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The inhibitory effects of the angiotensin-converting enzyme (ACE)-ANG II-angiotensin type 1 (AT₁) receptor axis on jejunal glucose uptake and the reduced expression of this system in type 1 diabetes mellitus (T1DM) have been documented previously. The ACE2-ANG-(1-7)-Mas receptor axis is thought to oppose the actions of the ACE-ANG II-AT₁ receptor axis in heart, liver, and kidney. However, the possible involvement of the ACE2-ANG-(1-7)-Mas receptor system on enhanced jejunal glucose transport in T1DM has yet to be determined. Rat everted jejunum and Caco-2 cells were used to determine the effects of ANG-(1-7) on glucose uptake and to study the ACE2-ANG-(1-7)-Mas receptor signaling pathway. Expression of target gene and protein in jejunal enterocytes and human Caco-2 cells were quantified using real-time PCR and Western blotting. T1DM increased jejunal protein and mRNA expression of ACE2 (by 59 and 173%, respectively) and Mas receptor (by 55 and 100%, respectively) in jejunum. One millimolar ANG-(1-7) reduced glucose uptake in jejunum and Caco-2 cells by 30.6 and 30.3%, respectively, effects that were abolished following addition of 1 μM A-779 (a Mas receptor blocker) or 1 μM GF-109203X (protein kinase C inhibitor) to incubation buffer for jejunum or Caco-2 cells, respectively. Finally, intravenous treatment of animals with ANG-(1-7) significantly improved oral glucose tolerance in T1DM but not control animals. In conclusion, enhanced activity of the ACE2-ANG-(1-7)-Mas receptor axis in jejunal enterocytes is likely to moderate the T1DM-induced increase in jejunal glucose uptake resulting from downregulation of the ACE-ANG II-AT₁ receptor axis. Therefore, altered activity of both ACE and ACE2 systems during diabetes will determine the overall rate of glucose transport across the jejunal epithelium.

There is increasing evidence that locally produced angiotensin AII (AII) regulates the function of many tissues, but the involvement of enterocyte-derived AII in the control of intestinal transport is unknown. This study examined whether there is a local renin-angiotensin system (RAS) in rat villus enterocytes and assessed the effects of AII on SGLT1-dependent glucose transport across the brush border membrane (BBM). Gene and protein expression of angiotensinogen, ACE, and AT(1) and AT(2) receptors were studied in jejunal and ileal enterocytes using immunocytochemistry, Western blotting and RT-PCR. Mucosal uptake of d-[(14)C]glucose by everted intestinal sleeves before and after addition of AII (0-100 nm) to the mucosal buffer was measured in the presence or absence of the AT(1) receptor antagonist losartan (1 microm). Immunocytochemistry revealed the expression of angiotensinogen, ACE, and AT(1) and AT(2) receptors in enterocytes; immunoreactivity of AT(1) receptor and angiotensinogen proteins was especially pronounced at the BBM. Expression of angiotensinogen and AT(1) and AT(2) receptors, but not ACE, was greater in the ileum than the jejunum. Addition of AII to mucosal buffer inhibited phlorizin-sensitive (SGLT1-dependent) jejunal glucose uptake in a rapid and dose-dependent manner and reduced the expression of SGLT1 at the BBM. Losartan attenuated the inhibitory action of AII on glucose uptake. AII did not affect jejunal uptake of l-leucine. The detection of RAS components at the enterocyte BBM, and the rapid inhibition of SGLT1-dependent glucose uptake by luminal AII suggest that AII secretion exerts autocrine control of intestinal glucose transport.

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.

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.

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.

Background: Increased jejunal glucose transport after ingestion of carbohydrate rich diets may reflect higher concentrations of lumenal glucose. Normal processing of carbohydrate causes wide fluctuations in glucose concentration in the jejunal lumen and this raises the question of whether the high lumenal concentrations seen at peak digestion affect glucose uptake., Aims: To study the effects of 30 minute exposure of rat jejunal mucosa to glucose on sodium-glucose transporter (SGLT1) mediated glucose transport across the brush border membrane., Methods: Jejunal mucosa was exposed in vitro or in vivo to 25 mM glucose or 25 mM mannitol for 30 minutes. In addition, isolated villus enterocytes were incubated with mannitol or glucose for the same time. Brush border membrane vesicles were isolated from these preparations and phlorizin sensitive 3H-D-glucose accumulation was measured., Results: Lumenal glucose in vivo significantly enhanced SGLT1 mediated glucose uptake by 49.2-57.2%. For jejunal loops in vitro, the increase was 32.0-85.2%. Kinetic analysis disclosed a 50% greater Vmax for glucose uptake in each preparation. The facilitated and passive components of uptake were, however, unaffected by prior exposure to glucose. Incubation of villus enterocytes with 25 mM glucose did not influence glucose uptake by brush border membranes. Finally, exposure of intact mucosa to 20 mM galactose, a nonmetabolised sugar also transported by SGLT1, did not alter glucose transport., Conclusions: Lumenal glucose promotes glucose transport by brush border membrane within 30 minutes. An intact mucosa is necessary for upregulation and evidence suggests that the response is mediated by locally acting mechanisms.

Rats treated with streptozotocin for 17 days were used to determine the cellular origin of enhanced brush border glucose transport in the diabetic small intestine. In the jejunum of both normal and diabetic rats, phlorizin-sensitive (SGLT1-mediated) glucose transport was shown, by section autoradiography, to take place in upper villus enterocytes. The distribution of brush border SGLT1 transporters along villi, determined using immunogold cytochemistry, was similar to that found for glucose uptake. Longer villi, supporting a larger number of absorbing enterocytes in the diabetic jejunum, appeared to be responsible for increased glucose uptake in this condition. SGLT1 protein and SGLT1-mediated glucose transport were undetectable in normal distal ileal villi. However, following treatment with streptozotocin, both SGLT1 protein and SGLT1-mediated glucose transport were found to be present in basal ileal villus enterocytes. SGLT1 protein and SGLT1-mediated glucose transport both increased during enterocyte migration to the villus tip. Cellular induction of the SGLT1 transporter, as well as longer villi contribute to enhanced glucose transport in diabetic rat distal ileum. Close correlation between the positional expression of SGLT1 protein and absorptive function suggests that transporter density is an important determinant for up-regulation of sodium-dependent glucose transport in diabetes.

The effect of infusion (2 h) of various sugars (20%, w/v) or early diabetes mellitus (2 h following injection of streptozotocin) on the potential difference (PD) across the brush border of rat jejunum has been studied in vitro. Infusion of glucose or galactose resulted in hyperpolarization of the brush-border PD (-55.2 and -54.9 mV respectively) compared with mannitol-infused animals (-47.7 mV). Infusion of fructose and alpha-methyl glucoside was without effect on PD. Ion substitution experiments showed that the glucose infusion-induced hyperpolarization was due either to a decrease in Cl- conductance and/or increased K+ conductance of the brush-border membrane. Na+ conductance appeared to be unaltered in these experiments. Since galactose infusion hyperpolarized PD without elevating plasma glucose or insulin levels, it seems that neither glucose nor insulin alone is responsible for the significantly greater PD. Treatment with streptozotocin produced glycosuria and hyperglycaemia within approximately 90 min and animals were used 30-60 min later. PD in streptozotocin-treated animals (-51.8 mV) was significantly greater than that measured in diluent-injected animals (-45.3 mV, P < 0.001). However, unlike the situation with sugar infusion, ion substitution experiments showed that the hyperpolarization seen in diabetic animals was due to a decreased Na+ conductance of the brush-border membrane. Since the magnitude of the brush-border PD dictates the electrochemical driving force for Na(+)-sugar cotransport, our data represent the first evidence for an effect of acute hyperglycaemia on the capacity for brush border sugar uptake. However, the enterocyte and systemic factors responsible for the membrane hyperpolarization are unclear.

Although the involvement of the adenylate cyclase system with glucose transport in the small intestine is poorly understood, there is increasing evidence that cyclic AMP stimulates sugar uptake. In order to study further the effects of cyclic AMP on this process, we have measured glucose accumulation by brush-border and basolateral membrane vesicles prepared from villus enterocytes following exposure of these cells to cyclic AMP and theophylline. Brush-border vesicles derived from enterocytes incubated with cyclic AMP and theophylline accumulated significantly more glucose over a wide range of sugar concentrations, suggesting a change in maximum velocity of the transport system. Glucose uptake by basolateral vesicles was increased at low, but not at high sugar concentrations. Incubation of isolated enterocytes with pancreatic glucagon at concentrations known to stimulate sugar transport by these cells significantly increased enterocyte levels of cyclic AMP. Treatment with glucagon or cyclic AMP resulted in significant hyperpolarization of the potential difference across the brush-border membrane, an important driving force for Na(+)-sugar cotransport. The response to glucagon and cyclic AMP appears to be caused by a decrease in Na+ permeability of the mucosal membrane. Taken together, these results suggest that cyclic AMP is a mediator of the actions of glucagon on enterocytes and provide further evidence for a role of cyclic AMP in the modulation of sugar transport across the intestinal enterocyte.

Acute hypoglycaemia enhances intestinal sugar uptake but the mechanisms involved are unknown. Results from the present study show increased galactose movement across the brush border and basolateral membranes of isolated upper, but not mid-villus, jejunal enterocytes 45 min after intravenous administration of insulin to rats at a level which reduced by half plasma glucose concentration. Incubation of upper villus cells from uninjected animals with insulin (100 mU ml-1) for 40 min was without effect on brush border or basolateral sugar transport. Insulin treatment of rats did not affect glucose uptake by brush border vesicles prepared from upper villus cells when the process was driven by an inwardly directed 100 mM sodium thiocyanate gradient. In contrast, glucose uptake using a 100 mM inwardly directed sodium chloride gradient was reduced by 49% following hypoglycaemia. It is concluded that the enhanced sugar uptake following insulin hypoglycaemia involves both brush border and basolateral membranes of only the most mature villus cells at the villus tip. Upregulation of Na(+)-sugar cotransport at the brush border is best explained by an increased electrochemical driving force for Na(+)-sugar cotransport rather than increased numbers of transporters. The transport response is not due to a direct effect of insulin on the enterocyte and the possible systemic factors involved are discussed.

Preparations of isolated rat jejunal enterocyte and brush-border and basolateral membrane vesicles have been used to study the effects of a 15 min exposure of upper and mid-villus enterocytes to pancreatic glucagon on the initial, unidirectional phlorhizin-sensitive (brush border) transport of galactose and phlorhizin-insensitive (basolateral) movement of the sugar. These acute effects of glucagon have been compared with responses following treatment of animals for 1 or 3 days with the hormone. Incubation of cells with glucagon significantly stimulated phlorhizin-sensitive uptake by 42 and 64% for upper and mid-villus cells, respectively. Glucagon, however, was without effect on phlorhizin-insensitive galactose uptake. This differential action of the hormone at the two cellular loci was confirmed by uptake data obtained using purified brush-border and basolateral membrane vesicles prepared from isolated cells. In contrast to the acute challenge with glucagon, treatment of animals for 3 days with the hormone significantly increased both phlorizin-sensitive (upper villus +31%, mid-villus +74%) and phlorizin-insensitive (upper villus +42%, mid-villus +53%) galactose uptake. Glucagon exposure of exposure of isolated cells from 3 days treated animals was without further effect on galactose uptake at the two membrane loci. These data represent the first evidence for a direct action of pancreatic glucagon on enterocyte sugar transport. Thus the hormone is likely to be important in the physiological control of sugar absorption in addition to its possible role in the modulation of transport during starvation and diabetes mellitus, conditions characterized by hyperglucanonaemia and enhanced intestinal sugar transport.

Bradykinin has been shown to stimulate sugar uptake by intact intestine, but the cellular locus of this action is unknown. In this study on rat we show that bradykinin (10(-7) M) stimulates both sodium-dependent glucose uptake at the brush-border membrane and carrier-mediated movement across the basolateral membrane of upper villus enterocytes. These results suggest a role for bradykinin in the local control of glucose transport though the mechanisms underlying these effects have still to be elucidated.

1. The effects of streptozotocin-induced diabetes mellitus on active jejunal glucose uptake in vivo, and on galactose movement across the brush-border (phlorhizin-sensitive) and basolateral (phlorhizin-insensitive) membranes of isolated upper and mid-villus enterocytes has been studied. 2. Chronic diabetes increased unidirectional phlorhizin-sensitive galactose uptake by mid-villus but not upper villus cells. In contrast, phlorhizin-insensitive uptake by both cell populations was enhanced by diabetes. 3. Diabetes increased glucose absorption in vivo by mechanisms which were unrelated to hyperphagia. Mucosal hyperplasia acting together with an epithelium containing a higher proportion of mature enterocytes is the most likely explanation for the response. 4. We conclude that, during diabetes, the mid-villus region is an important site of adaptation with functional changes occurring at both the brush-border and basolateral membranes. The increased hexose transport ability of the basolateral membrane is retained during cell transit along the villus.

The mechanisms involved in the increased Na(+)-dependent nutrient uptake across intestine of diabetic animals are poorly understood. Here we have studied the effect of acute (7d) and chronic (30-40d) diabetes on the autoradiographic localization of 3H-L-valine accumulation by rat jejunal villi and on enterocyte migration rate. In control rats, Na(+)-dependent valine uptake was confined to enterocytes on the upper 20-23% of the villus. In intestine from diabetic rats, however, this area was extended to occupy the upper 42-45% of an enlarged villus surface. Hyperphagia was not responsible for the expanded functional surface and systemic factors are therefore implicated in the adaptive response. Enterocyte migration rate was found to be unaffected by diabetes but an increased villus height in this condition resulted in an additional 13.5 h in enterocyte lifespan. These data are compatible with the hypothesis that during diabetes the earlier maturation of enterocyte absorptive function produces an epithelial surface containing a higher proportion of mature enterocytes.

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.

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.

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.

We report here the effects of a 72-h fast on the localisation of Na-dependent [3H]-valine uptake by rat small intestine. Starvation results in the earlier appearance of valine transport during cell migration and an enhanced accumulation of the amino acid at the villus tip.

The effects of feeding glucose, galactose, and alpha-methyl glucoside on the kinetics of absorption of these sugars in the jejunum of the fasting rat were assessed by recently developed chemical and electrical methods for characterizing the electrogenic active transfer mechanisms in vivo. The parameters of 'apparent Km' (an index of carrier affinity) and Vmax (an index of the maximum jejunal transfer capacity) were obtained from the kinetic data to describe quantitatively the active transport mechanisms in rats fed their normal solid diet, in rats deprived of solid food for three days, and in rats deprived of solid food but allowed ad libitum access to isotonic solutions of glucose, galactose, or alpha-methyl glucoside for up to three days. Feeding the sugars to fasting rats produced complex differential effects on the 'apparent Km's' and Vmax's of the various transport mechanisms. The changes, although complex, can best be explained by alterations in the transport mechanisms per se rather than by non-specific changes in intestinal morphology or metabolism. The differential effects induced by the feeding sugars are further evidence for the concept that jejunal enterocytes possess multiple carriers or mechansims for the absorption of actively transported sugars in vivo. The multiple forms of the sugar carriers (isocarriers?) MAY BE INVOLVED IN THE FACULTATIVE RESPONSES OF THE ENTEROCYTES TO CHANGES IN THE DIETARY LEVELS OF SUGAR AND MAY BE IMPORTANT IN UNDERSTANDING THE INFLUENCES OF DISEASE AND NUTRITIONAL STATUS ON ABSORPTION PROCESSES IN MAN.

The effects of perfusing solutions of different composition into the distal ileum of the rat, on glucose or galactose absorption from the jejunum has been determined in vivo. The rate of glucose absorption via the active, phlorhizin-sensitive pathway was increased significantly when either glucose or maltose were present in the lower ileum. In contrast, the presence of galactose, lactose and fructose produced only minimal effects on active glucose absorption. The phlorhizin-insensitive component of glucose absorption was unaffected by the presence of sugar in the distal region. Perfusion of glucose into the ileum enhanced the active uptake of the poorly metabolized hexose, galactose, from the jejunum. The administration of cycloheximide abolished the increase in active glucose absorption induced by distal maltose perfusion. The response to distal glucose (a) required the sugar to be perfused for a minimum of 3 h before increases in absorption became significant, and (b) was not dependent on distal glucose absorption by the phlorhizin-sensitive pathway. The possible mechanisms involved in this adaptive response are discussed. The enhancement of jejunal absorption may be important in man in conditions where glucose is present at abnormally high levels in the distal ileum.

Rats were pair-fed for 28-32 days a nutritionally adequate liquid control diet or an isocaloric diet containing ethanol (5% v/v). Brush-border membrane vesicles were prepared from jejunal mucosa and Na+-dependent D-glucose accumulation was measured using sugar concentrations of 0.2-1.0 mmol l-1. In separate experiments the effects of acute ethanol exposure on glucose uptake by vesicles prepared from chow-fed rats were determined. Chronic ethanol ingestion enhanced Na+-gradient-dependent glucose uptake. In contrast, incubation of vesicles with alcohol (4% v/v), reduced active uptake of the sugar.