Glucose metabolism in the mucosa of the small intestine. The effect of glucose on hexokinase activity.
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ABSTRACT:
SUBMITTER: Shakespeare P
PROVIDER: S-EPMC1187495 | biostudies-other | 1969 Jan
REPOSITORIES: biostudies-other
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Project description:1. The intracellular distribution of hexokinase activity was studied in the mucosa of rat and guinea-pig small intestine. In the rat 60% and in the guinea pig 45% of the hexokinase activity of homogenates were recovered in a total particulate fraction that contained only 5-17% of the homogenate activity of hexose phosphate isomerase, pyruvate kinase, lactate dehydrogenase and overall glycolysis (formation of lactate from glucose). 2. Fractionation of homogenates from guineapig small intestine showed that the particulate hexokinase activity was chiefly in the mitochondrial fraction with a small proportion in the nuclei plus brush-border fraction. 3. After chromatography of the particle-free supernatants on DEAE-cellulose, hexokinase types I and II were determined quantitatively. No evidence was obtained for the presence of hexokinase type III or glucokinase. In the preparations from guinea pigs, hexokinase types I and II amounted to 69% and 31% respectively of the eluted activity; the corresponding values for preparations from rats were 5.8% and 94.2%. 4. Total and specific hexokinase activities decreased significantly in homogenates and particle-free supernatants prepared from the intestinal mucosa of rats starved for 36hr. and increased again after re-feeding. The decrease in hexokinase activity in the particle-free supernatant from starved rats was chiefly due to a decrease in the type II enzyme.
| S-EPMC1186749 | biostudies-other
Project description:The effect of intubation of starved rats with solutions of glucose, 3-O-methylglucose, mannose, 2-deoxyglucose and sorbitol on the subcellular location of hexokinase in the mucosa of the small intestine was studied. Glucose, 3-O-methylglucose and mannose caused the soluble hexokinase activity to rise to a value similar to that found in fed animals, whereas sorbitol and 2-deoxyglucose caused smaller increases.
| S-EPMC1177568 | biostudies-other
Project description:1. The effect of perfusion on the activities of hexokinase and lactate dehydrogenase was studied in the proximal half of the small intestine of fed and starved rats. 2. Perfusion of preparations from starved rats with a medium containing glucose caused a significant increase in hexokinase activity of the particle-free supernatant. The increase in activity was observed as early as 5min after the start of perfusion and persisted for up to 66min of perfusion. No increase in hexokinase activity of the particle-free supernatant was observed when a medium containing mannitol was used. As a further control, preparations from fed rats were perfused under the same conditions. With the medium containing glucose, the hexokinase activity of the particle-free supernatant remained unchanged during the first 15min of perfusion and thereafter fell gradually until, after 66min of perfusion, 73% of the original activity was retained. 3. The activity of lactate dehydrogenase in the particle-free supernatant prepared from the proximal half of the untreated small intestine of starved rats was significantly lower than in corresponding preparations from fed animals. However, it did not change significantly on perfusion with media containing either mannitol or glucose. 4. The distribution of hexokinase activity between total particulate fraction and particle-free supernatant was measured in preparations from starved rats after perfusion for 5-10min. In preparations that had not been perfused the ratio of hexokinase activity in total particulate fraction/particle-free supernatant was significantly higher in starved than in fed animals. After perfusion with a medium containing glucose, the total homogenate activity had not changed significantly, whereas the ratio of hexokinase activity in total particulate fraction/particle-free supernatant decreased significantly and approached the value obtained with fed animals. 5. The results agree with the view that the glucose-dependent increase of hexokinase activity in the soluble cell compartment as observed in vivo and in vitro in the intestinal mucosa of starved rats is brought about by a release of hexokinase activity from a particulate subcellular structure(s).
| S-EPMC1185321 | biostudies-other
Project description:In common with other phosphofructokinase isoenzymes, phosphofructokinase in the epithelial cells of rat small-intestinal mucosa is activated by fructose 2,6-bisphosphate. However, fructose 2,6-bisphosphate was found not to be present in mucosa as judged by three criteria: (1) chromatography on Sephadex G-25 of crude mucosal extracts from fed rats did not result in a decrease, or indeed any change, in the activity of phosphofructokinase under suboptimal conditions at pH7; (2) ultrafiltrates of mucosal extracts did not possess any acid-labile activating activity when tested against chromatographed liver phosphofructokinase; (3) phosphofructokinase-2 activity was not detectable in mucosal extracts. Furthermore, the perfusion in vitro of isolated loops of jejunum or the incubation of mucosal scrapings from either fed rats or rats starved for 48 h showed that the activity of mucosal phosphofructokinase is not subject to short-term regulation by glucose. These observations are consistent with the view that phosphofructokinase is the rate-limiting enzyme of glycolysis in intestinal mucosa and account for the fact that the rate of glucose utilization by rat small intestine is not very responsive to changes in the concentration of glucose in the lumen.
| S-EPMC1153360 | biostudies-other
Project description:1. The occurrence of five enzymes of the pentose phosphate pathway in cell-free preparations of the mucosa of rat small intestine is described. These enzymes were found to be localized mainly in the supernatant fraction (6240000g-min.). 2. The properties of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were studied with respect to K(m) values for substrates and NADP(+), pH optima and the effects of p-chloromercuribenzoate and palmitoyl-CoA. Higher total and specific activities of these two dehydrogenases were noted in the proximal half of the small intestine of the rat than in the distal half. 3. The specific activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the mucosa of the small intestine of the rat, cat, rabbit and guinea pig were compared. 4. In the rat the specific activities of ribose 5-phosphate isomerase, transketolase and transaldolase were higher in the supernatant fractions from the intestinal mucosa than in those from the liver. 5. The role of the pentose phosphate pathway is discussed in relation to the metabolism of hexose phosphates in the intestinal mucosa.
| S-EPMC1270064 | biostudies-other
Project description:1. Lactic acid formation in supernatant fractions of homogenates of cat or rat small-intestinal mucosa was measured under optimum conditions with glucose, fructose, glucose 6-phosphate, fructose 1,6-diphosphate or 3-phosphoglycerate as substrate. 2. Between 80 and 107% of the glycolytic activity of the homogenate was recovered in these particle-free preparations when glucose, fructose, glucose 6-phosphate or fructose 1,6-diphosphate was used as substrate. 3. Evidence was obtained that hexokinase and phosphofructokinase were the rate-limiting enzymes in the initial sequence of glycolytic reactions. The limitation of rate by hexokinase was much more pronounced in preparations from the cat than in those from the rat. 4. With subcellular preparations from cat or rat small intestine lactic acid was also formed from ribose 5-phosphate and at rates similar to those observed with glucose. 5. A higher rate of glycolysis was observed with glucose 6-phosphate as substrate with preparations from the proximal half of the small intestine of the rat as compared with the distal half. 6. Mucosal preparations from rats starved for 24-48hr. exhibited only about one-quarter of the glycolytic activity of those of fed control groups. The decreased rate of formation of lactic acid from either glucose or fructose was mainly due to a decrease in the activity of hexokinase(s). The activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and a number of other enzymes were not significantly decreased by starvation. 7. The results are discussed in relation to metabolic control of glycolysis in other mammalian tissues.
| S-EPMC1265156 | biostudies-other
Project description:Glucose transport and metabolism by rat small intestine was investigated by using a preparation for the combined perfusion of the lumen and the vascular bed. 2. When 5 mM-glucose was present in the lumen, only 29% was transported unchanged to the vascular side. Lactate output into the vascular and luminal fluids accounted for a further 53% and 4% respectively of the glucose taken up from the lumen. 3. Glucose was readily taken up when added at 5 mM to the vascular compartment only. Vascular lactate output accounted for approx. 33% of the glucose uptake, and luminal lactate for 6%. 4. When 2 mM-glucose was added to the lumen with 5 mM-glucose in the vascular perfusate, there was no detectable net transport of glucose to the vascular side. However, of the glucose taken up from the lumen, 31% and 2% could be accounted for by increases in vascular and luminal lactate respectively. 5. When 10 mM-glucose was added to the lumen, with 5 mM-glucose in the vascular perfusate, 33% of the glucose disappearing from the lumen was transported to the vascular side. Extra lactate output to the vascular and lumen perfusates accounted for 50% and 9% respectively of the glucose uptake from the lumen. 6. These studies indicate that at low luminal glucose concentrations no sugar is transferred to the blood unchanged, and at sugar concentrations of 5--10 mM only 25--50% of the glucose leaving the lumen reaches the serosal side intact. Furthermore, the small intestine has a greater propensity to form lactate from luminal glucose than from vascular glucose.
| S-EPMC1152027 | biostudies-other
Project description:1. The submitochondrial localization of hexokinase activity in preparations of mitochondria from the small intestine of the guinea pig was studied by conventional methods. 2. Hexokinase activity in this tissue was predominantly associated with the outer mitochondrial membrane. 3. The inactivation of mitochondrial enzymes by trypsin in iso-osmotic and hypo-osmotic conditions was also used to determine the submitochondrial localization of hexokinase activity. 4. Hexokinase activity was found to be on the outside of the outer mitochondrial membrane. 5. It was shown that both type I and type II hexokinase activities are bound to the outside of the outer mitochondrial membrane. The types are present in the same ratio as that in which they occur in the cytosol of the cell. 6. Mitochondrial hexokinase from the small intestine did not show the latency phenomenon demonstrated by mitochondrial hexokinase from brain when subjected to a variety of treatments. However, hexokinase activity was solubilized from preparations of mitochondria from the small intestine by the same treatments as for mitochondrial hexokinase from brain. 7. The submitochondrial distribution of hexokinase activity in mitochondrial preparations from rat brain was determined by the trypsin inactivation method. 8. Hexokinase activity in preparations of mitochondria from rat brain was found on the outside of the outer membrane, between the mitochondrial membranes, and within the inner mitochondrial membrane. 9. Hexokinase from rat brain showed latency properties irrespective of its submitochondrial location.
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Project description:Increased glucose metabolism is considered as one of the most important metabolic alterations adapted by cancer cells in order to generate energy as well as high levels of glycolytic intermediates to support rapid proliferation. PH domain leucine-rich repeat protein phosphatase (PHLPP) belongs to a novel family of Ser/Thr protein phosphatases that function as tumor suppressors in various types of human cancer. Here we determined the role of PHLPP in regulating glucose metabolism in colon cancer cells. Knockdown of PHLPP increased the rate of glucose consumption and lactate production, whereas overexpression of PHLPP had the opposite effect. Bioenergetic analysis using Seahorse Extracelluar Flux Analyzer revealed that silencing PHLPP expression induced a glycolytic shift in colon cancer cells. Mechanistically, we found that PHLPP formed a complex with Akt and hexokinase 2 (HK2) in the mitochondrial fraction of colon cancer cells and knockdown of PHLPP enhanced Akt-mediated phosphorylation and mitochondrial localization of HK2. Depletion of HK2 expression or treating cells with Akt and HK2 inhibitors reversed PHLPP loss-induced increase in glycolysis. Furthermore, PHLPP knockdown cells became addicted to glucose as a major energy source in that glucose starvation significantly decreased cancer cell survival. As HK2 is the key enzyme that determines the direction and magnitude of glucose flux, our study identified PHLPP as a novel regulator of glucose metabolism by controlling HK2 activity in colon cancer cells.
| S-EPMC5253420 | biostudies-literature
Project description:The mammalian intestine displays two distinct patterns of mucosal organization. The small intestine contains mucosal epithelial invaginations (the crypts of Lieberkühn) that are continuous with evaginations (villi) into the lumen. The colon also contains crypts of Lieberkühn, but its epithelial surface is lined by flat surface cuffs. The epithelial cells of both organs communicate with the underlying mesenchyme through a basement membrane that is composed of a variety of extracellular matrix proteins, including members of the laminin family. The basement membranes of the small intestine and colon contain distinct laminin subtypes; notably, the villus basement membrane is rich in laminin alpha 5. Here, we show that the diminution of laminin alpha 5 in a mouse model led to a compensatory deposition of colonic laminins, which resulted in a transformation from a small intestinal to a colonic mucosal architecture. The alteration in mucosal architecture was associated with reduced levels of nuclear p27Kip1 - a cell-cycle regulator - and altered intestinal epithelial cell proliferation, migration and differentiation. Our results suggest that laminin alpha 5 has a crucial role in establishing and maintaining the specific mucosal pattern of the mouse small intestine.
| S-EPMC2587097 | biostudies-literature