Streptozotocin diabetes and sugar transport by rat ileal enterocytes: evidence for adaptation caused by an increased luminal nutrient load.

Preparations of villus enterocytes and brush border membrane vesicles have been used to study the effects of streptozotocin-induced diabetes mellitus in rats on sugar transport across the brush border and basolateral membranes of ileal epithelial cells. In isolated cells, diabetes increased Na(+)-dependent galactose transport across the brush border of mid-villus but not upper villus cells. Galactose transport across the basolateral membrane was, however, enhanced by diabetes in both cell populations. Kinetic analysis of vesicle data suggested the presence of two transporters for Na(+)-dependent glucose transport. Diabetes induced a 5-fold increase in both KT and Vmax of the high-affinity/low-capacity system together with a 2-fold increase in the Vmax of the low-affinity/high-capacity transporter. Glucose was almost undetectable in the lumen of the upper and lower ileum in control animals but was present at high levels (26.1 +/- 4.3 mM and 6.5 +/- 1.3 mM) in diabetic rats. The possible significance of these changes in luminal sugar concentration in relation to the adaptation of transport across ileal enterocytes is discussed.

Iron-transferrin binding to isolated guinea pig enterocytes and the regional localisation of intestinal iron transfer during ontogeny.

Neonatal animals are iron replete but in comparison with adults they display increased intestinal iron absorption. In order to examine possible mechanisms for this developmental adaptation we have measured the appearance of iron in peripheral blood following 30 min exposure of duodenal and ileal segments of adult and neonatal guinea pigs in vivo to 59Fe-ascorbate. Parallel experiments have determined the kinetics of 125I-labelled diferric transferrin binding to villus enterocytes isolated from duodenum and ileum. In adult animals the rate of appearance of 59Fe in peripheral blood was 11-fold greater following duodenal, compared to ileal exposure to the radioligand. No such regional difference was detected in the neonate. Isolated cells showed saturable binding of [125I]transferrin which was maximal between 30 and 60 min. The kinetics of specific transferrin binding by adult duodenal and ileal enterocytes were similar and were also not significantly different to respective values in neonatal duodenal and ileal cells. Thus, it is likely that increased iron absorption in the neonate is due in part to enhanced ileal iron transfer. The interaction of transferrin with its receptor, however, is not involved in this developmental change in uptake.

Developmental changes in mucosal iron binding proteins in the guinea pig. Expression of transferrin, H and L ferritin and binding of iron to a low molecular weight protein.

Neonatal animals show an increased intestinal iron absorption despite being iron replete. We have used the developing guinea pig to determine whether alterations in the expression of iron binding proteins in duodenal enterocytes can account for the difference in iron absorption between neonatal and adult guinea pigs. No mRNA for transferrin was detected in the enterocytes. H ferritin mRNA remained constant from birth into adulthood whilst there was a small increase (< 2-fold) in L ferritin mRNA. Western blotting showed a clear increase in immunoreactive ferritin, levels reaching those of the adult 19 days after birth. We have also demonstrated that in the neonate, 80% of newly absorbed iron in the enterocyte is associated with a low molecular weight fraction (LMW-IBF), whereas in the adult, ferritin and LMW-IBF bind iron to the same extent. We conclude that changes in expression of ferritin during ontogeny are accompanied by a change in distribution of iron binding between ferritin and LMW-IBF implicating these proteins in the regulation of iron absorption.

Transcriptional and translational control over sodium-glucose-linked transporter (SGLT1) gene expression in adult rat small intestine.

We have measured SGLT1 mRNA content and SGLT1-mediated glucose transport at different positions along the small intestine of control and streptozotocin diabetic rats and shown both parameters to be similar but higher in jejunal compared with ileal tissue. No such correlation was seen when comparing measurements of SGLT1 mRNA along jejunal villi with previous estimates of SGLT1 protein and SGLT1-mediated glucose transport [Debnam et al., Eur. J. Physiol. 430 (1995) 151-159]. This is the first time it has been possible to directly relate these three aspects of SGLT1 gene expression in a single species. Results are discussed in terms of a possible time rather than positional control over translation of SGLT1 mRNA.

Urinary excretion of hydroxycinnamates and flavonoids after oral and intravenous administration.

The urinary recoveries of the hydroxycinnamates, ferulic acid (3-methoxy, 4-hydroxy cinnamic acid), and chlorogenic acid (the quinic acid ester of 3,4-dihydroxycinnamic acid), and three structurally related flavonoids were studied in the rat. For the latter, the aglycone quercetin was compared with its 3-glucoside (isoquercitrin) and 3-rhamnoglucoside (rutin). Doses of 50 mg/kg were administered via the oral and intravenous routes and urine collected over the subsequent 24-h period. Reverse phase HPLC with photo-diode array detection was used to analyze the unchanged compound and their metabolites excreted in the urine. Ferulic acid and isoquercitrin were orally absorbed (5.4 and 0.48% of administered dose, respectively) and are therefore bioavailable. In contrast, neither unchanged chlorogenic acid, rutin, quercetin, nor the conjugated metabolites in the form of glucuronide or sulphate were detected in the urine after oral dosing. All the flavonoids studied produced low total urinary recoveries after intravenous administration, 9.2% for quercetin-3-rhamnoglucoside, 6.7% for the 3-glucoside, and 2.4% for the aglycone, indicating that extensive metabolism to low molecular weight compounds or excretion via other routes may be occurring. Overall it can be stated that renal excretion is not a major pathway of elimination for intact flavonoids and hydroxycinnamates in the rat.

Streptozotocin diabetes and the expression of GLUT1 at the brush border and basolateral membranes of intestinal enterocytes.

Changes in membrane expression of sodium-dependent glucose transporter (SGLT1) and glucose transporter isoform (GLUT2) protein have been implicated in the increased intestinal glucose transport in streptozotocin-diabetes. The possible involvement of GLUT1 in the transport response, however, has not previously been studied. Using confocal microscopy on tissue sections and Western blotting of purified brush border membrane (BBM) and basolateral membrane (BLM), we have examined enterocyte expression of GLUT1 in untreated and in 1 and 21 day streptozotocin diabetic rats. In control enterocytes, GLUT1 was absent at the BBM and detected at low levels at the BLM. Diabetes resulted in a 4- to 5-fold increased expression of GLUT1 at the BLM and the protein could also be readily detected at the BBM. Insulin treatment of diabetic rats increased GLUT1 level at the BBM but was without effect on expression of the protein at the BLM.

The small intestine can both absorb and glucuronidate luminal flavonoids.

We have studied the perfusion of the jejunum and ileum in an isolated rat intestine model with flavonoids and hydroxycinnamates and the influence of glycosylation on the subsequent metabolism. Flavone and flavonol glucosides and their corresponding aglycones are glucuronidated during transfer across the rat jejunum and ileum and this glucuronidation occurs without the need for gut microflora. Furthermore, this suggests the presence of glycosidases as well as UDP-glucuronyl transferase in the jejunum. In contrast, quercetin-3-glucoside and rutin are mainly absorbed unmetabolised. The results suggest that the more highly reducing phenolics are absorbed predominantly as glucuronides (96.5%+/-4.6) of the amount absorbed, whereas monophenolic hydroxycinnamates and monophenolic B-ring flavonoids are less predisposed to glucuronidation and higher levels of aglycone (88.1%+/-10.1) are detected on absorption through both the jejunum and ileum.

Effects of neomycin on galactose absorption across rat jejunum.

The effects of neomycin sulphate on galactose absorption have been studied using in vivo and in vitro preparations of rat small intestine. Neomycin (10(-3)M) produced an increase in the maximum transport capacity (Jmax) for the active component of absorption in vivo. The apparent Kt for absorption was unaffected. The antibiotic caused a dose-dependent increase in the potential difference across the mucosal membrane (Vm) measured in vitro, a maximal effect being seen at a concentration of 10(-4)M. Furthermore, the magnitude of the depolarization induced by the addition of galactose (4 mM) to the mucosal fluid was enhanced by neomycin (10(-4)M). Phlorhizin (10(-4)M) abolished the galactose-induced depolarization in both the absence and presence of the antibiotic. It is concluded that neomycin increases the electrical driving force for Na+ during Na+-coupled galactose entry into the enterocyte.

Hyperglucagonaemia: effects on active nutrient uptake by the rat jejunum.

The effects of chronic (72 h) glucagon treatment on active nutrient uptake by the rat jejunum have been determined using in-vitro electrophysiological and autoradiographic methods together with an in-vivo technique which measures absorption across a cannulated segment of upper jejunum. Glucagon caused a marked increase in the potential difference across the brush border membrane from a mean value of -47.6 mV under control conditions to -54.2 mV following treatment with the hormone (P less than 0.025). A similar hyperpolarization was also noted after 24 h glucagon administration. The magnitude of the depolarization induced by the addition of D-galactose (4 mmol/l) to the mucosal fluid was increased from 6.0 to 14.3 mV following 72 h glucagon treatment (P less than 0.05). Phloridzin (0.1 mmol/l) abolished the galactose-induced depolarization in both control and treated animals. Glucagon induced significant increases of 49.9 and 61.0% respectively for glucose and galactose absorption measured under in-vivo conditions. Autoradiographic studies revealed that following glucagon treatment, L-valine uptake occurred earlier during enterocyte migration along the villus. This resulted in an enhanced accumulation of the amino acid at the villus tip. We conclude that glucagon increases nutrient transport across the small intestine. The raised electrical gradient for Na+- coupled nutrient entry into the enterocyte is likely to be a major factor in the transport response.

Diabetes mellitus and the sodium electrochemical gradient across the brush border membrane of rat intestinal enterocytes.

The effects of chronic diabetes mellitus of 4-5 weeks duration on the potential difference across the brush border membrane of rat small intestine (Vm) and on Na+-dependent uptake of D-glucose by jejunal brush border vesicles have been studied. Diabetes increased Vm in the jejunum from a mean value of -47.2 mV in control tissue to -57.4 mV in diabetic tissue (P less than 0.001) but was without effect on ileal Vm. Measurements of Vm during ion-substitution experiments revealed that the conductance of Na+ of the jejunal brush border was reduced by diabetes, whilst K+ and Cl- permeabilities were unaltered. Uptake studies using brush border vesicles and an Na+-electrochemical gradient showed that diabetes caused a 56% increase in the initial rate of uptake of D-glucose but was without effect on the peak to equilibrium ratio. Taken together, data from these two studies suggest that the lower Na+ permeability of the brush border in diabetes enhances the electrical and chemical driving force for active Na+-dependent uptake of glucose by reducing glucose-independent movement of Na+ across this membrane. Finally, the possible humoral factors involved in this response to diabetes are discussed.

Early diabetes-induced changes in rat jejunal glucose transport and the response to insulin.

The effects of 1 day of streptozotocin-induced diabetes in rats on glucose transport across the brush border membrane (BBM) and basolateral membrane (BLM) prepared from jejunal enterocytes has been studied. The effects on glucose transport of treatment of diabetic animals with insulin to reduce to normal the elevated blood glucose levels has also been assessed. The maximum capacity (Vmax) for SGLT1-mediated glucose uptake by BBM vesicles was unaffected by diabetes or insulin treatment of diabetic rats. In contrast, Vmax for BLM glucose uptake was increased by 206% in diabetes, a response that could not be reversed by treatment with insulin. Western blotting of BBM for SGLT1 protein revealed a single band with a molecular weight of 73 kDa and the intensity of this band was unaffected by diabetes. However, an increased level of GLUT2 was noted in diabetic BLM and this was not a consequence of changes in glycaemic or insulin status. Diabetes hyperpolarised the BBM, implying an increased driving force for Na(+)-sugar co-transport but insulin treatment only partially reversed this enhanced potential difference. Benzamil (2 microns), an epithelial Na+ channel blocker, hyperpolarised the BBM of control but not diabetic enterocytes, implying that a reduced Na+ permeability was responsible for the diabetic hyperpolarisation. It was concluded that in early diabetes, before the onset of hyperphagia, a greater driving force for Na(+)-dependent BBM sugar transport together with increased GLUT2 activity at the BLM promotes sugar movement across the enterocyte. Possible triggers for the transport responses are discussed.

Autoradiographic localisation of opiate receptors in rat small intestine.

[3H]Naloxone and [3H]dihydromorphine are selective ligands for opiate receptors. Using an in vitro autoradiographic technique, binding of these ligands has been demonstrated to the villi and crypts in rat small intestine. These results indicate the presence of opiate receptor sites in the small intestine which suggests further a role for endogenous opiates in the transport functions of intestinal mucosa.

Regulation of renal function by the gastrointestinal tract: potential role of gut-derived peptides and hormones.

The concept of a regulatory link between the gastrointestinal tract and kidneys is not new. The idea that dietary intake and composition can affect renal function is perhaps self-evident, but defining this relationship, especially in terms of sensors and effectors, is proving more difficult. That the gastrointestinal tract can exert some control over renal function was strengthened by the early observation that oral ingestion of a sodium chloride load has a greater natriuretic effect than when the same amount is given intravenously. This effect was subsequently shown to be independent of changes in aldosterone and atrial natriuretic peptide, although not necessarily angiotensin-II. However, the nature of any intestinal natriuretic peptide remains uncertain, despite suggestions that various gut-derived hormones, including guanylin and uroguanylin, may be involved. There is also an emerging concept of gastrointestinal taste and taste-like receptor mechanisms present throughout the gastrointestinal tract, which may govern the excretion of other key electrolytes, including potassium and phosphate. The evidence for gut sensors of nutrients such as proteins, amino acids, glucose, and acid is now becoming more established. Thus, we can anticipate the existence and eventual characterization of several gut ion sensors.

GLUT2 protein at the rat proximal tubule brush border membrane correlates with protein kinase C (PKC)-betal and plasma glucose concentration.

AIMS/HYPOTHESIS: GLUT2 is the main renal glucose transporter upregulated by hyperglycaemia, when it becomes detectable at the brush border membrane (BBM). Since glucose-induced protein kinase C (PKC) activation in the kidney is linked to diabetic nephropathy, we investigated the effect of glycaemic status on the protein levels of PKC isoforms alpha, betaI, betaII, delta and epsilon in the proximal tubule, as well as the relationship between them and changes in GLUT2 production at the BBM. METHODS: Plasma glucose concentrations were modulated in rats by treatment with nicotinamide 15 min prior to induction of diabetes with streptozotocin. Levels of GLUT2 protein and PKC isoforms in BBM were measured by western blotting. Additionally, the role of calcium signalling and PKC activation on facilitative glucose transport was examined by measuring glucose uptake in BBM vesicles prepared from proximal tubules that had been incubated either with thapsigargin, which increases cytosolic calcium, or with the PKC activator phorbol 12-myristate,13-acetate (PMA). RESULTS: Thapsigargin and PMA enhanced GLUT-mediated glucose uptake, but had no effect on sodium-dependent glucose transport. Diabetes significantly increased the protein levels of GLUT2 and PKC-betaI at the BBM. Levels of GLUT2 and PKC-betaI correlated positively with plasma glucose concentration. Diabetes had no effect on BBM levels of alpha, betaII, delta or epsilon isoforms of PKC. CONCLUSIONS/INTERPRETATION: Enhanced GLUT2-mediated glucose transport across the proximal tubule BBM during diabetic hyperglycaemia is closely associated with increased PKC-betaI. Thus, altered levels of GLUT2 and PKC-betaI proteins in the BBM may be important factors in the pathogenic processes underlying diabetic renal injury.

Intestinal phosphate absorption in a model of chronic renal failure.

Hyperphosphatemia is an important consequence of chronic renal failure (CRF). Lowering of the plasma phosphate concentration is believed to be critical in the management of patients with CRF, especially those on dialysis. Reports of the effect of CRF on the intestinal handling of phosphate in vitro have been conflicting; but what happens in vivo has not been studied. What effect a reduction in the dietary phosphate intake has on intestinal phosphate absorption in CRF in vivo is unclear. In this study, we have used the in situ intestine loop technique to determine intestinal phosphate absorption in the 5/6-nephrectomy rat model of CRF under conditions of normal and restricted dietary phosphate intake. In this model of renal disease, we found that there is no significant change in the phosphate absorption in either the duodenum or jejunum regardless of the dietary phosphate intake. There was also no change in the expression of the messenger RNA of the major intestinal phosphate carrier the sodium-dependent-IIb transporter. Furthermore, we found no change in the intestinal villus length or in the location of phosphate uptake along the villus. Our results indicate that in CRF, unlike the kidney, there is no reduction in phosphate transport across the small intestine. This makes intestinal phosphate absorption a potential target in the prevention and treatment of hyperphosphatemia.

Non-haem iron transport in the rat proximal colon.

BACKGROUND: Only 10% of dietary iron is absorbed in the duodenum which implies that 90% (approximately 9 mg day(-1)) reaches the lower small intestine and colon. Therefore the purpose of this study was to assess the iron transport capacity of the rat proximal colon and to determine whether iron absorption is regulated by changes in dietary iron content. MATERIALS AND METHODS: Rats were fed for 14 days on either iron adequate (44 mg Fe kg(-1) diet) or iron-deficient (< 0.5 mg Fe kg(-1) diet) diets. The 59Fe transport across the colonic epithelium and its subsequent appearance in the blood were measured in vivo. In separate studies the colon was excised and used to measure divalent metal transporter expression. RESULTS: Divalent metal transporter (DMT1) was expressed at the apical membrane of the surface epithelium in rat proximal colon. In animals fed an iron-deficient diet, DMT1 mRNA and protein expression were increased. This was accompanied by a significant increase in tissue 59Fe uptake. CONCLUSIONS: The proximal colon can absorb non-haem iron from the intestinal lumen. The purpose of this mechanism remains to be elucidated.

Increased duodenal iron uptake and transfer in a rat model of chronic hypoxia is accompanied by reduced hepcidin expression.

BACKGROUND: Despite the requirement for increased iron delivery for erythropoiesis during hypoxia, there is very little information on how duodenal iron uptake and its transfer to the blood adapts to this condition. AIMS: To assess the effects of 30 days of chronic hypoxia in rats on luminal iron uptake and transfer of the metal to blood, together with gene expression of hepcidin, a proposed negative regulator of iron transport. METHODS: 59-Fe uptake by isolated duodenum and its transfer to blood by in vivo duodenal segments was measured after exposure of rats to room air or 10% oxygen for four weeks. Liver hepcidin expression was measured by real time reverse transcription-polymerase chain reaction. The effects of hypoxia on hepcidin gene expression by HepG2 cells was also determined. RESULTS: Hypoxia did not affect villus length but enhanced (+192.6%) luminal iron uptake by increasing the rate of uptake by all enterocytes, particularly those on the upper villus. Hypoxia promoted iron transfer to the blood but reduced mucosal iron accumulation in vivo by 66.7%. Hypoxia reduced expression of hepcidin mRNA in both rat liver and HepG2 cells. CONCLUSIONS: Prolonged hypoxia enhances iron transport from duodenal lumen to blood but the process is unable to fully meet the iron requirement for increased erythropoiesis. Reduced secretion of hepcidin may be pivotal to the changes in iron absorption. The processes responsible for suppression of hepcidin expression are unknown but are likely to involve a direct effect of hypoxia on hepatocytes.

Effects of fasting and glucagon on the kinetics of active hexose absorption across the rat distal ileum in vivo.

The effects of starvation for 1, 2 or 3 d and the administration of glucagon to fed rats on the kinetics of active glucose and galactose absorption across the distal ileum have been determined in vivo. Fasting caused reductions in 'apparent Kt' for glucose and galactose transport together with a decrease in Jmax for glucose but not galactose absorption. Treatment with glucagon produced decreases in Kt for the absorption of both hexoses and an increase in Jmax for glucose absorption. The Jmax for galactose uptake, however, was unaltered by glucagon administration. Villus size was unaltered by starvation of up to 3 d duration, but significantly decreased by glucagon treatment. The results suggest that chronically elevated plasma glucagon levels may be a factor in the change in kinetics of hexose absorption in the distal ileum evoked by fasting.

Effect of sodium concentration and plasma sugar concentration on hexose absorption by the rat jejunum in vivo. Further evidence of two transport mechanisms.

The sodium-dependency of both the saturable and non-saturable components of glucose and galactose absorption across the rat jejunum in vivo has been determined. The non-saturable process appears to be unaffected by sodium removal but when Na+ concentrations in the lumenal fluid are progressively reduced from 143 mM to 0 mM the Jmax for active absorption is greatly decreased. In a separate study these two components of glucose transport were further investigated using intravenous sugar infusion to modify the transepithelial sugar concentration gradient. When identical glucose concentrations were present in plasma and intestinal lumen, the reduction in glucose absorption was fully accounted for by the elimination of the non-saturable component from the overall absorptive process. Together, these observations can be interpreted as further evidence for the existence of at least two absorption processes in vivo. The implication for analysing results from experimental studies of intestinal sugar absorption in vivo is discussed.