metabolism in MLNs.

Effects of toothbrush hardness on in vitro wear and roughness of composite resins.

AIM: To investigate and compare the effects of toothbrushes with different hardness on abrasion and surface roughness of composite resins. MATERIALS AND METHODS: Toothbrushes (DENT. EX Slimhead II 33, Lion Dental Products Co. Ltd., Tokyo, Japan) marked as soft, medium and hard, were used to brush 10 beam-shaped specimens of each of three composites resins (Venus [VEN], Venus Diamond [VED] and Venus Pearl [VEP]; HeraeusKulzer) with standardized calcium carbonate slurry in a multistation testing machine (2N load, 60 Hz). After each of five cycles with 10k brushing strokes the wear depth and surface roughness of the specimens were determined. After completion of 50k strokes representative samples were inspected by SEM. Data were treated with ANOVA and regression analyses (p < 0.05). RESULTS: Abrasion of the composite resins increased linearly with increasing number of brushing cycles (r² > 0.9). Highest wear was recorded for VEN, lowest for VED. Hard brushes produced significantly higher wear on VEN and VEP, whereas no difference in wear by toothbrush type was detected for VED. Significantly highest surface roughness was found on VED specimens (Ra > 1.5 µm), the lowest one on VEN (Ra < 0.3 µm). VEN specimens showed increased numbers of pinhole defects when brushed with hard toothbrushes, surfaces of VEP were uniformly abraded without level differences between the prepolymerized fillers and the glass filler-loaded matrix, VED showed large glass fillers protruding over the main filler-loaded matrix portion under each condition. CONCLUSION: Abrasion and surface roughness of composite resins produced by toothbrushing with dentifrice depend mainly on the type of restorative resin. Hardness grades of toothbrushes have minor effects only on abrasion and surface roughness of composite resins. No relationship was found between abrasion and surface roughness. CLINICAL SIGNIFICANCE: The grade of the toothbrush used has minor effect on wear, texture and roughness of the composite resin.

Quantitative evaluation of the role of cysteine and methionine residues in the antioxidant activity of human serum albumin using recombinant mutants.

The importance of cysteine (Cys) and methionine (Met) residues for the antioxidant activity of human serum albumin (HSA) was investigated using recombinant HSA mutants, in which Cys34 and/or the six Met residues had been mutated to Ala. The scavenging activities of the mutants against five reactive oxygen and nitrogen species were evaluated by a chemiluminescence assay, electron paramagnetic resonance spectroscopy, or a HPLC-flow reactor assay. Our results showed that the contributions of Cys34 and the Met residues to the antioxidant activity of HSA were 61% and 29% against O(2)(•-), 68% and 61% against H(2)O(2), 38% and 6% against HO(•), 36% and 13% against HOCl, and 51% and 1% against (•)NO, respectively. Thus, the findings propose in a direct way that Cys34 plays a more important role than the Met residues in the antioxidant activity of HSA.

[Usefulness of an anti-mouse cerebellar tissue-derived antigen antibody test in predicting immunotherapy efficacy in patients with idiopathic cerebellar ataxia].

BACKGROUND: Autoimmune cerebellar ataxia (AICA) is a general term for diseases in which the cerebellum is damaged by an autoimmune mechanism. For the diagnosis of the AICA, anti-thyroid antibodies (anti-thyroid peroxidase antibody and anti-thyroglobulin antibody), anti-glutamic acid decarboxylase (GAD) antibodies, and anti-gliadin antibodies are measured. Immunotherapy is known to be effective for AICA, but some patients with effective immunotherapy lack autoantibodies associated with cerebellar ataxia. The purpose of this study was to clarify whether the effectiveness of immunotherapy in patients with suspected AICA could be predicted by anti-mouse cerebellar tissue-derived antigen antibody tests. METHODS: This study was conducted on 25 patients with idiopathic cerebellar ataxia (excluding multiple system atrophy, hereditary spinocerebellar degeneration, cancer-bearing patients, and patients taking phenytoin) who received immunotherapy from 2005 to 2016 at Tokyo Medical University Hachioji Medical Center. The patients were suspected of having AICA because they were positive for cerebellar ataxia-related autoantibodies (anti-thyroid antibody, anti-GAD antibody, anti-gliadin antibody, or anti-transglutaminase 6 antibody) or other autoantibodies. Antibodies that bind to mouse cerebellar tissue-derived antigens were defined as "anti-mouse cerebellar tissue-derived antigen antibodies" in this study, and their IgG-class antibodies were comprehensively measured using a slot blot. RESULTS: Anti-mouse cerebellar tissue-derived antigen antibody test results were correlated with immunotherapy efficacy. Furthermore, the combination of anti-mouse cerebellar tissue-derived antigen and anti-GAD antibody tests could predict the effectiveness of immunotherapy with 83% sensitivity and 100% specificity, while the combination of the anti-mouse cerebellar tissue-derived antigen, anti-GAD, and anti-gliadin (IgA class) antibody tests could predict the effectiveness of immunotherapy with 94% sensitivity and 86% specificity. CONCLUSION: Anti-mouse cerebellar tissue-derived antigen antibody tests could help to provide useful information for immunotherapy administration to patients with idiopathic cerebellar ataxia suspected to be AICA.

Regulated expression of acyl-CoA thioesterases in the differentiation of cultured rat brown adipocytes.

Acyl-CoA thioesterases (ACOTs) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and CoA-SH. In this study, we show that the expression profile of the ACOT isoforms changes remarkably during the differentiation of cultured rat brown adipocytes. Immunocytochemistry suggested that cytosolic ACOT1 was present in the preadipocytes, while mitochondrial ACOT2 was additionally expressed as the cells differentiated, concurrent with the accumulation of lipid droplets in the cytoplasm. Western blotting confirmed that, in contrast to ACOT1, the ACOT2 expression level was very low in the preadipocytes. However, after differentiation, the ACOT1 level fell to one-half of the baseline level and ACOT2 increased 18-fold. ACOT2 expression in the differentiated adipocytes was further enhanced by treatment with lipids or troglitazone. These changes in the ACOT2 expression level correlated well with changes in the expression of carnitine palmitoyltransferase 2, a mitochondrial ß-oxidation enzyme. These results indicate that, in differentiating brown adipocytes, cytosolic ACOT1 becomes downregulated as the cellular use of acyl-CoA increases, while mitochondrial ACOT2 is upregulated as the ß-oxidation capacity increases. ACOT isoform expression may be regulated during brown adipocyte differentiation to support the fat storage and combustion characteristics of this cell type.

Neuronal expression, cytosolic localization, and developmental regulation of the organic solute carrier partner 1 in the mouse brain.

Organic solute carrier partner 1 (OSCP1) is a mammalian, transporter-related protein that is able to facilitate the uptake of structurally diverse organic compounds into the cell when expressed in Xenopus laevis oocytes. This protein has been implicated in testicular handling of organic solutes because its mRNA expression is almost exclusive in the testis. However, in this study, we demonstrated significant expression of OSCP1 protein in mouse brain, the level of which was rather higher than that in the testis, although the corresponding mRNA expression was one-tenth of the testicular level. Immunohistochemistry revealed that OSCP1 was broadly distributed throughout the brain, and various neuronal cells were immunostained, including pyramidal cells in the cerebral cortex and hippocampus. However, there was no evidence of OSCP1 expression in glia. In primary cultures of cerebral cortical neurons, double-labeling immunofluorescence localized OSCP1 to the cytosol throughout the cell body and neurites including peri-synaptic regions. This was consistent with the subcellular fractionation of brain homogenates, in which OSCP1 was mainly recovered after centrifugation both in the cytosolic fraction and the particulate fraction containing synaptosomes. Immunoelectron microscopy of brain sections also demonstrated OSCP1 in the cytosol near synapses. In addition, it was revealed that changes in the expression level of OSCP1 correlated with neuronal maturation during postnatal development of mouse brain. These results indicate that OSCP1 may have a role in the brain indirectly mediating substrate uptake into the neurons in adult animals.

Sustained downregulation of YY1-associated protein-related protein gene expression in rat hippocampus induced by repeated electroconvulsive shock.

YY1AP-related protein (YARP) is a structural homolog of YY1-associated protein (YY1AP), which has a YY1-binding domain. During perinatal development, YARP mRNA expression is increased at a late stage of embryonic neurogenesis. It is not known whether YARP expression is regulated during adult neurogenesis. Electroconvulsive shock (ECS), a model for a highly effective depression treatment, is known to induce hippocampal neurogenesis after repeated treatment, so we employed ECS to measure the expression of YARP mRNA. Northern blots revealed significantly decreased expression of the YARP gene after repeated ECS but not single ECS. In situ hybridization clearly demonstrated a reduction of YARP mRNA expression in the CA (CA1, CA2, and CA3) subfields. Although clonic-tonic seizure was induced not only by ECS but also by injection of kainic acid to the striatum, the regulation of YARP mRNA expression was different between ECS and kainic acid. YARP mRNA was decreased only by the ECS method, suggesting that YARP expression is different at embryonic and adult neurogenic stage.

Upregulation of fatty acyl-CoA thioesterases in the heart and skeletal muscle of rats fed a high-fat diet.

In rodent models of diet-induced obesity, prolonged high-fat feeding increases the cellular uptake of fatty acids and causes lipotoxicity in the heart and skeletal muscle, where substrate overload to beta-oxidation generates mitochondrial stress. We examined the hypothesis that, because of its catalytic properties, acyl-CoA thioesterase (ACOT) would counteract these detrimental situations by modulating intracellular acyl-CoA levels. Rats were fed a low- or high-fat diet for up to 20 weeks, and the expressions of ACOT isoforms and fatty acid beta-oxidation enzymes were analyzed by western blotting. The expressions of ACOT1, ACOT2 and ACOT7 proteins in the heart and soleus muscle were significantly increased, by 2.0-7.6-fold, in rats fed the high-fat diet as compared with the low-fat diet group. These effects were accompanied by increases in carnitine palmitoyltransferase and acyl-CoA oxidase expression. However, ACOT was not induced in the extensor digitorum longus muscle or the liver. Subcellular fractionation of heart and soleus muscle homogenates confirmed expression of both the cytosolic and mitochondrial ACOT isoforms. These results underscore the functional relationship between ACOT and fatty acid oxidation, and suggest adaptive upregulation of ACOT to protect against fatty acid oversupply in the heart and skeletal muscle.

Eosinophils are required for the induction of bronchial hyperresponsiveness in a Th transfer model of BALB/c background.

BACKGROUND: Helper T (Th) cells are deeply involved in the pathophysiology of bronchial asthma, such as eosinophilic inflammation, bronchial hyperresponsiveness (BHR), airflow limitation and remodeling. It is still unclear whether Th cells contribute to BHR independently of eosinophilic inflammation. The double GATA (dblGATA) site is a high-affinity GATA-binding site in the GATA-1 promoter. dblGATA site-deficient (Delta dblGATA) mice lack eosinophils. METHOD: Ovalbumin (OVA)-reactive Th clones were transferred into Delta dblGATA and wild-type (WT) mice of BALB/c background. The number of eosinophils in the bronchoalveolar lavage fluid (BALF) and bronchial responsiveness to methacholine were examined after OVA challenge. RESULTS: The number of BALF eosinophils was significantly increased in WT mice, but not detectable in Delta dblGATA mice. BHR was also induced in WT mice, but significantly attenuated in Delta dblGATA mice. CONCLUSION: Eosinophils are involved in T-cell-mediated BHR.

Primary intrahepatic squamous cell carcinoma in a sika deer.

A white nodule was detected in the liver of a wild female sika deer. The nodule was histologically diagnosed as squamous cell carcinoma (SCC), and it transitioned into a hyperplastic and chronically inflamed intrahepatic bile duct showing Fasciola infection. Therefore, the tumor was demonstrated to have originated from the biliary epithelium of the intrahepatic bile duct. Hyperplastic and chronic inflammatory changes of the biliary epithelium might have contributed the carcinogenesis of the present case, as proposed in human primary intrahepatic SCC cases. To the best of our knowledge, this is the first reported case of primary intrahepatic SCC in an animal.

Intratesticular localization of the organic solute carrier protein, OSCP1, in spermatogenic cells in mice.

Organic solute carrier protein 1 (OSCP1) is a recently described human gene that facilitates the transport of various organic solutes into the cell, when expressed in frog eggs. In this study, we cloned a mouse ortholog of OSCP1 encoding 379 amino acid protein, with 94% homology to the human counterpart. The mouse OSCP1 mRNA was predominantly expressed in the testis, in which it was attributed to the spermatogenic cells, except the spermatogonia. Immunohistochemistry confirmed that OSCP1 protein is continuously expressed during spermatogenesis in a stage- and cell type-specific manner, in the leptotene spermatocytes at stage IX through step 15 spermatids. Subcellular fractionation of mouse testis homogenates indicated that OSCP1 is a 45-kDa cytosolic protein. Moreover, when green fluorescent protein-OSCP1 fusion constructs were transfected into cultured cells, the fluorescence localized evenly in the cytoplasm. These results suggest that mouse testis OSCP1 may indirectly mediate substrate uptake into meiotic and spermiogenic germ cells, within the cytosol.

[Cerebellar Ataxia and Autoantibodies].

The cerebellum is one of the main targets in the central nervous system for autoimmunity. Immune-mediated cerebellar ataxias include gluten ataxia, GAD antibody-associated cerebellar ataxia, Hashimoto's encephalopathy, and paraneoplastic cerebellar degeneration. Autoimmune cerebellar ataxia may be of either insidious or subacute onset, and vertigo or transient neurological symptoms occur in some patients before the onset of the disease, in contrast to spinocerebellar degeneration. If autoimmune cerebellar ataxia is suspected, early diagnosis and introduction of treatment are very important. For diagnosis, testing for gliadin antibody, TG6 antibody, GAD antibody, thyroid antibody, and anti-neuronal antibodies, including mGluR1, is useful. Magnetic resonance imaging voxel-based morphometry is also useful because it can detect cortical cerebellar atrophy of autoimmune cerebellar ataxia, different from spinocerebellar ataxia. As for treatment, it is important to remove autoimmune triggering factors (e.g.,dietary gluten or neoplasm). When the ataxia symptoms are causing hindrances in the daily life, it is worth considering immunotherapy including IVIg, steroid therapy and so on.

Chronic high-fat feeding impairs adaptive induction of mitochondrial fatty acid combustion-associated proteins in brown adipose tissue of mice.

Since brown adipose tissue (BAT) is involved in thermogenesis using fatty acids as a fuel, BAT activation is a potential strategy for treating obesity and diabetes. However, whether BAT fatty acid combusting capacity is preserved in these conditions has remained unclear. We therefore evaluated expression levels of fatty acid oxidation-associated enzymes and uncoupling protein 1 (Ucp1) in BAT by western blot using a diet-induced obesity C57BL/6J mouse model. In C57BL/6J mice fed a high-fat diet (HFD) over 2-4 weeks, carnitine palmitoyltransferase 2 (Cpt2), acyl-CoA thioesterase (Acot) 2, Acot11 and Ucp1 levels were significantly increased compared with baseline and control low-fat diet (LFD)-fed mice. Similar results were obtained in other mouse strains, including ddY, ICR and KK-Ay, but the magnitudes of the increase in Ucp1 level were much smaller than in C57BL/6J mice, with decreased Acot11 levels after HFD-feeding. In C57BL/6J mice, increased levels of these mitochondrial proteins declined to near baseline levels after a longer-term HFD-feeding (20 weeks), concurrent with the accumulation of unilocular, large lipid droplets in brown adipocytes. Extramitochondrial Acot11 and acyl-CoA oxidase remained elevated. Treatment of mice with Wy-14,643 also increased these proteins, but was less effective than 4 week-HFD, suggesting that mechanisms other than peroxisome proliferator-activated receptor α were also involved in the upregulation. These results suggest that BAT enhances its fatty acid combusting capacity in response to fat overload, however profound obesity deprives BAT of the responsiveness to fat, possibly via mitochondrial alteration.

Prevalence of Autoantibodies and the Efficacy of Immunotherapy for Autoimmune Cerebellar Ataxia.

OBJECTIVE: Autoimmune cerebellar ataxias were recently reported to be treatable. However, the proportion of patients with cortical cerebellar atrophy of unknown etiology with autoimmune-associated cerebellar ataxia and the actual effectiveness of immunotherapy in these diseases remain unknown. METHODS: We measured the level of autoantibodies (including anti-gliadin antibody, anti-glutamic acid decarboxylase (GAD) antibody, and anti-thyroid antibody) in 58 Japanese patients with cerebellar ataxia, excluding those with multiple system atrophy, hereditary spinocerebellar ataxia, cancer, or those who were receiving phenytoin, and the efficacy of immunotherapy was assessed. RESULTS: Thirty-one of 58 (53%) patients were positive for anti-GAD antibody, anti-gliadin antibody, or anti-thyroid antibody. Seven of the 12 anti-gliadin antibody-positive patients, three of the four anti-GAD antibody-positive patients, and three of the six anti-thyroid antibody-positive patients responded well to immunotherapy, indicating that 59% of patients with ataxia-associated antibody-positive cerebellar ataxia undergoing immunotherapy responded well. CONCLUSION: Some patients with cerebellar ataxia have autoimmune conditions and diagnosing autoimmune cerebellar ataxia is therefore an important component in the care of patients with this disease entity.

Eight patients with Williams syndrome and craniosynostosis.

Williams syndrome (WS) is a well-known genetic syndrome caused by a microdeletion on chromosome 7q11.23 encompassing the elastin gene. It is characterized by distinctive facies, congenital cardiovascular malformations, intellectual disabilities, and various other manifestations. Some patients were reported with craniosynostosis. Here, we report 8 WS cases diagnosed with craniosynostosis using three-dimensional cranial computed tomography. These findings suggest that craniosynostosis may occur more frequently in WS patients than expected.

Characterization of mouse homolog of brain acyl-CoA hydrolase: molecular cloning and neuronal localization.

Acyl-CoA hydrolase could provide a mechanism via its potency to modulate cellular concentrations of acyl-CoAs for the regulation of various cellular events including fatty acid metabolism and gene expression. However, only limited evidence of this is available. To better understand the physiological role of this enzyme, we characterized a mouse brain acyl-CoA hydrolase, mBACH. The cloned cDNA for mBACH encoded a 338-amino-acid polypeptide with >95% identity to the human and rat homologs, indicating that the BACH gene is highly conserved among species. This was supported by the similarity in genomic organization of the BACH gene between humans and mice. Bacterially expressed mBACH was highly active against long-chain acyl-CoAs with a relatively broad specificity for chain length. While palmitoyl-CoA hydrolase activity was widely distributed in mouse tissues, it was marked in the brain, consistent with mBACH being almost exclusively distributed in this tissue, where >80% of the enzyme activity was explained by mBACH present in the cytosol. Immunohistochemistry demonstrated a neuronal localization of mBACH in both the central and peripheral nervous systems. In neurons, mBACH was distributed throughout the cell body and neurites. Although four isoforms except mBACH itself, that may be generated by the alternative use of exons of a single mBACH gene, were cloned, their mRNA levels in the brain were estimated to be negligible. However, a 50-kDa polypeptide besides the major one of 43-kDa seemed to be translated from the mBACH mRNA with differential in-frame ATG triplets used as the initiation codon. These findings will contribute to the functional analysis of the BACH gene using mice including genetic studies.

Expression of acyl-CoA hydrolase in the developing mouse brain.

Brain acyl-CoA hydrolase (BACH) is a cytosolic enzyme responsible for the brain long-chain acyl-CoA thioesterase activity, that is the highest in the body. BACH was detected in the mouse brain as early as embryonic day (E) 11.5 by immunoblotting. The level of the major isoform (43-kDa) was low until E12.5, but promptly elevated to a peak 7 days after birth. Thereafter, it declined somewhat and reached a steady-state level in adulthood. These changes in BACH expression were approximately reflected in the palmitoyl-CoA hydrolyzing activity in the developing mouse brain, and the time course was quite similar to that of microtubule-associated protein 2 (MAP2) expression. In immunohistochemistry of E14.5 embryo brains, cells expressing BACH almost coincided with the cells committed to the neuronal lineage, which expressed MAP2 but not nestin. These results indicate that BACH expression is induced during embryogenesis in association with neuronal differentiation, and persists after terminal differentiation into neurons in postnatal stages, resulting in the constitutive high expression of BACH in the adult brain in a neuron-specific manner.

Hypoglycemic effects of clove (Syzygium aromaticum flower buds) on genetically diabetic KK-Ay mice and identification of the active ingredients.

Clove (Syzygium aromaticum flower buds) EtOH extract significantly suppressed an increase in blood glucose level in type 2 diabetic KK-A(y) mice. In-vitro evaluation showed the extract had human peroxisome proliferator-activated receptor (PPAR)-γ ligand-binding activity in a GAL4-PPAR-γ chimera assay. Bioassay-guided fractionation of the EtOH extract resulted in the isolation of eight compounds, of which dehydrodieugenol (2) and dehydrodieugenol B (3) had potent PPAR-γ ligand-binding activities, whereas oleanolic acid (4), a major constituent in the EtOH extract, had moderate activity. Furthermore, 2 and 3 were shown to stimulate 3T3-L1 preadipocyte differentiation through PPAR-γ activation. These results indicate that clove has potential as a functional food ingredient for the prevention of type 2 diabetes and that 2-4 mainly contribute to its hypoglycemic effects via PPAR-γ activation.