male wistar rats weighing 220240 g and male c57bl/6j mice weighing 2025 g were from centre elevage janvier , le genest - st . the animals had free access to tap water and standard food and were treated in accordance with european community guidelines concerning care and use of laboratory animals . gavages of conscious rats or mice with 1 g of glucose per kilogram of body weight were performed after a 16-h fast using a d - glucose solution without ( control ) or with relm-. the total bolus volume for mice and rats was 0.25 ml and 1 ml , respectively , and the amount of glucose in each bolus was adjusted for the animal weight . the recombinant murine relm- ( 18 kda ; peprotech , neuilly - sur - seine , france ) used was highly purified by high - performance liquid chromatography and was endotoxin - free . relm- , at a final concentration of 0.1 and 1 nmol / l , results in dose range of 0.010.1 g / kg of body weight . relm- is a stable molecule as high amounts of the homodimer form have been detected in mice and human stools ( 21 ) . preliminary experiments showed that the peptide given by gavage was found in proximal small intestine after 5 min with a recovery of 70% . before starting the ogtt , blood samples were taken from the tail and fasting blood glucose levels ( milligram per deciliter ) were determined ( accu - chek ; roche diagnostic , meylan , france ) . the bleeds were further taken at 15 , 30 , 60 , and 120 min after oral glucose administration . rats were fasted for 16 h and were killed by pentobarbital overdose . the proximal jejunum was dissected out and four adjacent samples were mounted in ussing chambers as described previously ( 22 ) . the tissues were bathed with 4 ml of krebs - ringer bicarbonate ( krb ) solution ( ph 7.4 ) with 10 mmol / l glucose at 37c . in the solution bathing the mucosal side of the tissue , glucose electrogenic ion transport was monitored continuously as short - circuit current ( isc ) by using an automated voltage clamp apparatus ( dvc 1000 ; wpi , aston , england ) linked through a maclab 8 to a macintosh computer . nmol / l ) was added in the mucosal bath 2 min before a glucose challenge . carbachol ( 100 mmol / l ) was added at the end of each experiment as a control . further , similar tests were performed with relm- incubated overnight at 4c with a rabbit polyclonal antibody raised against relm-. results were expressed as the intensity of the isc ( a / cm ) or as the percentage of the peak isc obtained after glucose challenge ( measured after 3 min ) over basal isc ( measured just before the addition of glucose ) . jejunal loops ( 4 cm long ) were prepared and 0.5 ml of krb solution without ( control ) or with 1 nmol / l relm- was inserted inside the jejunal lumen . the jejunal loops were incubated for 15 min in oxygenized krb at 37c and conditions were maintained during hexose transport assay . the corresponding jejunal loops were filled with 1 ml of krb solution without ( control ) or with 1 nmol / l relm- and containing 0.02 ci / ml of the isotopic tracer d-[1-c ] glucose ( 49.5 mci / mmol ) and glucose to obtain a final concentration of 10 , 30 , and 100 mmol / l . similarly , we studied the paracellular transport with 30 mmol / l mannitol and the isotopic tracer d-[1-c ] mannitol ( 59 mci / mmol ) at 0.2 ci / ml . all the jejunal loops were incubated in 10 ml of krb solution during the indicated time . the radioactivity was measured in the collected samples of serosal krb solution and used to calculate glucose or mannitol transport as picomoles per milligram of jejunal protein . five independent experiments were performed and significance is expressed as * p < 0.05,**p < 0.01 . rats were anesthetized by pentobarbital and laparotomized for in situ experiments . three jejunal segments ( 5 cm long ) were tied and filled with 3 ml of krb without ( control ) or with 1 nmol / l relm-. after 3 min of in situ incubation , 3 ml of 60 mmol / l glucose solution were injected in the lumen to obtain a final concentration of 30 mmol / l . after a further 3 min , these loops were removed and opened along the mesenteric border and the mucosa was scraped off on ice with a glass blade . total cell protein extracts and bbms were prepared from the scrapings as previously described ( 23 ) , and enrichment was estimated by determination of alkaline phosphatase activity ( 20-fold increase of activity in bbms ) . solubilized proteins were resolved by electrophoresis on 10% sds - page gels and transferred onto nitrocellulose membranes for immunoblot analysis . the following rabbit antibodies were used at a 1:1,000 dilution : sglt-1 ( ab 1352 ; chemicon international , temecula , ca ) , glut2 , pkc ii ( sc-9117 , sc-210 ; santa cruz biotechnology , tebu - bio , france ) , phospho - ampk- ( thr172 ) , ampk- ( 23a3 ) , and phospho - pkc ( pan ( 2531 , 2603 , 190d10 ; cell signaling technology , ozyme , france ) . the intensity of the specific immunoreactive bands was quantified using national institutes of health image ( scion , frederick , md ) . uptake experiments were performed using rat intestinal everted rings as previously described ( 24 ) . groups of eight intestinal rings were incubated at 37c for 15 min in oxygenized krb buffer in the absence ( control ) and the presence of relm- ( 1 nmol / l ) and cytochalasin b as indicated . then the rings were incubated for 2 min in an uptake solution corresponding to a krb buffer containing 30 mmol / l glucose and 0.1 ci / ml of the isotopic tracer d-[1-c ] glucose . after adding the uptake solution , the rings were washed in ice - cold krb solution , and radioactivity incorporated in the tissue was quantified by liquid scintillation . data were not corrected for extracellular substrate because relm- was found not to affect paracellular diffusion . total protein from homogenized tissue with a dounce homogenizer was determined with bicinchoninic acid reagent from pierce ( thermo scientific , brebires , france ) . recombinant murine relm- was purchased from peprotech ( neuilly - sur - seine , france ) . d-[1-c ] mannitol was from ge healthcare amersham biosciences ( les ulis , france ) , d-[1-c ] glucose was from perkin elmer ( boston , ma ) , and compound c from merck sharp & dohme - chibret ( paris , france ) . all other chemical reagents were purchased from sigma ( st . louis , mo ) . one - way anova with turkey - kramer multiple comparisons post hoc test was performed using graphpad prism version 4.0 for windows ( graphpad software , san diego , ca ) . gavages of conscious rats or mice with 1 g of glucose per kilogram of body weight were performed after a 16-h fast using a d - glucose solution without ( control ) or with relm-. the total bolus volume for mice and rats was 0.25 ml and 1 ml , respectively , and the amount of glucose in each bolus was adjusted for the animal weight . the recombinant murine relm- ( 18 kda ; peprotech , neuilly - sur - seine , france ) used was highly purified by high - performance liquid chromatography and was endotoxin - free . relm- , at a final concentration of 0.1 and 1 nmol / l , results in dose range of 0.010.1 g / kg of body weight . relm- is a stable molecule as high amounts of the homodimer form have been detected in mice and human stools ( 21 ) . preliminary experiments showed that the peptide given by gavage was found in proximal small intestine after 5 min with a recovery of 70% . before starting the ogtt , blood samples were taken from the tail and fasting blood glucose levels ( milligram per deciliter ) were determined ( accu - chek ; roche diagnostic , meylan , france ) . the bleeds were further taken at 15 , 30 , 60 , and 120 min after oral glucose administration . rats were fasted for 16 h and were killed by pentobarbital overdose . the proximal jejunum was dissected out and four adjacent samples were mounted in ussing chambers as described previously ( 22 ) . the tissues were bathed with 4 ml of krebs - ringer bicarbonate ( krb ) solution ( ph 7.4 ) with 10 mmol / l glucose at 37c . in the solution bathing the mucosal side of the tissue , glucose was replaced by mannitol . both solutions electrogenic ion transport was monitored continuously as short - circuit current ( isc ) by using an automated voltage clamp apparatus ( dvc 1000 ; wpi , aston , england ) linked through a maclab 8 to a macintosh computer . krb alone ( vehicle ) or containing relm- ( 0.0011 nmol / l ) was added in the mucosal bath 2 min before a glucose challenge . carbachol ( 100 mmol / l ) was added at the end of each experiment as a control . further , similar tests were performed with relm- incubated overnight at 4c with a rabbit polyclonal antibody raised against relm-. results were expressed as the intensity of the isc ( a / cm ) or as the percentage of the peak isc obtained after glucose challenge ( measured after 3 min ) over basal isc ( measured just before the addition of glucose ) . animals were fasted for 16 h and were killed by pentobarbital overdose . the proximal jejunum was dissected out and rinsed in cold saline solution . jejunal loops ( 4 cm long ) were prepared and 0.5 ml of krb solution without ( control ) or with 1 nmol / l relm- was inserted inside the jejunal lumen . the jejunal loops were incubated for 15 min in oxygenized krb at 37c and conditions were maintained during hexose transport assay . the corresponding jejunal loops were filled with 1 ml of krb solution without ( control ) or with 1 nmol / l relm- and containing 0.02 ci / ml of the isotopic tracer d-[1-c ] glucose ( 49.5 mci / mmol ) and glucose to obtain a final concentration of 10 , 30 , and 100 mmol / l . similarly , we studied the paracellular transport with 30 mmol / l mannitol and the isotopic tracer d-[1-c ] mannitol ( 59 mci / mmol ) at 0.2 ci / ml . all the jejunal loops were incubated in 10 ml of krb solution during the indicated time . the radioactivity was measured in the collected samples of serosal krb solution and used to calculate glucose or mannitol transport as picomoles per milligram of jejunal protein . five independent experiments were performed and significance is expressed as * p < 0.05,**p < 0.01 . three jejunal segments ( 5 cm long ) were tied and filled with 3 ml of krb without ( control ) or with 1 nmol / l relm-. after 3 min of in situ incubation , 3 ml of 60 mmol / l glucose solution were injected in the lumen to obtain a final concentration of 30 mmol / l . after a further 3 min , these loops were removed and opened along the mesenteric border and the mucosa was scraped off on ice with a glass blade . total cell protein extracts and bbms were prepared from the scrapings as previously described ( 23 ) , and enrichment was estimated by determination of alkaline phosphatase activity ( 20-fold increase of activity in bbms ) . solubilized proteins were resolved by electrophoresis on 10% sds - page gels and transferred onto nitrocellulose membranes for immunoblot analysis . the following rabbit antibodies were used at a 1:1,000 dilution : sglt-1 ( ab 1352 ; chemicon international , temecula , ca ) , glut2 , pkc ii ( sc-9117 , sc-210 ; santa cruz biotechnology , tebu - bio , france ) , phospho - ampk- ( thr172 ) , ampk- ( 23a3 ) , and phospho - pkc ( pan ( 2531 , 2603 , 190d10 ; cell signaling technology , ozyme , france ) . the intensity of the specific immunoreactive bands was quantified using national institutes of health image ( scion , frederick , md ) . uptake experiments were performed using rat intestinal everted rings as previously described ( 24 ) . groups of eight intestinal rings were incubated at 37c for 15 min in oxygenized krb buffer in the absence ( control ) and the presence of relm- ( 1 nmol / l ) and cytochalasin b as indicated . then the rings were incubated for 2 min in an uptake solution corresponding to a krb buffer containing 30 mmol / l glucose and 0.1 ci / ml of the isotopic tracer d-[1-c ] glucose . after adding the uptake solution , the rings were washed in ice - cold krb solution , and radioactivity incorporated in the tissue was quantified by liquid scintillation . data were not corrected for extracellular substrate because relm- was found not to affect paracellular diffusion . total protein from homogenized tissue with a dounce homogenizer was determined with bicinchoninic acid reagent from pierce ( thermo scientific , brebires , france ) . recombinant murine relm- was purchased from peprotech ( neuilly - sur - seine , france ) . d-[1-c ] mannitol was from ge healthcare amersham biosciences ( les ulis , france ) , d-[1-c ] glucose was from perkin elmer ( boston , ma ) , and compound c from merck sharp & dohme - chibret ( paris , france ) . all other chemical reagents were purchased from sigma ( st . louis , mo ) . one - way anova with turkey - kramer multiple comparisons post hoc test was performed using graphpad prism version 4.0 for windows ( graphpad software , san diego , ca ) . the area under the curve was significantly ( p < 0.05 ) increased in mice when relm- was used at 1 nmol / l and the two doses used ( 0.1 and 1 nmol / l ) were effective in rats ( fig . 1a and b , insets ) . a similar 15% increase in blood glucose as compared with control was observed in both mice and rats . these data show that luminal administration of exogenous relm- is active in vivo as previously described ( 6 ) . ogtt ( 1 g / kg ) was performed in overnight - fasted mice ( a ) or rats ( b ) with a 15% d - glucose solution without (  , dotted lines ) or with relm- (  , 0.1 nmol / l ;  , 1 nmol / l ) . glucose concentration was determined in blood samples from the tail and is expressed as milligram per deciliter over the time points ( minutes ) . nmol / l (  ) and 1 nmol / l (  ) relm-. the route of glucose entry can involve active na - dependent glut ( sglt-1 ) or diffusive transporter glut2 . we used the ussing chamber to characterize the effect of mucosal relm- on rat intestinal active glucose transport ( sglt-1 ) . rat jejunal mucosa mounted in the chamber was allowed to reach a steady state ( usually 40 min ) . addition of 10 mmol / l glucose in the mucosal bath induced a rise in isc ( maximum after 3 min ) representing an increase in sglt-1 activity . addition of relm- to the mucosal side 2 min before glucose challenge induced a marked and dose - dependent inhibition of glucose - induced isc . 2a , addition of relm- to the mucosal side inhibited glucose transport by jejunal mucosa in a concentration - dependent manner . maximal inhibition was achieved with 0.1 nmol / l relm-. the concentration that produced a half - maximal inhibition ( ic50 ) of glucose transport was 3 further , an overnight incubation of 0.1 nmol / l relm- with an antibody raised against relm- countered the inhibition of glucose - induced isc by relm- as shown in fig . next , we investigated if the observed inhibition by relm- was associated with an altered abundance of sglt-1 in jejunal bbms . a typical immunoblot of sglt-1 protein in bbms after glucose challenge in the presence or the absence of relm- is shown in fig . the mean densitometry of three separate blots shows that glucose increased the amount of sglt-1 protein in bbms by 1.5-fold as compared with control . this increase is reduced by half in the presence of relm-. effect of luminal relm- on glucose - induced isc . rat jejunal mucosa was mounted in ussing chamber and the increase in isc was studied at steady state . electrogenic ( na ) transport was followed as an index of the active glucose transport by cotransporter sglt-1 . relm- was added in the mucosal bath 2 min before challenging the tissues with 10 mmol / l glucose . b : the effect of 0.1 nmol / l relm- after an overnight incubation with an antibody against relm-. c : representative sglt-1 immunoblot of solubilized bbms . bbms were prepared from rat jejunum loops incubated in situ with and without luminal relm- during 6 min . densitometric analysis of immunoreactive bands was performed using national institutes of health image analysis program . the densitometry represents the amount of sglt-1 relative to -actin and is representative of at least three separate experiments . each point of the isc study represents the means  se of four to eight noncumulative values from five separate experiments . significant differences from control , * p < 0.05 , * * p < 0.01 . we isolated rat jejunum and performed in vitro studies of transmural glucose transport to directly evaluate the effect of relm- on intestinal transport . as shown in fig . 3a , luminal glucose in jejunal loops significantly increased net mucosal to serosal glucose flux . a dose - dependent effect on glucose transport was observed with increasing glucose concentrations 10 , 30 , and 100 mmol / l . further , relm- , at 1 nmol / l , significantly enhanced the jejunal transport of 10 , 30 , and 100 mmol / l glucose but not that of 30 mmol / l mannitol . thus , increased jejunal glucose transport induced by relm- is unlikely to have been caused by changes in paracellular permeability . this is in line with histological studies of the jejunum tissues used in these experiments that did not show any visible mucosal deterioration ( data not shown ) . further , experiments were performed to identify if the effect of relm- implicated potential downstream kinases , pkc , and ampk , which are known as key effectors of intestinal glucose transport ( 6,20,23,25 ) . the ampk inhibitor , compound c , and the pkc inhibitor , chelerythrine , inhibited the 30 mmol / l glucose - induced jejunal glucose transport as shown , respectively , in fig . 3b and c. the insets represent the area under the curve of the 30-min glucose transport kinetics . relm- significantly increased glucose transport by approximately twofold and this effect was blunted by compound c and chelerythrine as shown , respectively , in fig . 3b and c. these results indicate a likely involvement of pkc and ampk in the luminal effect of relm- on glucose uptake . transmural transport of glucose or mannitol was performed in jejunal sacs from adult wistar rats . / l relm- (  ) or vehicle (  ) in oxygenized krb buffer with glucose at 10 , 30 , and 100 mmol / l and the isotopic tracer d-[1-c ] glucose . the radioactivity measured in the collected samples was used to calculate glucose transport as picomoles per milligram of jejunal protein per minute . the kinetics of glucose ( 30 mmol / l ) transmural transport is shown in the absence (  , dotted line ) and in the presence (  ) of 1 nmol / l relm- in b and c. b : the ampk inhibitor ( compound c or cc ) was incubated without (  ) or with (  ) relm-. c : similarly , the pkc inhibitor ( chelerythrine chloride or ccl ) is incubated without (  ) and with (  ) relm-. the insets show the corresponding area under the curve . the data are representative of the means  se of at least four individual experiments , * p < 0.05 ; * * p < 0.01 vs. control . the above results prompted us to assess by in situ experiments the cellular effects of relm- on ampk and pkc . jejunal segments were injected with a krb solution alone or containing 30 mmol / l glucose in the absence and the presence of 1 nmol / l relm-. the mucosal scrapings of jejunum were examined by western blot analysis . the results indicated that glucose as well as relm- stimulated ampk phosphorylation over control values as shown in fig . 4a . the corresponding mean densitometric analysis indicates that relm- induced a threefold increase in ampk phosphorylation . a further increase in the phosphorylation of ampk occurred when glucose and relm- were added together . similarly , relm- induced a threefold increase in pkc phosphorylation in line with our data ( 6 ) in the mouse colon ( data not shown ) . this prompted us to study the translocation of cytosolic pkc ii of enterocytes to bbms , a mechanism that is associated with intestinal glucose transport ( 20,23 ) . we performed western blot analysis to determine the expression of pkc ii in bbms obtained from intestinal segments that were incubated with glucose ( 30 mmol / l ) or relm- ( 1 nmol / l ) as described above . 4b , relm- or glucose induced , respectively , 1.5- or 2-fold increase of pkc ii at the bbms as compared with control values . a further increase ( 2.7-fold compared with the control ) of pkc ii at the bbms these results suggest that relm- stimulates the phosphorylation of ampk and pkc as well as an increased shift of pkc ii to the bbms of rat jejunal tissue . the loops were filled with krb buffer with or without 1 nmol / l relm-. after 3 min , glucose ( 30 mmol / l ) was added in this mucosal bath . after a further 3 min , loops were excised and kept on ice before scraping off the mucosa . total protein extraction and bbms preparation were performed immediately as described in research design and methods . representative immunoblots for phospho - ampk ( a ) in mucosal extracts and pkc ii ( b ) in bbms are shown . we explored whether relm- could enhance the glucose - induced glut2 translocation to the apical membrane of enterocytes . to this end , we measured glucose uptake in rat everted jejunal rings in the presence of relm- and without or with cytochalasin b , a competitive inhibitor of glut2 ( 26 ) . 5a , cytochalasin b inhibited glucose uptake by 50% in agreement with the implication of glut2 in glucose uptake ( 27,28 ) . relm- enhanced glucose uptake by twofold and this effect was also strongly inhibited by cytochalasin b. further , we performed western blot analysis to determine the expression of glut2 in bbms obtained from intestinal segments that had been incubated with relm- under the same conditions as above . we observed that glucose or relm- alone induced , respectively , a four- or threefold increase in the amount of glut2 found in the brush - border fraction , respectively ( fig . 5b ) . when glucose and relm- were added together , a further increase in the amount of glut2 was observed ( fivefold as compared with control ) . this suggests that relm- enhanced glucose uptake is a result of an increased insertion of glut2 into the bbms . a : rat jejunal everted rings were incubated in oxygenized krb buffer without ( control ) or with 1 the rings were incubated for 2 min in a krb buffer containing 30 mmol / l glucose and 0.1 ci / ml of the isotopic tracer d-[1-c ] glucose ) . the radioactivity incorporated in the tissue b : rat jejunal loops were treated 6 min with a mucosal bath containing 30 mmol / l glucose with or without 1 nmol / l relm- and bbms were prepared as described in research design and methods . a representative western blot analysis of the corresponding protein lysates with glut2 antibody is shown . densitometric analysis of immunoblots indicates an increase of glut2 in the presence of glucose or relm-. the data are expressed relative to -actin and is representative of at least three separate experiments . significantly different from control , * p < 0.05 . the area under the curve was significantly ( p < 0.05 ) increased in mice when relm- was used at 1 nmol / l and the two doses used ( 0.1 and 1 nmol / l ) were effective in rats ( fig . 1a and b , insets ) . a similar 15% increase in blood glucose as compared with control was observed in both mice and rats . these data show that luminal administration of exogenous relm- is active in vivo as previously described ( 6 ) . ogtt ( 1 g / kg ) was performed in overnight - fasted mice ( a ) or rats ( b ) with a 15% d - glucose solution without (  , dotted lines ) or with relm- (  , 0.1 nmol / l ;  , 1 nmol / l ) . glucose concentration was determined in blood samples from the tail and is expressed as milligram per deciliter over the time points ( minutes ) . the route of glucose entry can involve active na - dependent glut ( sglt-1 ) or diffusive transporter glut2 . we used the ussing chamber to characterize the effect of mucosal relm- on rat intestinal active glucose transport ( sglt-1 ) . rat jejunal mucosa mounted in the chamber was allowed to reach a steady state ( usually 40 min ) . addition of 10 mmol / l glucose in the mucosal bath induced a rise in isc ( maximum after 3 min ) representing an increase in sglt-1 activity . addition of relm- to the mucosal side 2 min before glucose challenge induced a marked and dose - dependent inhibition of glucose - induced isc . 2a , addition of relm- to the mucosal side inhibited glucose transport by jejunal mucosa in a concentration - dependent manner . maximal inhibition was achieved with 0.1 nmol / l relm-. the concentration that produced a half - maximal inhibition ( ic50 ) of glucose transport was 3 further , an overnight incubation of 0.1 nmol / l relm- with an antibody raised against relm- countered the inhibition of glucose - induced isc by relm- as shown in fig . next , we investigated if the observed inhibition by relm- was associated with an altered abundance of sglt-1 in jejunal bbms . a typical immunoblot of sglt-1 protein in bbms after glucose challenge in the presence or the absence of relm- is shown in fig . the mean densitometry of three separate blots shows that glucose increased the amount of sglt-1 protein in bbms by 1.5-fold as compared with control . this increase is reduced by half in the presence of relm-. effect of luminal relm- on glucose - induced isc . rat jejunal mucosa was mounted in ussing chamber and the increase in isc was studied at steady state . electrogenic ( na ) transport was followed as an index of the active glucose transport by cotransporter sglt-1 . relm- was added in the mucosal bath 2 min before challenging the tissues with 10 mmol / l glucose . values for isc were standardized to control values and expressed as percentage of controls . a : dose - dependent inhibition . b : the effect of 0.1 nmol / l relm- after an overnight incubation with an antibody against relm-. c : representative sglt-1 immunoblot of solubilized bbms . bbms were prepared from rat jejunum loops incubated in situ with and without luminal relm- during 6 min . densitometric analysis of immunoreactive bands was performed using national institutes of health image analysis program . the densitometry represents the amount of sglt-1 relative to -actin and is representative of at least three separate experiments . each point of the isc study represents the means  se of four to eight noncumulative values from five separate experiments . significant differences from control , * p < 0.05 , * * p < 0.01 . we isolated rat jejunum and performed in vitro studies of transmural glucose transport to directly evaluate the effect of relm- on intestinal transport . as shown in fig . 3a , luminal glucose in jejunal loops significantly increased net mucosal to serosal glucose flux . a dose - dependent effect on glucose transport was observed with increasing glucose concentrations 10 , 30 , and 100 mmol / l . further , relm- , at 1 nmol / l , significantly enhanced the jejunal transport of 10 , 30 , and 100 mmol / l glucose but not that of 30 mmol / l mannitol . thus , increased jejunal glucose transport induced by relm- is unlikely to have been caused by changes in paracellular permeability . this is in line with histological studies of the jejunum tissues used in these experiments that did not show any visible mucosal deterioration ( data not shown ) . further , experiments were performed to identify if the effect of relm- implicated potential downstream kinases , pkc , and ampk , which are known as key effectors of intestinal glucose transport ( 6,20,23,25 ) . the ampk inhibitor , compound c , and the pkc inhibitor , chelerythrine , inhibited the 30 mmol / l glucose - induced jejunal glucose transport as shown , respectively , in fig . 3b and c. the insets represent the area under the curve of the 30-min glucose transport kinetics . relm- significantly increased glucose transport by approximately twofold and this effect was blunted by compound c and chelerythrine as shown , respectively , in fig . 3b and c. these results indicate a likely involvement of pkc and ampk in the luminal effect of relm- on glucose uptake . transmural transport of glucose or mannitol was performed in jejunal sacs from adult wistar rats . / l relm- (  ) or vehicle (  ) in oxygenized krb buffer with glucose at 10 , 30 , and 100 mmol / l and the isotopic tracer d-[1-c ] glucose . the radioactivity measured in the collected samples was used to calculate glucose transport as picomoles per milligram of jejunal protein per minute . the kinetics of glucose ( 30 mmol / l ) transmural transport is shown in the absence (  , dotted line ) and in the presence (  ) of 1 nmol / l relm- in b and c. b : the ampk inhibitor ( compound c or cc ) was incubated without (  ) or with (  ) relm-. c : similarly , the pkc inhibitor ( chelerythrine chloride or ccl ) is incubated without (  ) and with (  ) relm-. the insets show the corresponding area under the curve . the data are representative of the means  se of at least four individual experiments , * p < 0.05 ; * * p < 0.01 vs. control . the above results prompted us to assess by in situ experiments the cellular effects of relm- on ampk and pkc . jejunal segments were injected with a krb solution alone or containing 30 mmol / l glucose in the absence and the presence of 1 nmol / l relm-. the mucosal scrapings of jejunum were examined by western blot analysis . the results indicated that glucose as well as relm- stimulated ampk phosphorylation over control values as shown in fig . 4a . the corresponding mean densitometric analysis indicates that relm- induced a threefold increase in ampk phosphorylation . a further increase in the phosphorylation of ampk occurred when glucose and relm- were added together . similarly , relm- induced a threefold increase in pkc phosphorylation in line with our data ( 6 ) in the mouse colon ( data not shown ) . this prompted us to study the translocation of cytosolic pkc ii of enterocytes to bbms , a mechanism that is associated with intestinal glucose transport ( 20,23 ) . we performed western blot analysis to determine the expression of pkc ii in bbms obtained from intestinal segments that were incubated with glucose ( 30 mmol / l ) or relm- ( 1 nmol / l ) as described above . 4b , relm- or glucose induced , respectively , 1.5- or 2-fold increase of pkc ii at the bbms as compared with control values . a further increase ( 2.7-fold compared with the control ) of pkc ii at the bbms these results suggest that relm- stimulates the phosphorylation of ampk and pkc as well as an increased shift of pkc ii to the bbms of rat jejunal tissue . the loops were filled with krb buffer with or without 1 nmol / l relm-. after 3 min , glucose ( 30 mmol / l ) was added in this mucosal bath . after a further 3 min total protein extraction and bbms preparation were performed immediately as described in research design and methods . representative immunoblots for phospho - ampk ( a ) in mucosal extracts and pkc ii ( b ) in bbms are shown . the data are representative of three separate experiments . significantly different from control , * p < 0.05 . we explored whether relm- could enhance the glucose - induced glut2 translocation to the apical membrane of enterocytes . to this end , we measured glucose uptake in rat everted jejunal rings in the presence of relm- and without or with cytochalasin b , a competitive inhibitor of glut2 ( 26 ) . 5a , cytochalasin b inhibited glucose uptake by 50% in agreement with the implication of glut2 in glucose uptake ( 27,28 ) . relm- enhanced glucose uptake by twofold and this effect was also strongly inhibited by cytochalasin b. further , we performed western blot analysis to determine the expression of glut2 in bbms obtained from intestinal segments that had been incubated with relm- under the same conditions as above . we observed that glucose or relm- alone induced , respectively , a four- or threefold increase in the amount of glut2 found in the brush - border fraction , respectively ( fig . 5b ) . when glucose and relm- were added together , a further increase in the amount of glut2 was observed ( fivefold as compared with control ) . this suggests that relm- enhanced glucose uptake is a result of an increased insertion of glut2 into the bbms . a : rat jejunal everted rings were incubated in oxygenized krb buffer without ( control ) or with 1 nmol / l relm-. the presence of cytochalasin b is indicated . the rings were incubated for 2 min in a krb buffer containing 30 mmol / l glucose and 0.1 ci / ml of the isotopic tracer d-[1-c ] glucose ) . the radioactivity incorporated in the tissue b : rat jejunal loops were treated 6 min with a mucosal bath containing 30 mmol / l glucose with or without 1 nmol / l relm- and bbms were prepared as described in research design and methods . a representative western blot analysis of the corresponding protein lysates with glut2 antibody is shown . densitometric analysis of immunoblots indicates an increase of glut2 in the presence of glucose or relm-. the data are expressed relative to -actin and is representative of at least three separate experiments . significantly different from control , * p < 0.05 . in the present study , we show that relm- , a resistin - like molecule , can directly increase jejunal absorption of glucose in the rat . several lines of evidence suggest that transepithelial transport of glucose in the small intestine can be mediated by an active absorption through na / glucose cotransporter ( sglt-1 ) as well as by a diffusive component glut2 at the apical membrane ( 27 ) . we found that relm- inhibited the activity of sglt-1 , whereas enhancing the presence of glut2 at the bbm of enterocytes . moreover , the increased jejunal glucose transport induced by relm- was inhibited by cytochalasin b in agreement with a functional role of glut2 . the underlying molecular mechanism involves the activation of pkc ii and ampk kinases as described for such reciprocal regulation of glucose transporters ( 20 ) . the acute treatment of rat jejunum with relm- had no significant effect on passive mannitol movement suggesting that relm- increases glucose transport by the use of transporters . we found that mucosal relm- inhibited the activity and the translocation of cytosolic sglt-1 to cell membranes . this effect was blunted by an antibody raised against relm-. other peptides such as leptin , angiotensin ii , and cck-8 have also been shown to inhibit sglt-1 activity ( 14,16,28 ) . we show that in contrast to ckk-8 , which seems to have no effect on glut2 ( 28 ) , relm- can induce glut2 translocation . indeed , in response to relm- the amount of glut2 was increased in the bbms . similarly , a rapid insertion of glut2 to apical membrane in response to another gut - peptide , glp-2 , has been reported ( 29,30 ) . taken together , these findings suggest the involvement of glut2 in relm- stimulated glucose uptake . the mechanisms responsible for relm- effect may involve the activation of pkc ( 6 ) and ampk ( 25 ) that have been shown to regulate jejunal glucose transporters ( 20 ) . this is sustained by the report that luminal epidermal growth factor increases jejunal glucose transport in rabbit through pkc activity ( 31 ) . we found that chelerythrine , an inhibitor of pkc , blocked relm- stimulation of glucose uptake . the effect of relm- is accompanied by an increase in the amount of pkc ii at the bbms . the activation of pkc has been shown to inhibit sglt-1 mediated transport of hexoses ( 14,32 ) . in oocytes expressing rat and rabbit sglt-1 , the activation of pkc decreases the maximum rate of transport for both isoforms . this change is accompanied by proportional change in the number of sglt-1 molecules at the plasma membrane , indicating that pkc regulates endocytosis of the vesicles containing the transporter ( 33 ) . further , sglt-1 contains a consensus site of pkc phosphorylation and thus pkc phosphorylation of the transporter could control its activity ( 34 ) . these data are in line with our results showing that relm- inhibits sglt-1 activity and its translocation to bbms in rat small intestine . the activation of ampk has been shown to downregulate sglt-1dependent glucose transport but also to enhance glut2 translocation to the apical membrane of the jejunum ( 20 ) . this effect of ampk leads to an increased glucose uptake in jejunum ( 20 ) as well as in muscle ( 35 ) . interestingly , several hormones have been shown to regulate ampk in a strictly tissue - specific manner ( 36 ) . we demonstrate that the compound c blocked relm- stimulation of glucose transport in rat intestine suggesting the involvement of ampk . humans and rodents express two isoforms of the catalytic subunit ( 1 , 2 ) that form the heterotrimeric complex ampk (  ,  , and  ) known to serve as a regulator of energy balance ( 37 ) . we showed that relm- could increase the phosphorylation of the conserved threonine residue ( thr-172 ) of 1 in the jejunal mucosa that is crucial for the ampk activity . as evoked above , activated pkc decreases the number of sglt-1 transporters but can also activate the translocation of glut2 to bbms as described elsewhere ( 23 ) . thus , luminal relm- can directly enhance glucose transport by mustering glut2 at bbms through pkc and ampk activation . this inverse regulation of sglt-1 and glut2 by luminal relm- may be important when enterocytes require energy as shown in stress - induced pathology ( 38 ) . the energy sensor molecule ampk as well as pkc can increase the glut2 energy - independent pathway to override that of sglt-1 that requires energy ( 20,32 ) . in agreement with this concept , the sglt-1-mediated absorption of nutrients such as galactose and glucose is decreased during either systemic ( 39 ) or intestinal inflammation ( 40 ) . further , to meet the increased metabolic demand of inflamed tissue , it has been shown that proinflammatory cytokines ( interleukin [ il]-8 , il-6 , and il-1 ) can increase jejunal absorption of glucose without changes in bbm sglt-1 content ( 41 ) . an increased expression of relm- has been described in the intestine during jejunal inflammation ( 19,42,43 ) . the expression of relm- is increased by several proinflammatory cytokines and by lipopolysaccharide ( 9,21 ) and they may act together to modulate intestinal glucose absorption . thus , the enhanced glucose absorption by relm- in response to inflammatory stimulus may contribute to the associated energy demand . obese and insulin - resistant rodent models that are characterized by a low - grade inflammation are associated with an increased expression of the gut - derived relm- ( 8) . expression of relm- has been shown in rat ( 18 ) and mice ( 19 ) proximal intestine and the peptide may act locally in a paracrine manner or as a circulating hormone linking the gut to the liver . when given by gavage , it is possible that relm- could be partly absorbed by the small intestine to reach the blood as demonstrated for leptin ( 44 ) . in favor of a hormonal effect , the infusion of intestinal relm- in mice has been shown to promote a marked increase in the rate of hepatic glucose production ( 7 ) . we observed that acute relm- administration in the intestinal lumen of rodents resulted in an increased glycemia in ogtt . this could result from the observed increased intestinal glucose absorption and an acute hepatic insulin resistance ( 7 ) . this is in line with a local intestinal and distant action of this gut peptide . as to whether relm- may also counteract the described insulin inhibition of intestinal sugar absorption remains to be established ( 45 ) . there may be different relm- thresholds in insulin target tissues as described for resistin ( 46 ) . a better understanding of relm- action on different tissues could emerge if putative relm- receptors were identified . in this context , the effect of relm- may even involve rapid neuronal activation because relm family members relm- ( 47 ) and relm- ( 4 ) can bind to neurons . other than short - regulation , relm- can have a chronic effect as shown in diet - induced metabolic disorders ( 25 ) . transgenic mice over - expressing circulating relm- exhibited significant hyperglycemia , hyperlipedemia when fed on high - fat diet . in conclusion further studies may reveal if this process in conjunction with an adverse nutritional and inflammatory status , can participate in the onset of diabetes .
objectivean increased expression of relm- ( resistin - like molecule- ) , a gut - derived hormone , is observed in animal models of insulin resistance / obesity and intestinal inflammation . 
 intestinal sugar absorption is modulated by dietary environment and hormones / cytokines . 
 the aim of this study was to investigate the effect of relm- on intestinal glucose absorption.research design and methodsoral glucose tolerance test was performed in mice and rats in the presence and the absence of relm-. the relm- action on glucose transport in rat jejunal sacs , everted rings , and mucosal strips was explored as well as downstream kinases modulating sglt-1 and glut2 glucose transporters.resultsoral glucose tolerance test carried out in rodents showed that oral administration of relm- increased glycemia . 
 studies in rat jejunal tissue indicated that mucosal relm- promoted absorption of glucose from the gut lumen . 
 relm- had no effect on paracellular mannitol transport , suggesting a transporter - mediated transcellular mechanism . in studies with jejunal mucosa mounted in ussing chamber , 
 luminal relm- inhibited sglt-1 activity in line with a diminished sglt-1 abundance in brush border membranes ( bbms ) . 
 further , the potentiating effect of relm- on jejunal glucose uptake was associated with an increased abundance of glut2 at bbms . 
 the effects of relm- were associated with an increased amount of protein kinase c ii in bbms and an increased phosphorylation of amp - activated protein kinase ( ampk).conclusionsthe regulation of sglt-1 and glut2 by relm- expands the role of gut hormones in short - term ampk / protein kinase c mediated control of energy balance .