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Volume 314, number 3,4G6-470 FEBS 11919 December 1992 6 1992 Federation of European Biochemical Societies 00145793/92/%5.00

Role of liver-type glucose transporter (GLUT2) in transport across the basolateral membrane in rat jejunum

Ken-ichi Miyamoto”, Toshimitsu Takagib, Takeru Fujiib, Tomoko Matsubarab, Kyoko Ha&, Yutaka Taketani”, Tatsuzo Oka”, Hisanori Minami” and Yukihiro Nakabou”

“Depurtntettt of Ntrtrition, Scllool of Medicirta, Unirwsity of Tokokushirttu,Kwmtoto-Clto3, Tokusltirna 770, Jupm and “Laboratory of Cell Biology, Teikoktr Sei_wku Co., Sanbortnturw, Kagawa 567, Jupun

Received 5 November 1992

To obtain information on the regulution of glucose transport across the bosolater;tl membrane (BLM) of intestinal upithelinl cells, WCmeasured the number of~H]cytochalasin I3 binding sites and the level of liver-type glucose tmnsportcr (GLUT2) protein in the BLM in the jejunum of rats (i) with diabetes {ii) given a high-carbohydrate diet or (iii) with cxperimcntal hyperglycemia (12 h infusion of a high-glucose solution). A glucose uptake and the number orb-glucose inhibitublu [3H]cytochnlasin B binding sites in DLM vesicles were significantly increased in all three conditions. Western blot analysis showed that the amount of GLUT2 protein in BLM vesicles was increased in rats with dinbctcs and those given a high-carbohydrate diet, but not in ~hosc with experimental hyperglycemia. These results suggest that there is a mechanism for rapid regulation of glucose transport in the BLM thnt does not depend on change in the amount of GLUT2.

D-Glucose transport; Basolateral membrane; GLUT’; Diubetcs; High-glucose infusion: Rat intestine

1. INTRODUCTION the jejunum of acute diabetic rats the level of GLUT2 mRNA changed in parallel with the D-glucose transport Glucose is transported across the small intestine by activity, whereas the level of SGLTl, which is located a two step process; the first step is entry of glucose into in the BBM, did not change [8]. These results suggest the cpithelium across the brush border membrane that the induction of glucose transport occurs in the (BBM), and the second is its efflux from cells across the BLM of intestinal epithelial cells, not in the BBM. basolateral membrane (BLM) into the blood [l]. The Recently facilitative glucose transport across the molecular mechanism of glucose transport across the ELM was found to be induced by experimental hyper- BBM via the Na”-dependent glucose co-transporter glycemia (infusion of high-glucose solution) or a high- (SGLTI) has been well characterized by Wright. et al. glucose diet [lo-131. However, nothing is yet known [2], but little is known about glucose transport across about the regulation of the GLUT2 protein in the BLM the BLM. of intestinal epithelial cells. Therefore, in the present Recently, cDNAs for 5 facilitative glucose trans- study, we investigated the levels of GLUT2 protein in porters (GLUTI-5) have been isolated [3]. GLUT2 has rats with diabetes, those given high-carbohydrate diet been shown to be present in the liver, small intestine, and those with experimental hyperglycemia (12 11infu- kidney and the islets of Langerhans [4,5]. Thorens et al. sion of high-glucose solution). Our results clearly indi- suggested that, as GLUT2 has a relative high K,, for cate differential mechanisms for the regulation of baso- glucose, it may be required for glucose sensing by/3-cells lateral glucose transport in rat small intestine. in islets. This glucose transporter is also present in the BLM of intestinal epithelial cells, and is responsible for 2. MATERIALS AND METHODS the release of glucose across the BLM [4-63. Gould et al. found that the K, value for glucose of GLUT2 ex- 2.1. E.xperilrtertraldidxres pressed in Xerlopus oocytes is consistent with that re- Male Sprague-Duwley rats (Japan SLC, Shizuoka, Japan), weigh- ing lGO-180 g, were used. The animals were housed in stainless-steel ported by others for BLM preparations from rat smell cuges and given a carbohydrate-free diet (24% protein. 55% fat, 9% intestine [7]. cellulose, 4% mineral mixture and 1% vitamins), for 2 weeks before Previously we showed that the SGLTI, GLUT2 and experiments, because the level of dietary sugar significantly affects the GLUTS genes are expressed in rat jejunum, and that in level ofliver-type glucose transporter (GLUT2) mRNAin ratjejunum [9], Most diet components were purchased from Wnko Chemical In- dustries (Osaka, Japan). The animals hnd free access to water and were Corrcspandeme address: K.-i. Miyamoto, Department of Nutrition, mitinlilimxi m&r it i2-B ii&~t:ciiWk CjdC at 24°C. Anirna!s ivcre made School of Medicine, University of Tokushima, KuramotoCho3. To- diabetic by intraperitoneal injection of 65 mg/kg streptozocin (STZ) kushima 770, Japan. freshly dissolved in 100 mM citrate buffer (pi-i 4.5). Control rats

466 Volume 314, number 3 FEBS LETTERS December 1992 received a similar injection of citrate buffer only. Animals were given 2.8. hfurrrials a cnrbohydrate-free diet and were killed 10 days after STZ injection, t@H]glucose, [‘H]cytochalnsinB, [“‘Clurca and [‘351]protcinA were as described previously [S]. purchased from Amersham. Cytochalnsin B. cytochalasin E, [email protected] case and D-sorbitol were from Sigma Chemical Co.

Intravenous infusion experiments were carried out as described by Mocnz and Cheeseman [lO,l2]. Rats (160-180 g) maintained on a 3. RESULTS carbohydrate-l&z diet for 2 weeks were anesthetized with pcntobarbi- tal and a t&c was inserted into the jugular vein and pushed into the Membranes isolated from rat jejunum were analyzed heart. The other end of the tube was brought out through an incision for a marker enzyme of the BLM. Their specific activity in the back. On the day of use, animals were placed in a small cage of Na*,K*-ATPase was about 16-fold that of the crude and connected to an infusion tube, and a solution of 50% glucose wns infused at 6.0 ml/h. After infusion for 12 h, the animals were killed and extract (crude homogenate, 9.4 1: 1.8 humol FQ/hlmg the jejunum WBSused for preparation of the BLM. protein; BLM, 150.9 $ 19.6 pmol POJhlmg protein). GLUT2 antiserum reacted with a band of 61 kDa pro- 2.3. if&h-cutbohydru~c d&r Animals (I SO-170 g) were maintained for 2 weeks on R carbohy- drate-f’rc- diet and then given a 55% glucose diet (55% gluco5c in place of 55% fat) for 5 days before experiments 1141. A 2.4. Preporatio,z of BLMJrunl iutcrtirtalcpirldiul cdh Rats wcrc anesthetized by intraperitoneal injection of pntobarbi- tal. the abdomen was opened and the porlal vein was cut. Then warm (37OC) I50 mM phosphate-buffered 0.9% saline. conruining 30 mM EDTA; pH 7.4, wns injected into the left ventricle, and the small intestine 1x15 removed and inverted over a glass rod. Cells :vcre re- leased by shaking, suspended in cold sucrose burer, and collected by low-sped ccntrifugation [IS]. BLM vesicles were prepared by diffcr- cntial centrifugalion and final purification on a Percoll gradient, as describrti by Scalera et al. [IO]. The final BLM pellet showed a 16.fold increase in spccitic activity of Na’,K+-ATPase over that of tbc crude preparation. There was no significant difference in tbc Na*,K+-ATP- nse activities in BLM preparations from rnts with hyperglycemia and diabetes and those fed a high-carbohydmte diet.

2.5. Glucose uprake cm1 cytociralnsh 6 billing assoy in the BLM Olucosc transport and binding orcytochalasin B to the BLM from rat jejunum were measured by the methods of Maenr and Cheeseman [Ill. Membranes were incubated for I5 min with the ligand in the presence of 2 ,uM cytochalasin E to reduc% non-specific binding. The membranes were pelleted by ccntrifuzation for 30 min and the supr- natant was removed before dissolving the pellet in sodium hydroxide. For assay of the glucose-inhroiublc cytochalasin I3 binding, incuba- tions were performed with cit!ler 1.25 M o-glucose or 1.25 M sorbital in the medium. The amount ofcytochalasin B bound to BLM vesicles was calculated after correction for unbound [‘M]cytochalasin nssoci- ated with the pellet using [“‘Cluren as a non-binding aqueous space marker. Binding constants were calculated using a computer program. In experiments on glucose uptake, 10 ,ul of vesicles (10 mg protein/ ml) was incubated with 20~1 of uptake medium for 3 sand the reaction was stopped with I.455 ~1 of ice-cold buffer containing 0.2 mM phlo- retin. The mixture was promptly Altered through a 0.45 pm pore size filter, which was then washed twice with 5 ml oT stop solution [11,12].

2.7. wL?slrrll l!lIolrirl.g GLUT2 untibody was kindly provided by B. Thorens [17]. This antibody was further purified as the IgG fraction by DE-52 cellulose Fig. 1. Western blot analyses of GLUT2 and sucrase in the brush- column chromatography. Membranes were rcsuspcnded in Laemmli border (BBM) and basoluterai membrane (BLM) of rat jcjunum. (A) sample buffer containing 5% SDS and separated on a 10% SDS- Sucrasc, (B) GLUT2. Intestinal BBM were prepared by the method polyacrylamidc gel 1181.Elcctrotrnnsfer to nitrorrllulose filters was of Macnr and Cheeseman [IO].The membrane from r;ltsmall intestine performed for 6 h. The Rlters were washed at room temperature for was purified in the presence of the protease inhibitors, aprotinin, 10 min in TBST (20 mM Tris. III-I 7.4. 150 mhl N&I. and 0.02% phcnylmethylsulfonyl fluoride, N-p-tosyllysine chloromcthyl ketone, Twccn 2ii). The fiitcrs were blocked with 0.5% fat-free dry milk, and f?-tosyl-L-phsnyhlaninechloromethyl ketoneand Futhan [5.26]. After incubated with anti-pcptidc antibodies (IgG fraction) against rat scpnration by SDS-PAGE, the proteins were tmnsferred el=tro- GLUTZ. They were thep wnshcd with TBST at room temperature, and phoretically to a nitrocellulosc filter by the method of Towbin et al. incubated with iii’ cpm oip lz5f-:abc:cd protcir, =JmI TBST, .C:nrll ..,,..y 122. The filter was incubated with anti-GLUT2 and anti-sucrase anti- they were washed for 60 min in TEST and exposed to Kodak XAR body for 2 h, and then incubated with proxidas+coupled gOat anti- Alms at -7OOC. Quanlificadon was performed by scanning densitom- rabbit IgG [5,28]. The bound antibody was located by staining with etry. 4-chloro naphthoi. Volume 314, number 3 FEBS LETTERS December 1992

imental hyperglycemia (B), and those given a high-car- bohydrate diet (C). The number of binding sites were clearly increased in all three groups, and these increases A paralleled changes in the I’,,, for transport [lo-131. The I’mun values for n=glucose-inhibitable cytochalasin B binding in the groups with diabetes and hyperglycemia and those given a high-carbohydrate diet were 30.2 $ 5.1 pmol/mg protein (n = 6), 29.5 f 4.6 pmol/mg protein (n = 6) and 36.5 f 6.6 pmohmg protein (11= 7), the control values being 15.7 f 3.2 pmollmg protein, 14.3 2 4.8 pmollmg protein and 19.1 f 3.2 pmol/mg protein, respectively. The binding affinities of these groups were I 10 20 30 40 the same: diabetes, 124.7 & 12.9 @I (control, 119.8 z!~ ttODNDC~lc6/ au protein ) 2 1.8 PM); hyperglycemia, 119.5 1: 21.8 ,uM (control, 0 110.6 f 11.7); high-carbohydrate diet, 121.3 f 20.8 ,uM (control, 120.6 z!z17.5 PM). The uptakes of 50 mM glucose in these animals are shown in Fig. 3. ~-Glucose transport across the BLM vesicle was significantly in- creased in all test groups relative to that of the control. This was largely a consequence of an increased V,,,, not a reduced KI [IO-i 31. An increase in [‘H]cytochalasin i3 binding was consistent with the induction of transport observed in ELM preparations. Previously we found that the level of GLUT2 mRNA changed in parallel with D-galactose transport activity in the jejunum of diabetic rats [S]. As shown in Fig. 4A, the amount of GLUT2 protein increased about 3.0-fold in the BLM vesicles obtained from diabetic rats 10 days after STZ injection. The blood glucose level increased to a maximum 3 days after STZ injection [S]. As shown in Fig. 4C, a high-glucose diet increased in the intestinal level of GLUT2 protein about 2.6-fold after 5 days. The observation that GLUT2 protein increased in rats fed a high-carbohydrate diet is consistent with the results

11491Dhm~lc~/ m protein 1 Fig. 2. Scatchard plots of D-glucose-inhibitable [“I-l]cytochnlasin E3 binding to purified jejunnl BLM prepared from rats with hypurglyce- mia, and dinbctcs and those fed a high-carbohydrate. D-Glucose-in- hibitnble [“H]cytochalasin B binding was determined as described in section 2. The data are taken from two typical expcrimcnts in which membranes from 8 rats were pooled, and points represent meana for triplicate determinations. (A) Diabetes, (B) hyperglycemia, (C) high- carbohydrate. (0) Control group. (a) !cst group. --Dfsbetc& hcrglyccmia Bigh-eark&dratc tcin in the BLM from ral intestinal epithelial cells, but Fig. 3. Glucose uptake across BLM vesicles prcpdred from rats wilh not with any protein in the brush-border or microsomal hyperglycemiu and diirbetes and those fed a high-carbohydrate diet. membranes (Fig. 1). This observation that the molecu- The uptake of glucose into vesicles was initiated by mining 10 yl of vesicles with 20 ~1 of uptake mdia containing b-glucose I- a-[‘H]glu- lar weight of intestinal GLUT2 is 61 kDa is consistent case [I 1,121. Uptuke was initiated by vortcxing the tube and. at the with a report of Thorens et al. [17]. conclusion of the uptake period, 1,455 ~1 of ice-cold stop solution was Western blotting for sucrase as a marker enzyme of rapidly injected into the mixture. Glucose held by the vesicles was br>. ..I..mh_ho&r " .rrmrnt)ram=c..w... .1.._" $_Rr?rJ) ._*w...__rmwal~rl +l~t dhe determined as dcscribcd in section 2. A correction for passive pcrme- preparations were not contaminated with BBM. ittic:: was mndc by subtracting the uptake of the same concentrations of L-glucose. The bars indicate the SD. from the mean (II = 6). (A) Fig. 2 shows the data for [‘H]cytochalasin B binding Diabetes, (II) hyperglycemia, (C) high-carbohydrate. (0) Control to BLM prepared from rats with diabetes (A) and exper- group, (m) lc51 group. Volume 314, number 3 FEBS LETTERS December 1992

A Dirbcter rapid type [I 3,211. In the present study, we also ob- ccc123456 served a rapid response of intestinal glucose transport in rats with experimental hyperglycemia (12 h-infusion of high-glucose solution). In these rats, unlike in those with diabetes and those on a high carbohydrate diet, -- -6l increase in the number of [3M]cytochalasin B binding sites in the BLM was not associated by increase in the amount of intestinal GLUT2 protein. This difference Bmtrglpcmim B was not due to differences in the extents of contamina- CC 1234 tion of the BLM preparations, because there were no significant differences in the specific activities of marker enzymes for the BLM in preparations from the three groups (data not shown). Other glucose transporter isoforms might be associ- tligh-c8rmydrnto ated with the glucose transport activity in the BLM in C c c c 123456 rats with hyperglycemia, because the cytochalasin B binding assay measured the total number of trans- porters irrespective of their isoform [23]. Cheeseman et al. observed rapid regulation of the glucose transporter in BLM vesicles by [3H]cytochalasin B binding assay Fig. 4. Western blot of GLUT2 protein in BLM vesicles from rats with hyperglycemia, and diabetes and those fed high-carbohydrate diet. ilo- 31. lnfiision of high-giucosc solution for 6 h stimu- Purified membranes (SO~goTmembrane protein/lane) were subjected lated glucose transport @-fold) and increased cytocha- to SDS-108 PAGE and trnnskrred to nylon filters. The filters were lasin B binding to 1.8-fold that of non-infused animals. then incubated with anti-peptidc antibody of rat GLUT2 ;Ind Izsl- However, vesicles prepared from animals 4 h after in labeled protein A and subjected to autoradiography. (A) Diabetes. (B) hyperglycemia. (C) high-carbohydrate diet. Lane C, control group; vivo injection of cycloheximide showed 80% reduction lanes l-6, test group. in glucose transport with no significant change in the cytochalasin B binding [12]. The increase in transport resulting from hyperglycemia appears to be due to in- crease in the functional activity of existing carriers in the obtained in the cytocalasin B binding experiments [!3]. membrane followed by recruitment of new carriers, as Glucose infusion resulted in significant hyperglycemia in fat cells [24]. They suggest that newly recruited carri- (12-h infusion of glucose. plasma gluccse = 296 f 30 ers could have a lower K, and a higher turnover rate. mg/ml; saline infusion, 160 & 19 mdml). The effect of We performed RNA-blot analyses of rat small intcs- a 12-h intravenous infusion of glucose on the level of tine with GLUT!, -3, -4 and -5 cDNA, but could detect intestinal GLUT2 protein is shown in Fig. 4B. The no signal except that for GLUT5 mRNA [8,9]. Re- GLUT2 protein level was not increased by the 12-h cently, Davidson et al. showed thal GLUT5 protein is infusion of glucose compared with that of saline infu- located in the BBM in human small intestine [25]. We sion, although [31-l]cytochalasin l3 binding increased sig- could not detect this transporter in the BLM in rat nificantly in these rats (Fig. 2B). We examined whether jejunum with rabbit anti-GLUT5 antibody (RaGLUT- GLUT2 was present in other membrane fractions in SAP, East Acres, Biogenesis, Bourncmoulh, UK). Since hyperg@emic rats jqjunum but detected no im- the affinity for glucose transport and cytochalasin B munoreactive band of the protein in brush-border or binding in the BLM of :*with hyperglycemia is simi- microsomal p:.embrane preparations. Therefore, we lar to those in ratr .I diabetes and those given a concluded that the GLUT2 protein level did not in- high-carbohydrate i ,, we suggest that the functional crease over the control level in rats with hyperglycemia. glucose transporter in Lhe BLM of rals with hyperglyce- mia may have a low affinity for glucose, like that of GLUTZ. 4. DISCUSSION As the changes in transport capacity in the small Numerous studies have shown that the transport ca- intestinal BLM are not nearly as marked as those in pacity of the small intestine is altered by a variety of adipocytes, they may correspond to a physical response conditions such as change in dietary conditions, lacta- occurring shortly after the start of a meal that increases tion, resection, and disease [lo-!3,19-221. Many of the absorption of a very important nutrient [!2]. The these adaptational responses take several days and are interesting finding in the present study was that there after? exp!aiEed by alterations in intestinal morphology was a mechanism for rapid regulation of glucose trans- [l]. However, there are reports that the response of port that does not depend on change in the amount of glucose transport to hyperglycemia is a much more GLUT2.

4G9 Volume 314, number 3 FEBS LETTERS December 1992

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