Regulatory mechanisms of glucose absorption in the mouse proximal small intestine during fasting and feeding.

Fasting is known to alter the function of various organs and the mechanisms of glucose metabolism, which affect health outcomes and slow aging. However, it remains unclear how fasting and feeding affects glucose absorption function in the small intestine. We studied the effects of the fasting and feeding on glucose-induced short-circuit current (Isc) in vitro using an Ussing chamber technique. Glucose-induced Isc by SGLT1 was observed in the ileum, but little or no Isc was observed in the jejunum in ad libitum-fed mice. However, in mice fasted for 24-48 h, in addition to the ileum, robust glucose-induced Isc was observed over time in the jejunum. The expression of SGLT1 in the brush border membranes was significantly decreased in the jejunum under fed conditions compared to 48 h fasting, as analyzed by western blotting. Additionally, when mice were fed a 60% high glucose diet for 3 days, the increase in glucose-induced Isc was observed only in the ileum, and totally suppressed in the jejunum. An increase in Na+ permeability between epithelial cells was concomitantly observed in the jejunum of fasted mice. Transepithelial glucose flux was assessed using a non-metabolizable glucose analog, 14C-methyl α-D-glucopyranoside glucose (MGP). Regardless of whether fed or fasted, no glucose diffusion mechanism was observed. Fasting increased the SGLT1-mediated MGP flux in the jejunum. In conclusion, segment-dependent up- and down-regulation mechanisms during fasting and feeding are important for efficient glucose absorption once the fast is broken. Additionally, these mechanisms may play a crucial role in the small intestine's ability to autoregulate glucose absorption, preventing acute hyperglycemia when large amounts of glucose are ingested.

The effect of claudin-15 deletion on cationic selectivity and transport in paracellular pathways of the cecum and large intestine.

The large intestine plays a pivotal role in water and electrolyte balance. Paracellular transport may play a role in ion transport mechanisms in the cecum and large intestine; however, these molecular mechanisms and their physiological roles have not been fully studied. Claudin-15 forms a cation channel in tight junctions in the small intestine, but its role in the cecum and large intestine has not been investigated. This study aimed to explore the physiological role of claudin-15 in the cecum and large intestine using claudin-15 (Cldn15) KO mice. Electrical conductance, short-circuit current, Na+ flux, and dilution potential were assessed in isolated tissue preparations mounted in Ussing chambers. The induced short-circuit current of short-chain fatty acids, which are fermentative products in the intestinal tract, was also measured. Compared to wild type mice, the electrical conductance and paracellular Na+ flux was decreased in the cecum, but not the middle large intestine, while in both the cecum and the middle large intestine, paracellular Na+ permeability was decreased in Cldn15 KO mice. These results suggest that claudin-15 is responsible for Na+ permeability in the tight junctions of the cecum and large intestine and decreased Na+ permeability in the cecum may cause impaired absorption function.

Na+-dependent intestinal glucose absorption mechanisms and its luminal Na+ homeostasis across metamorphosis from tadpoles to frogs.

The abrupt morphological changes of the intestine during metamorphosis have been detailed in frogs. The features of intestinal metamorphosis are shortening of the intestine and remodeling of the intestinal epithelium. It is believed that the purpose of the morphological changes of the intestine is adaptation from aquatic herbivorous to carnivorous life. However, little is known about the physiological importance of these morphological changes. To elucidate the functional changes during metamorphosis, we measured luminal Na+ concentrations and Na+-dependent glucose uptake in tadpoles and adult African clawed frogs Xenopus laevis. The small intestine was isolated and divided into four segments in length, the luminal contents collected for analysis of ion concentration by ion chromatography. Phlorizin-sensitive glucose-induced short-circuit current (ΔIsc) was measured in intestinal preparations mounted in Ussing chambers. Although dietary sodium intake was extremely low in tadpoles, luminal Na+ concentration gradually increased along the proximal to the middle part of the intestine (>70 mM), and this Na+ concentration was comparable with that of carnivorous adult frogs. The increment of glucose-induced ΔIsc was observed in tadpole intestine. We also measured the ΔIsc induced by acetic acid, which is the major short-chain fatty acid produced by fermentation. The expression levels of mRNA for Na+-dependent glucose transporter 1 and tight junction protein claudin-15 in each intestinal segment was measured. These results suggest that luminal Na+ homeostasis is important and luminal Na+ is kept at a high concentration for Na+-dependent nutrient absorption mechanisms.

Reconstitution of functional tight junctions with individual claudin subtypes in epithelial cells.

The claudin family of membrane proteins is responsible for the backbone structure and function of tight junctions (TJs), which regulate the paracellular permeability of epithelia. It is thought that each claudin subtype has its own unique function and the combination of expressed subtypes determines the permeability property of each epithelium. However, many issues remain unsolved in regard to claudin functions, including the detailed functional differences between claudin subtypes and the effect of the combinations of specific claudin subtypes on the structure and function of TJs. To address these issues, it would be useful to have a way of reconstituting TJs containing only the claudin subtype(s) of interest in epithelial cells. In this study, we attempted to reconstitute TJs of individual claudin subtypes in TJ-deficient MDCK cells, designated as claudin quinKO cells, which were previously established from MDCK II cells by deleting the genes of claudin-1, -2, -3, -4, and -7. Exogenous expression of each of claudin-1, -2, -3, -4, and -7 in claudin quinKO cells resulted in the reconstitution of functional TJs. These TJs did not contain claudin-12 and -16, which are endogenously expressed in claudin quinKO cells. Furthermore, overexpression of neither claudin-12 nor claudin-16 resulted in the reconstitution of TJs, demonstrating the existence of claudin subtypes lacking TJ-forming activity in epithelial cells. Exogenous expression of the channel-forming claudin-2, -10a, -10b, and -15 reconstituted TJs with reported paracellular channel properties, demonstrating that these claudin subtypes form paracellular channels by themselves without interaction with other subtypes. Thus, the reconstitution of TJs in claudin quinKO cells is advantageous for further investigation of claudin functions.Key words: tight junction, claudin, paracellular permeability, epithelial barrier.

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique.

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog skin. Since then, this technique has been applied to many different tissues to study the physiological parameters of transport across membranes. The Ussing chamber method is preferable to other methods because native tissue can be used, making it more applicable to what is happening in vivo. However, because native tissue is used, throughput is low, time is limited, and tissue preparation requires skill and training. These chambers have been used to study specific transporter proteins in various tissues, understand disease pathophysiology such as in Cystic Fibrosis, study drug transport and uptake, and especially contributed to the understanding of nutrient transport in the intestine. Given the whole epithelial transport process of a tissue, not only transepithelial pathways, but also paracellular pathways are important. Tight junctions are a key determinant of tissue specific paracellular permeability across the intestine. In this article, the Ussing chamber technique will be used to assess paracellular permselectivity of ions by measuring transepithelial conductance and dilution potentials.

Angulin-2/ILDR1, a tricellular tight junction protein, does not affect water transport in the mouse large intestine.

Angulin-2/ILDR1 is a member of the angulin protein family, which is exclusively expressed at tricellular tight junctions in epithelia. Tricellular tight junctions are found where three cells meet and where three bicellular tight junction strands converge. Tricellular tight junctions are thought to be important for paracellular permeability of ions and water in epithelial tissues. It was recently reported that angulin-2/ILDR1 knockout mice have water transport abnormalities in the kidney. Since angulin-2/ILDR1 is the main tricellular tight junction protein in the large intestine, the goal of this research was to examine the effect of angulin-2/ILDR1 knockout on large intestinal paracellular water transport. We found that Ildr1 knockout mice showed no detectable phenotype other than deafness. In addition, paracellular transport as assessed by Ussing chamber was unchanged in Ildr1 knockout mice. However, we found that in the colon and the kidney of Ildr1 knockout mice, another tricellular tight junction protein, angulin-1/LSR, changes its expression pattern. We propose that with this replacement in tissue localization, angulin-1/LSR compensates for the loss of angulin-2/ILDR1 and maintains the barrier and function of the epithelia in the large intestine as well as the kidney.

Na+-Coupled Nutrient Cotransport Induced Luminal Negative Potential and Claudin-15 Play an Important Role in Paracellular Na+ Recycling in Mouse Small Intestine.

Many nutrients are absorbed via Na+ cotransport systems, and therefore it is predicted that nutrient absorption mechanisms require a large amount of luminal Na+. It is thought that Na+ diffuses back into the lumen via paracellular pathways to support Na+ cotransport absorption. However, direct experimental evidence in support of this mechanism has not been shown. To elucidate this, we took advantage of claudin-15 deficient (cldn15-/-) mice, which have been shown to have decreased paracellular Na+ permeability. We measured glucose-induced currents (ΔIsc) under open- and short-circuit conditions and simultaneously measured changes in unidirectional 22Na+ fluxes (ΔJNa) in Ussing chambers. Under short-circuit conditions, application of glucose resulted in an increase in ΔIsc and unidirectional mucosal to serosal 22Na+ (∆JNaMS) flux in both wild-type and cldn15-/- mice. However, under open-circuit conditions, ΔIsc was observed but ∆JNaMS was strongly inhibited in wild-type but not in cldn15-/- mice. In addition, in the duodenum of mice treated with cholera toxin, paracellular Na+ conductance was decreased and glucose-induced ∆JNaMS increment was observed under open-circuit conditions. We concluded that the Na+ which is absorbed by Na+-dependent glucose cotransport is recycled back into the lumen via paracellular Na+ conductance through claudin-15, which is driven by Na+ cotransport induced luminal negativity.