The relationship between carotenoid accumulation and the expression of carotenoid biosynthetic genes during fruit maturation was investigated in three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). We cloned the cDNAs for phytoene synthase (CitPSY), phytoene desaturase (CitPDS), ζ-carotene (car) desaturase (CitZDS), carotenoid isomerase (CitCRTISO), lycopene β-cyclase (CitLCYb), β-ring hydroxylase (CitHYb), zeaxanthin (zea) epoxidase (CitZEP), and lycopene ϵ-cyclase (CitLCYe) from Satsuma mandarin, which shared high identities in nucleotide sequences with Valencia orange, Lisbon lemon, and other plant species. With the transition of peel color from green to orange, the change from β,ϵ-carotenoid (α-car and lutein) accumulation to β,β-carotenoid (β-car, β-cryptoxanthin, zea, and violaxanthin) accumulation was observed in the flavedos of Satsuma mandarin and Valencia orange, accompanying the disappearance of CitLCYe transcripts and the increase in CitLCYb transcripts. Even in green fruit, high levels of β,ϵ-carotenoids and CitLCYe transcripts were not observed in the juice sacs. As fruit maturation progressed in Satsuma mandarin and Valencia orange, a simultaneous increase in the expression of genes (CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP) led to massive β,β-xanthophyll (β-cryptoxanthin, zea, and violaxanthin) accumulation in both the flavedo and juice sacs. The gene expression of CitCRTISO was kept low or decreased in the flavedo during massive β,β-xanthophyll accumulation. In the flavedo of Lisbon lemon and Satsuma mandarin, massive accumulation of phytoene was observed with a decrease in the transcript level for CitPDS. Thus, the carotenoid accumulation during citrus fruit maturation was highly regulated by the coordination of the expression among carotenoid biosynthetic genes. In this paper, the mechanism leading to diversity in β,β-xanthophyll compositions between Satsuma mandarin and Valencia orange was also discussed on the basis of the substrate specificity of β-ring hydroxylase and the balance of expression between upstream synthesis genes (CitPSY, CitPDS, CitZDS, and CitLCYb) and downstream synthesis genes (CitHYb and CitZEP).

The bioavailability of dietary phytochemicals may be influenced by the food matrix in which they are consumed. In this study the impact of a full-fat yogurt on the bioavailability and metabolism of orange juice flavanones was investigated. Human plasma and urine were collected over a 24 h period after the consumption of 250 mL of orange juice containing a total of 168 micromol of hesperetin-7-O-rutinoside and 12 micromol of naringenin-7-O-rutinoside, with and without 150 mL of full-fat yogurt. The juice also contained 1 g of paracetamol and 5 g of lactulose. HPLC-MS(2) analysis revealed the accumulation of hesperetin-7-O-glucuronide, and an unassigned hesperetin-O-glucuronide metabolite in plasma reached a peak concentration (C(max)) of 924 +/- 224 nmol/L, 4.4 +/- 0.5 h (T(max)) after orange juice ingestion. The T(max) is indicative of absorption in the colon. When the juice was consumed with yogurt, neither the C(max) at 661 +/- 170 nmol/L nor the T(max) at 5.1 +/- 0.4 h were significantly different from those obtained with juice alone. The two hesperetin glucuronides were also excreted in urine along with a third hesperetin-O-glucuronide, two hesperetin-O-glucuronide-O-sulfates, a hesperetin-O-diglucuronide, a naringenin-O-diglucuronide, and, tentatively identified, naringenin-7-O-glucuronide and naringenin-4'-O-glucuronide. This indicates the occurrence of substantial, postabsorption, phase II metabolism prior to urinary excretion. The quantity of flavanone metabolites excreted 0-5 h after orange juice ingestion was significantly reduced by yogurt, but over the full 0-24 h urine collection period, the amounts excreted, corresponding to ca. 7.0% of intake, were not affected by the addition of yogurt to the drink. Nor did yogurt have a significant effect on gastric emptying, as determined by plasma paracetamol levels, or on the mouth to cecum transit time of the head of the meal, assessed by measurement of lactulose-derived breath hydrogen. There is also a discussion of the merits of studies of the absorption and metabolism of flavanones based on direct analysis of metabolites by HPLC-MS and the more traditional indirect approach where samples are treated with a mollusc glucuronidase/sulfatase preparation prior to HPLC analysis of the released aglycones.

The Rho family GTPase Rac1 has been implicated in the regulation of glucose uptake in myoblast cell lines. However, no evidence for the role of Rac1 has been provided by a mouse model. The purpose of this study is to test the involvement of Rac1 in insulin action in mouse skeletal muscle. Intravenous administration of insulin indeed elicited Rac1 activation in gastrocnemius muscle, suggesting the involvement of Rac1 in this signaling pathway. We then examined whether insulin-stimulated translocation of the facilitative glucose transporter GLUT4 from its storage sites to the skeletal muscle sarcolemma depends on Rac1. We show that ectopic expression of constitutively activated Rac1, as well as intravenous administration of insulin, caused translocation of GLUT4 to the gastrocnemius muscle sarcolemma, as revealed by immunofluorescent staining of a transiently expressed exofacial epitope-tagged GLUT4 reporter. Of particular note, insulin-dependent, but not constitutively activated Rac1-induced, GLUT4 translocation was markedly suppressed in skeletal muscle-specific rac1-knockout mice compared to control mice. Immunogold electron microscopic analysis of endogenous GLUT4 gave similar results. Collectively, we propose a critical role of Rac1 in insulin-dependent GLUT4 translocation to the skeletal muscle sarcolemma, which has heretofore been predicted solely by cell culture studies.