Metabolic Profiling for Evaluating the Dipeptidyl Peptidase-IV Inhibitory Potency of Diverse Green Tea Cultivars and Determining Bioactivity-Related Ingredients and Combinations.

There are numerous cultivars of tea (Camellia sinensis L.), but the differences in their anti-hyperglycemic-related effects are largely unknown. The inhibition of the dipeptidyl peptidase (DPP)-IV enzyme plays an essential role in controlling hyperglycemia in diabetes by blocking the degradation of incretin hormones, which is necessary for insulin secretion. In this study, we examined the DPP-IV inhibitory activity of leaf extracts from diverse Japanese green tea cultivars. The inhibitory rates differed among tea extracts. Metabolic profiling (MP), using liquid chromatography-mass spectrometry, of all cultivars revealed compositional differences among cultivars according to their DPP-IV inhibitory capacity. Epigallocatechin-3-O-(3-O-methyl)gallate, kaempferol-3-O-rutinoside, myricetin-3-O-glucoside/galactoside, and theogallin were newly identified as DPP-IV inhibitors. The bioactivity of a tea extract was potentiated by adding these ingredients in combination. Our results show that MP is a useful approach for evaluating the DPP-IV inhibitory potency of green tea and for determining bioactivity-related ingredients and combinations.

The combined effect of green tea and α-glucosyl hesperidin in preventing obesity: a randomized placebo-controlled clinical trial.

Green tea, a widely consumed beverage in Asia, contains green tea catechins effective against obesity, especially epigallocatechin-3-O-gallate (EGCG), but must be consumed in an impractically huge amount daily to elicit its biological effect. Meanwhile, citrus polyphenols have various physiological effects that could enhance EGCG functionality. Here we investigated the antiobesity effect of a combination of EGCG and α-glucosyl hesperidin, a citrus polyphenol, at doses that have not been previously reported to exert antiobesity effects by themselves in any clinical trial. In a randomized, placebo-controlled, double-blinded, and parallel-group-designed clinical trial, 60 healthy Japanese males and females aged 30-75 years consumed green tea combined with α-glucosyl hesperidin (GT-gH), which contained 178 mg α-glucosyl hesperidin and 146 mg EGCG, for 12 weeks. Physical, hematological, blood biochemical, and urine examinations showed that GT-gH is safe to use. At week 12, GT-gH prevented weight gain and reduced body mass index (BMI) compared with the placebo. Especially in those aged < 50 years, triglyceride and body fat percentage decreased at week 6, visceral fat level and body fat percentage decreased at week 12; body weight, BMI, and blood LDL/HDL ratio also decreased. In conclusion, taking GT-gH prevents weight gain, and the antiobesity effect of GT-gH was more pronounced in people aged < 50 years.

Arabidopsis Bax inhibitor-1 promotes sphingolipid synthesis during cold stress by interacting with ceramide-modifying enzymes.

Bax inhibitor-1 (BI-1) is a widely conserved cell death suppressor localized in the endoplasmic reticulum membrane. Our previous results revealed that Arabidopsis BI-1 (AtBI-1) interacts with not only Arabidopsis cytochrome b 5 (Cb5), an electron transfer protein, but also a Cb5-like domain (Cb5LD)-containing protein, Saccharomyces cerevisiae fatty acid 2-hydroxylase 1, which 2-hydroxylates sphingolipid fatty acids. We have now found that AtBI-1 binds Arabidopsis sphingolipid Δ8 long-chain base (LCB) desaturases AtSLD1 and AtSLD2, which are Cb5LD-containing proteins. The expression of both AtBI-1 and AtSLD1 was increased by cold exposure. However, different phenotypes were observed in response to cold treatment between an atbi-1 mutant and a sld1sld2 double mutant. To elucidate the reasons behind the difference, we analyzed sphingolipids and found that unsaturated LCBs in atbi-1 were not altered compared to wild type, whereas almost all LCBs in sld1sld2 were saturated, suggesting that AtBI-1 may not be necessary for the desaturation of LCBs. On the other hand, the sphingolipid content in wild type increased in response to low temperature, whereas total sphingolipid levels in atbi-1 were unaltered. In addition, the ceramide-modifying enzymes AtFAH1, sphingolipid base hydroxylase 2 (AtSBH2), acyl lipid desaturase 2 (AtADS2) and AtSLD1 were highly expressed under cold stress, and all are likely to be related to AtBI-1 function. These findings suggest that AtBI-1 contributes to synthesis of sphingolipids during cold stress by interacting with AtSLD1, AtFAH1, AtSBH2 and AtADS2.

Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/NEDD8-activating enzyme in Arabidopsis seedlings.

SMALL ACIDIC PROTEIN 1 (SMAP1) functions upstream of the degradation of AUX/IAA-proteins in the response to 2,4-dichlorophenoxyacetic acid and physically interacts with the COP9 SIGNALOSOME (CSN). Also, its function is linked to RELATED TO UBIQUITIN (RUB) modification. To further investigate the relationship between SMAP1 and the RUB modification system, we examined the effect of MLN4924, an inhibitor of RUB/NEDD8-activating E1 enzyme, on the growth of Arabidopsis thaliana. We found that the anti-auxin resistant 1 mutants, which lack SMAP1, are more sensitive to MLN4924 than wild type and that SMAP1 is responsible for this hypersensitivity. This new evidence supports our previous speculation that SMAP1 acts in Cullin-RING ubiquitin E3 ligase regulated signaling processes via its interaction with components associated with the RUB modification system.

SMALL ACIDIC PROTEIN1 acts with RUB modification components, the COP9 signalosome, and AXR1 to regulate growth and development of Arabidopsis.

Previously, a dysfunction of the SMALL ACIDIC PROTEIN1 (SMAP1) gene was identified as the cause of the anti-auxin resistant1 (aar1) mutant of Arabidopsis (Arabidopsis thaliana). SMAP1 is involved in the response pathway of synthetic auxin, 2,4-dichlorophenoxyacetic acid, and functions upstream of the auxin/indole-3-acetic acid protein degradation step in auxin signaling. However, the exact mechanism by which SMAP1 functions in auxin signaling remains unknown. Here, we demonstrate that SMAP1 is required for normal plant growth and development and the root response to indole-3-acetic acid or methyl jasmonate in the auxin resistant1 (axr1) mutation background. Deletion analysis and green fluorescent protein/glutathione S-transferase pull-down assays showed that SMAP1 physically interacts with the CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME (CSN) via the SMAP1 F/D region. The extremely dwarf phenotype of the aar1-1 csn5a-1 double mutant confirms the functional role of SMAP1 in plant growth and development under limiting CSN functionality. Our findings suggest that SMAP1 is involved in the auxin response and possibly in other cullin-RING ubiquitin ligase-regulated signaling processes via its interaction with components associated with RELATED TO UBIQUITIN modification.

A gene encoding SMALL ACIDIC PROTEIN 2 potentially mediates the response to synthetic auxin, 2,4-dichlorophenoxyacetic acid, in Arabidopsis thaliana.

The SMALL ACIDIC PROTEIN 2 (SMAP2) gene is a paralogue of the SMAP1 gene that mediates the response to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) in the root of Arabidopsis thaliana. Their encoded proteins, SMAP1 and SMAP2, are similar in calculated molecular weight and isoelectric point, and in having a highly conserved phenylalanine and aspartic acid-rich domain. RNA expression analysis showed that SMAP1 mRNA is present throughout the plant body while SMAP2 mRNA is restricted to siliques and anthers. Over-expression of the SMAP2 gene, as well as SMAP1, by 35S cauliflower mosaic virus promoter restored sensitivity to 2,4-D in the 2,4-D-resistant mutant, aar1, which is defective in SMAP1 function. The results suggest that SMAP2 has an ability to mediate the 2,4-D response and is expressed only in restricted tissues.

A small acidic protein 1 (SMAP1) mediates responses of the Arabidopsis root to the synthetic auxin 2,4-dichlorophenoxyacetic acid.

2,4-dichlorophenoxyacetic acid (2,4-D), a chemical analogue of indole-3-acetic acid (IAA), is widely used as a growth regulator and exogenous source of auxin. Because 2,4-D evokes physiological and molecular responses similar to those evoked by IAA, it is believed that they share a common response pathway. Here, we show that a mutant, antiauxin resistant1 (aar1), identified in a screen for resistance to the anti-auxin p-chlorophenoxy-isobutyric acid (PCIB), is resistant to 2,4-D, yet nevertheless responds like the wild-type to IAA and 1-napthaleneacetic acid in root elongation and lateral root induction assays. That the aar1 mutation alters 2,4-D responsiveness specifically was confirmed by analysis of GUS expression in the DR5:GUS and HS:AXR3NT-GUS backgrounds, as well as by real-time PCR quantification of IAA11 expression. The two characterized aar1 alleles both harbor multi-gene deletions; however, 2,4-D responsiveness was restored by transformation with one of the genes missing in both alleles, and the 2,4-D-resistant phenotype was reproduced by decreasing the expression of the same gene in the wild-type using an RNAi construct. The gene encodes a small, acidic protein (SMAP1) with unknown function and present in plants, animals and invertebrates but not in fungi or prokaryotes. Taken together, these results suggest that SMAP1 is a regulatory component that mediates responses to 2,4-D, and that responses to 2,4-D and IAA are partially distinct.