The effect of gastric inhibitory polypeptide on intestinal glucose absorption and intestinal motility in mice

Gastric inhibitory polypeptide (GIP) is released from the small intestine upon meal ingestion and increases insulin secretion from pancreatic β cells. Although the GIP receptor is known to be expressed in small intestine, the effects of GIP in small intestine are not fully understood. This study was designed to clarify the effect of GIP on intestinal glucose absorption and intestinal motility. Intestinal glucose absorption in vivo was measured by single-pass perfusion method. Incorporation of [¹⁴C]-glucose into everted jejunal rings in vitro was used to evaluate the effect of GIP on sodium-glucose co-transporter (SGLT). Motility of small intestine was measured by intestinal transit after oral administration of a non-absorbed marker. Intraperitoneal administration of GIP inhibited glucose absorption in wild-type mice in a concentration-dependent manner, showing maximum decrease at the dosage of 50 nmol/kg body weight. In glucagon-like-peptide-1 (GLP-1) receptor-deficient mice, GIP inhibited glucose absorption as in wild-type mice. In vitro examination of [¹⁴C]-glucose uptake revealed that 100 nM GIP did not change SGLT-dependent glucose uptake in wild-type mice. After intraperitoneal administration of GIP (50 nmol/kg body weight), small intestinal transit was inhibited to 40% in both wild-type and GLP-1 receptor-deficient mice. Furthermore, a somatostatin receptor antagonist, cyclosomatostatin, reduced the inhibitory effect of GIP on both intestinal transit and glucose absorption in wild-type mice. These results demonstrate that exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility through a somatostatin-mediated pathway rather than through a GLP-1-mediated pathway.     

In vivo, villin is required for Ca(2+)-dependent F-actin disruption in intestinal brush borders.

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca(2+)-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca(2+) differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca(2+), whereas Ca(2+) had no effect in villin-null isolates. Moreover, increase in intracellular Ca(2+) by serosal carbachol or mucosal Ca(2+) ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 +/- 9.6%, compared with wild-type mice, 70 +/- 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.     

Intestinal NaCl transport in NHE2 and NHE3 knockout mice

Sodium/proton exchangers [Na(+)/H(+) (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na(+) and Cl(-) flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(-)], and NHE3 knockout [NHE3(-)] mice. Under basal conditions, NHE(+) and NHE2(-) jejuna had similar rates of net Na(+) (approximately 6 microeq/cm(2) x h) and Cl(-) (approximately 3 microeq/cm(2) x h) absorption. In contrast, NHE3(-) jejuna had reduced net Na(+) absorption (approximately 2 microeq/cm(2) x h) but absorbed Cl(-) at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net Na(+) and Cl(-) absorption in all genotypes. Studies of the Na(+) absorptive flux (J) indicated that J in NHE(+) jejunum was not sensitive to 1 microM EIPA, whereas J in NHE3(-) jejunum was equally sensitive to 1 and 100 microM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMP(i)) abolished net NaCl absorption and stimulated electrogenic Cl(-) secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(-) and NHE3(-) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na(+) absorption; 2) an amiloride-sensitive Na(+) transporter partially compensates for Na(+) absorption in NHE3(-) jejunum; 3) cAMP(i) stimulation abolishes net Na(+) absorption in NHE(+), NHE2(-), and NHE3(-) jejunum; and 4) electroneutral Cl(-) absorption is not directly dependent on either NHE2 or NHE3.     

Enhancement of rat intestinal calcium absorption by vanadate.

Vanadate alters intestinal transport and may have a role in regulating cell function. To determine whether it influences calcium absorption, we tested the effects of acute and chronic vanadate administration on calcium absorption using single-pass perfusion of jejunal and ileal segments of the in vivo rat intestine. Acute vanadate administration increased the lumen-to-mucosa and net fluxes of calcium in both the jejunum and ileum. The increase was largely due to an enhancement of the saturable fluxes of calcium and was observed at 10(-4) M concentration of vanadate, but not at higher or lower concentrations of the oxyanion, except at the highest concentration used, 10(-2) M, where calcium absorption was inhibited. Chronic vanadate administration caused, on the other hand, no changes in calcium absorption. We have demonstrated previously that rat intestinal (Na+ + K+)-ATPase is inhibited by vanadate, an effect that could raise cell sodium and increase the efflux of sodium across the brush border membrane. The results suggest that the vanadate enhancement of calcium absorption may be related to an increased entry of calcium into the mucosa, possibly as a result of an augmented exchange through the Na+/Ca+ antiport system. Alternatively, vanadate may influence access to a calcium channel in the mucosal membrane of the intestinal epithelium, leading to the observed increase in absorption.     

Suppressor of cytokine signaling-2 limits intestinal growth and enterotrophic actions of IGF-I in vivo

Suppressors of cytokine signaling (SOCS) typically limit cytokine receptor signaling via the JAK-STAT pathway. Considerable evidence demonstrates that SOCS2 limits growth hormone (GH) action on body and organ growth. Biochemical evidence that SOCS2 binds to the IGF-I receptor (IGF-IR) supports the novel possibility that SOCS2 limits IGF-I action. The current study tested the hypothesis that SOCS2 normally limits basal or IGF-I-induced intestinal growth and limits IGF-IR signaling in intestinal epithelial cells. Intestinal growth was assessed in mice homozygous for SOCS2 gene deletion (SOCS2 null) and wild-type (WT) littermates at different ages and in response to infused IGF-I or vehicle or EGF and vehicle. The effects of SOCS2 on IGF-IR signaling were examined in ex vivo cultures of SOCS2 null and WT intestine and Caco-2 cells. Compared with WT, SOCS2 null mice showed significantly enhanced small intestine and colon growth, mucosal mass, and crypt cell proliferation and decreases in radiation-induced crypt apoptosis in jejunum. SOCS2 null mice showed significantly greater growth responses to IGF-I in small intestine and colon. IGF-I-stimulated activation of IGF-IR and downstream signaling intermediates were enhanced in the intestine of SOCS2 null mice and were decreased by SOCS2 overexpression in Caco-2 cells. SOCS2 bound directly to the endogenous IGF-IR in Caco-2 cells. The intestine of SOCS2 null mice also showed enhanced growth responses to infused EGF. We conclude that SOCS2 normally limits basal and IGF-I- and EGF-induced intestinal growth in vivo and has novel inhibitory effects on the IGF-IR tyrosine kinase pathway in intestinal epithelial cells.     

Net fluxes of electrolytes in the rat intestine infected with Moniliformis dubius (Acanthocephala).

The effect of Moniliformis dubius on fluxes of Na+, K+, Cl-, and HCO3-in the rat intestine was determined using a conventional in vivo single-pass perfusion technique. Results for ion and water movements in the uninfected gut were in agreement with previous studies. In the parasitized intestine the jejunal pH was significantly lower than that in control animals, matching the restriction of the parasites to this region of the small intestine. While parasitism did not affect Na+ transport in the distal ileum, Na+ absorption was reduced (pH 7.0), or secretion enhanced (pH 6.0), in the two proximal regions. Cl-absorption was reduced in the distal ileum, but secretion was enhanced in the other two segments. Parasitism also enhanced K+ secretion in all segments. Net H2O absorption was reduced at pH 7.0; at pH 7.0; at pH 6.0 net secretion was also reduced. These changes clearly indicate that a parasite restricted to the jejunum may significantly affect the absorptive and secretory activity of the intestine distal to the site of infection. The results are discussed in the light of current concepts of electrolyte transport. The effect of the parasites on mucosal function distal to their site of attachment is discussed in terms of the release by the parasite of toxin-like substances, changes in the physical-chemical characteristics of the intestinal lumen, and interference with neurohormonal control of gastrointestinal function.     

IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis

Trichinella spiralis infection elicits a vigorous IgE response and pronounced intestinal and splenic mastocytosis in mice. Since IgE both activates mast cells (MC) and promotes their survival in culture, we examined its role in MC responses and parasite elimination in T. spiralis-infected mice. During primary infection, wild-type but not IgE-deficient (IgE(-/-)) BALB/c mice mounted a strong IgE response peaking 14 days into infection. The splenic mastocytosis observed in BALB/c mice following infection with T. spiralis was significantly diminished in IgE(-/-) mice while eosinophil responses were not diminished in either the blood or jejunum. Similar levels of peripheral blood eosinophilia and jejunal mastocytosis occurred in wild-type and IgE-deficient animals. Despite the normal MC response in the small intestine, serum levels of mouse MC protease-1 also were lower in parasite-infected IgE(-/-) animals and these animals were slower to eliminate the adult worms from the small intestine. The number of T. spiralis larvae present in the skeletal muscle of IgE(-/-) mice 28 days after primary infection was about twice that in BALB/c controls, and the fraction of larvae that was necrotic was reduced in the IgE-deficient animals. An intense deposition of IgE in and around the muscle larvae was observed in wild-type but not in IgE null mice. We conclude that IgE promotes parasite expulsion from the gut following T. spiralis infection and participates in the response to larval stages of the parasite. Furthermore, our observations support a role for IgE in the regulation of MC homeostasis in vivo.     

Lack of the intestinal Muc1 mucin impairs cholesterol uptake and absorption but not fatty acid uptake in Muc1-/- mice

Before cholesterol and fatty acid molecules in the small intestinal lumen can interact with their possible transporters for uptake and absorption, they must pass through a diffusion barrier, which may modify the kinetics of nutrient assimilation. This barrier includes an unstirred water layer and a surface mucous coat, which is located at the intestinal lumen-membrane interface. In the present study, we investigated whether disruption of the mucin gene (Muc)1 may influence intestinal uptake and absorption of cholesterol and fatty acid in male Muc1(-/-) mice. The wild-type mice displayed relatively high levels of Muc1, Muc2, Muc3, and Muc4 mRNAs and relatively low levels of Muc5ac and Muc5b mRNAs in the small intestine. The absence of Muc1 mRNA and protein in the small intestines of Muc1(-/-) mice confirmed complete knockout of the Muc1 gene, but the mRNA expression for other mucin genes remained unchanged. Intestinal uptake and absorption of cholesterol but not palmitic acid were significantly reduced in Muc1(-/-) mice compared with the wild-type mice. However, knockout of the Muc1 gene did not impair either expression levels of the genes that encode intestinal sterol efflux transporters Abcg5 and Abcg8 and fatty acid transporter Fatp4 or small intestinal transit rates. We conclude that physiological levels of the epithelial mucin produced by the Muc1 gene are necessary for normal intestinal uptake and absorption of cholesterol in mice. Our study implies that because cholesterol absorption efficiency is reduced by approximately 50% in Muc1-deficient mice, there may be one or more additional pathways for cholesterol absorption.     

Electroneutral sodium absorption and electrogenic anion secretion across murine small intestine are regulated in parallel

Electrolyte transport processes of small intestinal epithelia maintain a balance between hydration of the luminal contents and systemic fluid homeostasis. Under basal conditions, electroneutral Na(+) absorption mediated by Na(+)/H(+) exchanger 3 (NHE3) predominates; under stimulated conditions, increased anion secretion mediated by CFTR occurs concurrently with inhibition of Na(+) absorption. Homeostatic adjustments to diseases that chronically affect the activity of one transporter (e.g., cystic fibrosis) may include adaptations in the opposing transport process to prevent enterosystemic fluid imbalance. To test this hypothesis, we measured electrogenic anion secretion (indexed by the short-circuit current) across NHE3-null [NHE3(-)] murine small intestine and electroneutral Na(+) absorption (by radioisotopic flux analysis) across small intestine of mice with gene-targeted disruptions of the anion secretory pathway, i.e., CFTR-null [CFTR(-)] or Na(+)-K(+)-2Cl(-) cotransporter-null [NKCC1(-)]. Protein expression of NHE3 and CFTR in the intestinal epithelia was measured by immunoblotting. In NHE3(-), compared with wild-type small intestine, maximal and bumetanide-sensitive anion secretion following cAMP stimulation was significantly reduced, and there was a corresponding decrease in CFTR protein expression. In CFTR(-) and NKCC1(-) intestine, Na(+) absorption was significantly reduced compared with wild-type. NHE3 protein expression was decreased in the CFTR(-) intestine but was unchanged in the NKCC1(-) intestine, indicating that factors independent of expression also downregulate NHE3 activity. Together, these data support the concept that absorptive and secretory processes determining NaCl and water movement across the intestinal epithelium are regulated in parallel to maintain balance between the systemic fluid volume and hydration of the luminal contents.     

Absorption of 3-oxy-methyl-D-glucose by chicken cecum and jejunum in vivo

Jejunal and cecal 3-oxy-methyl-D-glucose (3-OMG) absorption was studied in 4- to 8-week-old chickens by an in vivo perfusion technique (perfusion rate 1.5 ml/min). Total and phloridzin-insensitive 3-OMG absorption was tested for lumenal substrate concentrations ranging from 1.25 to 20 mmol/l. The estimated apparent Michaelis constants in jejunum and cecum were 5.1 and 4.0 mmol/l (Lineweaver-Burk method) and 3.2 and 3.1 mmol/l (visual inspection method), respectively. Vmax were similar in both segments with either method, about 0.3 mumol/cm2 X 5 min. Passive permeability coefficients were the same in both regions (about 45 l/cm2 X 5 min X 10(3)). The transport properties of the cecal epithelium in vivo suggest a role of these intestinal segments in the absorption of nutrients originated from digestive processes.     

Pyridoxal 5'-phosphate disappearance from perfused rat jejunal segments: correlation with perfusate alkaline phosphatase and water absorption

The relative effects of perfusate alkaline phosphatase activity and net water absorption on 2 microM pyridoxal 5'-phosphate (PLP) luminal disappearance from rat jejunum were studied in a single-pass, in vivo perfused intestinal segment model. Perfusate consisted of unlabeled PLP in buffer (pH = 7.4). Net water flux was monitored by inclusion of [3H]polyethylene glycol. PLP was measured by the [14C]tyrosine apodecarboxylase assay. Single and multiple regression analysis of results during perfusion of 2 microM PLP in Krebs bicarbonate buffer demonstrated no correlation between perfusate alkaline phosphatase activity and net water absorption and significant correlations between PLP luminal disappearance and both perfusate alkaline phosphatase activity and net water absorption. Correlation for the latter was improved when disappearance results were corrected for variations in perfusate alkaline phosphatase activity. When perfusate buffers were selected to yield divergent rates of net water absorption, the one associated with greater net water absorption was also associated with greater PLP disappearance. That this could not be explained by changes in perfusate alkaline phosphatase activity was demonstrated both by assessment of the rate of decay of PLP added in vitro to exited perfusate incubated at 37 degrees C and by measurement of alkaline phosphatase activity under conditions defined by the buffers using a modified spectrophotometric assay. Conclusions were: (1) In vivo PLP luminal disappearance correlates significantly with both perfusate alkaline phosphatase activity and net water absorption; (2) these two factors appear to act as independent variables; and (3) future studies on PLP intestinal absorption will need to take both of these variables into account in the interpretation of results.     

Effects of calcium and sugars on intestinal manganese absorption

An in vivo luminal perfusion technique was used to investigate the influence of Ca, Mg, lactose, and glucose on Mn absorption in different segments of the rat intestine. Mn absorption was determined by measuring disappearance of⁵⁴Mn activity from the perfusion solution containing 0.1 or 0.01 mmol/L Mn. Na and water absorption were also determined. Mn absorption decreased during the first 30 min of perfusion to reach a steady state thereafter. Ca (1 mmol/L) inhibited Mn absorption in the proximal jejunum and in the colon, whereas Mn absorption was increased by Ca in the distal jejunum. Mg (1 mmol/L), lactose, and glucose (25 mmol/L each) had no effect on Mn absorption in the jejunum. These results can be explained by a direct interaction of Mn and Ca during transcellular Ca transport in the proximal jejunum and colon. The reason for the stimulatory effect of Ca in the distal jejunum is unknown.     

Adaptation of electrolytes and fluid transport in rat small and large intestine after distal small bowel resection

Na+, Cl- and water transport were studied in jejunum, caecum and colon after either 50% or 80% of small bowel resection (SBR). Four weeks after surgery, dry and wet weights, net absorption in vivo of sodium, chloride and water were determined. There was a significant intestinal growth after 50% or 80% SBR except for the colon which only showed increased tissue mass after 80% SBR. Net transport was stimulated both, per organ and per unit mass. In the small intestine and caecum both organ growth and changes in cell function appear to be involved in the adaptive response, regardless the extent of the small intestine resected. In the colon, compensatory growth appear to contribute to the adaptive response only after 80% SBR, whilst the transport function of the colonocytes seems to be stimulated after both types of SBR.     

Apolipoprotein A-IV regulates chylomicron metabolism-mechanism and function

Dietary fat is an important mediator of atherosclerosis and obesity. Despite its importance in mediating metabolic disease, there is still much unknown about dietary fat absorption in the intestine and especially the detailed biological roles of intestinal apolipoproteins involved in that process. We were specifically interested in determining the physiological role of the intestinal apolipoprotein A-IV (A-IV) using A-IV knockout (KO) mice. A-IV is stimulated by fat absorption in the intestine and is secreted on nascent chylomicrons into intestinal lymph. We found that A-IV KO mice had reduced plasma triglyceride (TG) and cholesterol levels and that this hypolipidemia persisted on a high-fat diet. A-IV KO did not cause abnormal intestinal lipid absorption, food intake, or adiposity. Additionally, A-IV KO did not cause abnormal liver TG and cholesterol metabolism, as assessed by measuring hepatic lipid content, lipogenic and cholesterol synthetic gene expression, and in vivo VLDL secretion. Instead, A-IV KO resulted in the secretion of larger chylomicrons from the intestine into the lymph, and those chylomicrons were cleared from the plasma more slowly than wild-type chylomicrons. These data suggest that A-IV has a previously unknown role in mediating the metabolism of chylomicrons, and therefore may be important in regulating plasma lipid metabolism.     

Calbindin D9k is not required for 1,25-dihydroxyvitamin D3-mediated Ca2+ absorption in small intestine

The exact role of calbindin D9k in vitamin D-mediated calcium absorption has been debated but remains unsettled. In 129/OlaHsd mice, calbindin D9k was found highest in duodenum (36-50%) and kidney (24-34%) followed by stomach, lung and uterus. Age does not affect the relative distribution of calbindin D9k but it does decline with age in duodenum of both male and female 129/Ola mice. Recently, we produced a null calbindin D9k mutant 129/OlaHsd mouse; this mouse proved to be indistinguishable from the wild-type in phenotype and in a serum calcium level regardless of age or gender. We have now examined directly whether the mutant mouse can absorb calcium from the intestine in response to the active form of vitamin D. The calbindin D9k null mutant mouse is fully able to absorb calcium from the intestine in response to 1,25-dihydroxyvitamin D3. It is, therefore, clear that calbindin D9k is not required for vitamin D-induced intestinal calcium absorption.     

Increased hepatobiliary and fecal cholesterol excretion upon activation of the liver X receptor is independent of ABCA1

The ATP-binding cassette transporter ABCA1 is essential for high density lipoprotein (HDL) formation and considered rate-controlling for reverse cholesterol transport. Expression of the Abca1 gene is under control of the liver X receptor (LXR). We have evaluated effects of LXR activation by the synthetic agonist T0901317 on hepatic and intestinal cholesterol metabolism in C57BL/6J and DBA/1 wild-type mice and in ABCA1-deficient DBA/1 mice. In wild-type mice, T0901317 increased expression of Abca1 in liver and intestine, which was associated with an approximately 60% rise in HDL. Biliary cholesterol excretion rose 2.7-fold upon treatment, and fecal neutral sterol output was increased by 150-300%. Plasma cholesterol levels also increased in treated Abca1(-/-) mice (+120%), but exclusively in very low density lipoprotein-sized fractions. Despite the absence of HDL, hepatobiliary cholesterol output was stimulated upon LXR activation in Abca1(-/-) mice, leading to a 250% increase in the biliary cholesterol/phospholipid ratio. Most importantly, fecal neutral sterol loss was induced to a similar extent (+300%) by the LXR agonist in DBA/1 wild-type and Abca1(-/-) mice. Expression of Abcg5 and Abcg8, recently implicated in biliary excretion of cholesterol and its intestinal absorption, was induced in T0901317-treated mice. Thus, activation of LXR in mice leads to enhanced hepatobiliary cholesterol secretion and fecal neutral sterol loss independent of (ABCA1-mediated) elevation of HDL and the presence of ABCA1 in liver and intestine.