Edwin R. Price

Digestive Adaptations of Aerial Lifestyles

Flying vertebrates (birds and bats) are under selective pressure to reduce the size of the gut and the mass of the digesta it carries. Compared with similar-sized nonflying mammals, birds and bats have smaller intestines and shorter retention times. We review evidence that birds and bats have lower spare digestive capacity and partially compensate for smaller intestines with increased paracellular nutrient absorption.

Changes in Fatty Acid Composition During Starvation in Vertebrates: Mechanisms and Questions

Animals often rely heavily on stored lipids as a fuel source during extended periods of fasting/starvation; this results in notable decreases in lipid content during the fast. Additionally, the composition of stored lipids often changes during periods of fasting, although the reasons for these compositional changes have not been fully explored. We examine the changes in fatty acid composition that occur during starvation through the lens of two important processes: (1) changes in the triacylglycerol to phospholipid ratio and (2) selective mobilization and oxidation of particular fatty acids. As triacylglycerols are oxidized, the ratio of triacylglycerols to phospholipids should decrease, resulting in higher overall proportions of polyunsaturated fatty acids, which are more abundant in phospholipids. Selective mobilization of fatty acids results in the preferential oxidation of short-chained and highly unsaturated fatty acids, the proportions of which should therefore decrease during starvation. In general, decreases in the triacylglycerol to phospholipid ratio appear to explain observed changes in fatty acid composition of whole animals and some tissues. On the other hand, selective mobilization of fatty acids can explain many of the compositional changes observed in adipose tissue. Together, these two processes should be considered when seeking to identify exceptional species or examples of unique lipid regulation. One notable exception is hibernating mammals, which do not exhibit standard selective mobilization patterns, possibly in order to conserve certain essential polyunsaturated fatty acids during their hibernation fast.

Paracellular absorption in laboratory mice: Molecule size-dependent but low capacity

Water-soluble nutrients are absorbed by the small intestine via transcellular and paracellular processes. The capacity for paracellular absorption seems lower in nonfliers than in fliers, although that conclusion rests largely on a comparison of relatively larger nonflying mammals (>155g) and relatively smaller flying birds (<155g). We report on paracellular absorption in laboratory mice, the smallest nonflying mammal species studied to date. Using a standard pharmacokinetic technique, we measured the extent of absorption (fractional absorption=f) of inert carbohydrate probes: L-arabinose (M(r)=150.13Da) and cellobiose (342.3) that are absorbed exclusively by the paracellular route, and 3-O-methyl D-glucose (3OMD-glucose) (M(r)=194) absorbed both paracellularly and transcellularly. f was measured accurately in urine collection trials of 5-10h duration. Absorption of 3OMD-glucose by mice was essentially complete (f=0.95±0.07) and much higher than that for L-arabinose (f=0.21±0.02), indicating that in mice, like other nonflying mammals, >80% of glucose is absorbed by mediated process(es) rather than the passive, paracellular route. As in all other vertebrates, absorption of cellobiose (f=0.13±0.02) was even lower than that for L-arabinose, suggesting an equivalent molecular size cut-off for flying and nonflying animals and thus a comparable effective TJ aperture. An important ecological implication is that smaller water-soluble plant secondary metabolites that have been shown to be absorbed by the paracellular path in cell culture, such as phenolics and alkaloids, might be absorbed in substantial amounts by bats and small birds relative to nonflying mammals such as mice.

Paracellular nutrient absorption is higher in bats than rodents: integrating from intact animals to the molecular level.

Flying vertebrates have been hypothesized to rely heavily on paracellular absorption of nutrients to compensate for having smaller intestines than non-flyers. We tested this hypothesis in an insectivorous bat (Myotis lucifugus) and two insect-eating rodents (Onychomys leucogaster and Peromyscus leucopus). In intact animals, the fractional absorption of orally dosed l-arabinose (Mr 150) was 82% in M. lucifugus, which was more than twice that of the rodents. Absorption of creatinine (Mr 113) was greater than 50% for all species and did not differ between M. lucifugus and the rodents. We also conducted intestinal luminal perfusions on anesthetized animals. Absorption of l-arabinose per nominal surface area in M. lucifugus was nearly double that of the rodents, while absorption of creatinine was not different among species. Using an everted sleeve preparation, we demonstrated that high concentrations of l-arabinose and creatinine did not inhibit their own uptake, validating their use as passive, paracellular probes. Histological measurements indicated that M. lucifugus has more cells, and presumably more tight junctions, per nominal surface area than P. leucopus. This seems unlikely to explain entirely the higher absorption of l-arabinose in M. lucifugus during perfusions, because l-arabinose absorption normalized to the number of enterocytes was still double that of P. leucopus. As an alternative, we investigated tight junction gene expression. M. lucifugus had higher expression of claudin-1 and claudin-15, and lower expression of claudin-2 relative to P. leucopus. Expression of claudin-7 and occludin did not differ among species. Taken together, our results support the hypothesis that bats have evolved higher paracellular nutrient absorption than non-flying animals, and that this phenomenon might be driven by both histological characteristics and differences in tight junction gene expression.

Cold exposure increases intestinal paracellular permeability to nutrients in the mouse

SUMMARY In situations of increased energy demand and food intake, animals can often acclimate within several days. The intestine generally responds to elevated digestive demand by increasing in size. However, there is likely a limit to how quickly the intestine can grow to meet the new demand. We investigated the immediate and longer-term changes to intestinal properties of the mouse when suddenly exposed to 4°C. We hypothesized that paracellular permeability to nutrients would increase as part of an immediate response to elevated absorptive demand. We measured absorption of l-arabinose, intestinal size and gene expression of several tight junction proteins (claudin-2, claudin-4, claudin-15 and ZO-1) at three time points: pre-exposure, and after 1 day and 2 weeks of cold exposure. Cold exposure increased food intake by 62% after 2 weeks but intake was not significantly increased after 1 day. Intestinal wet mass was elevated after 1 day and throughout the experiment. Absorption of arabinose rose by 20% after 1 day in the cold and was 33% higher after 2 weeks. Expression of claudin-2 increased after 1 day of cold exposure, but there were no changes in expression of any claudin genes when normalized to ZO-1 expression. Our results indicate that intestinal mass can respond rapidly to increased energy demand and that increased paracellular permeability is also part of that response. Increased paracellular permeability may be a consequence of enterocyte hyperplasia, resulting in more tight junctions across which molecules can absorb.

Intestinal perfusion indicates high reliance on paracellular nutrient absorption in an insectivorous bat Tadarida brasiliensis

Flying vertebrates have been hypothesized to have a high capacity for paracellular absorption of nutrients. This could be due to high permeability of the intestines to nutrient-sized molecules (i.e., in the size range of amino acids and glucose, MW 75-180 Da). We performed intestinal luminal perfusions of an insectivorous bat, Tadarida brasiliensis. Using radio-labeled molecules, we measured the uptake of two nutrients absorbed by paracellular and transporter-mediated mechanisms (L-proline, MW 115 Da, and D-glucose, MW 180 Da) and two carbohydrates that have no mediated transport (L-arabinose, MW 150 Da, and lactulose, MW 342 Da). Absorption of lactulose (0.61±0.06 nmol min(-1) cm(-1)) was significantly lower than that of the smaller arabinose (1.09±0.04 nmol min(-1) cm(-1)). Glucose absorption was significantly lower than that of proline at both nutrient concentrations (10mM and 75 mM). Using the absorption of arabinose to estimate the portion of proline absorption that is paracellular, we calculated that 25.1±3.0% to 66.2±7.8% of proline absorption is not transporter-mediated (varying proline from 1 mM to 75 mM). These results confirm our predictions that 1) paracellular absorption is molecule size selective, 2) absorption of proline would be greater than glucose absorption in an insectivore, and 3) paracellular absorption represents a large fraction of total nutrient absorption in bats.

High paracellular nutrient absorption in intact bats is associated with high paracellular permeability in perfused intestinal segments

Water-soluble nutrients are absorbed by the small intestine via transcellular and paracellular mechanisms. Based on a few previous studies, the capacity for paracellular nutrient absorption seems greater in flying mammals than in nonflying mammals, but there has been little investigation of the mechanisms driving this difference. Therefore, we studied three species each of bats (Artibeus lituratus, Sturnira lilium and Carollia perspicillata) and nonflying mammals (Akodon montensis, Mus musculus and Rattus norvegicus). Using standard pharmacokinetic techniques in intact animals, we confirmed the greater paracellular nutrient absorption in the fliers, comparing one species in each group. Then we conducted in situ intestinal perfusions on individuals of all species. In both approaches, we measured the absorption of 3OMD-glucose, a nonmetabolizable glucose analog absorbed both paracellularly and transcellularly, as well as l-arabinose, which has no mediated transport. Fractional absorption of l-arabinose was three times higher in the bat (S. lilium: 1.2±0.24) than in the rodent (A. montensis: 0.35±0.04), whereas fractional absorption of 3OMD-glucose was complete in both species (1.46±0.4 and 0.97±0.12, respectively). In agreement, bats exhibited two to 12 times higher l-arabinose clearance per square centimeter nominal surface area than rodents in intestinal perfusions. Using l-arabinose, we estimated that the contribution of the paracellular pathway to total glucose absorption was higher in all three bats (109–137%) than in the rodents (13–39%). These findings contribute to an emerging picture that reliance on the paracellular pathway for nutrient absorption is much greater in bats relative to nonflying mammals and that this difference is driven by differences in intestinal permeability to nutrient-sized molecules.

Intestinal paracellular absorption is necessary to support the sugar oxidation cascade in nectarivorous bats.

ABSTRACT We made the first measurements of the capacity for paracellular nutrient absorption in intact nectarivorous bats. Leptonycteris yerbabuenae (20u2005g mass) were injected with or fed inert carbohydrate probes l-rhamnose and d(+)-cellobiose, which are absorbed exclusively by the paracellular route, and 3-O-methyl-d-glucose (3OMD-glucose), which is absorbed both paracellularly and transcellularly. Using a standard pharmacokinetic technique, we collected blood samples for 2u2005h after probe administration. As predicted, fractional absorption (f) of paracellular probes declined with increasing Mr in the order of rhamnose (f=0.71)>cellobiose (f=0.23). Absorption of 3OMD-glucose was complete (f=0.85; not different from unity). Integrating our data with those for glucose absorption and oxidation in another nectarivorous bat, we conclude that passive paracellular absorption of glucose is extensive in nectarivorous bat species, as in other bats and small birds, and necessary to support high glucose fluxes hypothesized for the sugar oxidation cascade. Highlighted Article: Passive paracellular absorption of glucose is extensive in nectarivorous bats and necessary to support high glucose fluxes hypothesized for the sugar oxidation cascade.

A Comparison of mucosal surface area and villous histology in small intestines of the Brazilian free-tailed bat (Tadarida brasiliensis) and the mouse (Mus musculus)

Studies on birds have led to the hypothesis that increased intestinal absorption between enterocytes (paracellular) evolved as a compensation for smaller intestinal size in fliers, which was perhaps selected to minimize the mass of digesta carried. This hypothesis predicts that bats will also exhibit relatively reduced intestinal size and high paracellular absorption, compared with nonflying mammals. Published studies on three bat species indicate relatively high paracellular absorption. One mechanism for increasing paracellular absorption per cm2 small intestine (SI) is increased number of tight junctions (TJs) across which paracellular absorption occurs. To our knowledge, we provide the first comparative analysis of enterocyte size and number in flying and nonflying mammals. Intestines of insectivorous bats Tadarida brasiliensis were compared with Mus musculus using hematoxylin and eosin staining method. Bats had shorter and narrower SIs than mice, and after correction for body size difference by normalizing to mass3/4, the bats had 40% less nominal surface area than the mouse, as predicted. Villous enhancement of surface area was 90% greater in the bat than in the mouse, mainly because of longer villi and a greater density of villi in bat intestines. Bat and mouse were similar in enterocyte diameter. Bats exceeded mice by 54.4% in villous area per cm length SI and by 95% in number of enterocytes per cm2 of the nominal surface area of the SI. Therefore, an increased density of TJs per cm2 SI may be a mechanistic explanation that helps to understand the high paracellular absorption observed in bats compared to nonflying mammals. J. Morphol. 276:102–108, 2015.

Claudin gene expression patterns do not associate with interspecific differences in paracellular nutrient absorption.

Bats exhibit higher paracellular absorption of glucose-sized molecules than non-flying mammals, a phenomenon that may be driven by higher permeability of the intestinal tight junctions. The various claudins, occludin, and other proteins making up the tight junctions are thought to determine their permeability properties. Here we show that absorption of the paracellular probe l-arabinose is higher in a bat (Eptesicus fuscus) than in a vole (Microtus pennsylvanicus) or a hedgehog (Atelerix albiventris). Furthermore, histological measurements demonstrated that hedgehogs have many more enterocytes in their intestines, suggesting that bats cannot have higher absorption of arabinose simply by having more tight junctions. We therefore investigated the mRNA levels of several claudins and occludin, because these proteins may affect permeability of tight junctions to macronutrients. To assess the expression levels of claudins per tight junction, we normalized the mRNA levels of the claudins to the constitutively expressed tight junction protein ZO-1, and combined these with measurements previously made in a bat and a rodent to determine if there were among-species differences. Although expression ratios of several genes varied among species, there was not a consistent difference between bats and non-flyers in the expression ratio of any particular gene. Protein expression patterns may differ from mRNA expression patterns, and might better explain differences among species in arabinose absorption.