Flavonoids have differential effects on glucose absorption in rats (Rattus norvegicus) and American robins (Turdis migratorius).

Mounting evidence suggests that small birds rely largely on non-mediated intestinal absorption of glucose through the paracellular pathway, while non-flying mammals rely on mediated absorption across the enterocyte membranes by using glucose transporters SGLT-1 and GLUT-2. Relying on non-mediated transport of glucose may decrease its absorption rate at low glucose concentrations but may release small birds from the effects of glucose transport inhibitors. We evaluated transport by using flavonoids known to inhibit glucose transport in vitro. Quercetin, isoquercetrin, and phloridzin were tested in rats (Rattus norvegicus) and robins (Turdis migratirius), and naringenin, naringenin-7-glucoside, genistein, epigallocatechin gallate (EGCG), and phloretin were used only in rats. By using a pharmacokinetic approach that involves serial blood collection and area under the curve calculations, we determined the bioavailability of 3-0-methyl D-glucose, the non-metabolized analogue of D-glucose. Six of the eight flavonoids tested in rats significantly decreased the absorption of 3-0-methyl D-glucose, while none of the flavonoids tested in robins significantly decreased the bioavailability of 3-0-methyl D-glucose. We conclude that flavonoids effectively decrease glucose absorption in rats, which rely on mediated absorption of glucose, but that flavonoids do not have an effect in robins, which rely on non-mediated absorption of glucose.

Assessment of radiolabeled D-glucose and the nonmetabolizable analog 3-O-methyl-D-glucose as tools for in vivo absorption studies.

3-O-methyl-D-glucose has been extensively used as a proxy for d-glucose uptake. This nonmetabolizable analog has lower affinity for transporters, potentially leading to underestimates of glucose absorption rates as well as overestimates of the nutritional significance of passive uptake. Here we sought to precisely quantify the bias, if any, incurred when using 3-O-methyl-D-glucose by comparing relative absorption rates with D-glucose in vivo in a seasonally frugivorous bird, the American robin. By simultaneously administering these D-glucose probes with L-glucose--the latter absorbed only via nonmediated mechanisms and the former absorbed by both mediated and nonmediated mechanisms--using common pharmacokinetic procedures, we were able to estimate the nutritional significance of paracellular uptake in this species. The relative absorption rate of 3-O-methyl-D-glucose calculated over the initial absorptive phase was not significantly different from that of D-glucose, indicating that the former provides reasonable estimates of glucose absorption rates in vivo. The ratio of L-glucose to D-glucose cumulative fractional absorption indicates that around 60% of total glucose absorption in American robins is paracellular and showed no apparent bias in using 3-O-methyl-D-glucose when averaged over the entire initial absorptive phase. Although the absorption and elimination kinetics of radiolabeled D-glucose were appropriate for pharmacokinetic analysis in this study, because of the potential for interspecific differences in loss to catabolism, it should be used in vivo with caution and with independent verification of absorption efficiency.

Is alpha-pinene a substrate for permeability-glycoprotein in wood rats?

alpha-Pinene is the dominant monoterpene in Juniperus monosperma. Wood rat species in the genus Neotoma that consume J. monosperma vary in their inclusion of it in their wild diet and in their tolerance of whole J. monosperma or alpha-pinene in laboratory feeding trials. A proposed mechanism for variable tolerance is a difference in absorption of alpha-pinene from the small intestine that is mediated by the intestinal transporter permeability glycoprotein (Pgp). To determine if alpha-pinene is a Pgp substrate, we tested whether it can competitively inhibit Pgp and thereby increase the accumulation of a known Pgp substrate (digoxin) in (1) everted sleeves of small intestine from Neotoma stephensi, a juniper specialist, N. albigula, a sympatric generalist that consumes juniper, N. cinerea, a more distantly related generalist, and Sprague-Dawley rats, and (2) in Caco-2 cells that over express Pgp. We also measured Pgp ATPase phosphate production in transfected insect membrane vesicles exposed to alpha-pinene. We found no significant increase in digoxin accumulation with competitive inhibition experiments, and no increase in phosphate production with transfected membranes, at any concentration of alpha-pinene up to 100 muM. To test whether other compounds in juniper affect Pgp activity, we acclimated five N. stephensi to a juniper diet for 5 d, but found no significant effect compared to animals on control diet. Our data suggest that alpha-pinene is not a Pgp substrate.

A new method to measure intestinal activity of P-glycoprotein in avian and mammalian species.

Permeability-glycoprotein (Pgp) actively exports numerous potentially toxic compounds once they diffuse into the cell membrane of intestinal epithelial cells. We adapted the everted sleeve technique to make the first measures of intestinal Pgp function in an avian species (chicken) and in wild mammalian species, and compared them to laboratory rats. Tissues maintained both structural and functional integrity, and our method offers advantages over other in vitro techniques by using smaller intestinal sections (1 cm), and shorter incubation times (8-12 min). To determine Pgp function, we compared accumulation of [(3)H]-digoxin in sleeves incubated in Ringer solution with and without a transport-saturating concentration of a competitive inhibitor, cyclosporin A. We demonstrated significant variation in Pgp activity within individuals along the intestine, between populations fed different diets, and between species (laboratory rats had one-third to one-fifth the Pgp activity of wild rodents). In chicken, we also tested the effect of natural metabolites on digoxin accumulation. We found that among flavonoids, genistein (200 microM), found in soy and other legumes, but not quercetin (10, 30, 100, 330 microM) or the 3-beta-glycoside isoquercetrin (100 microM), significantly increased digoxin accumulation. Among fungal metabolites, sterigmatocystin (5 microM), but not aflatoxin B1 (5 microM), significantly increased digoxin accumulation.

Intestinal capacity of P-glycoprotein is higher in the juniper specialist, Neotoma stephensi, than the sympatric generalist, Neotoma albigula.

Permeability-glycoprotein (Pgp) is a membrane-bound, ATP-dependent, transport protein that excludes many cytotoxic compounds including plant metabolites and pollutants from the barrier epithelia of many tissues including the small intestine. We hypothesized that intestinal Pgp capacity would be higher in Neotoma stephensi, a specialist on Juniperus monosperma known to be high in plant toxins, than the sympatric generalist, Neotoma albigula, which consumes juniper in the field, but is unable to tolerate a high juniper diet. We measured Pgp activity as the difference in accumulation of a known Pgp substrate, digoxin, between everted sections of small intestine exposed to ethanol vehicle control and a maximal level of a known competitive inhibitor of Pgp, cyclosporin A. We estimated intestinal capacity by averaging Pgp activity along the intestine and multiplying by total small intestine mass. These first measures of Pgp in wild mammals show a significant difference among species with the juniper specialist, N. stephensi, exhibiting a 2.4 fold higher capacity than the generalist, N. albigula. This result suggests that Pgp may play a role in the ability of N. stephensi to tolerate juniper.