Development of Wistar rat model of insulin resistance

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1 PO ox 2345, eijing , Chin World J Gstroenterol 2005;11(24): World Journl of Gstroenterology ISSN wjg@wjgnet.com ELSEVIER 2005 The WJG Press nd Elsevier Inc. All rights reserved. Development of Wistr rt model of insulin resistnce ASIC RESEARCH Jing Ai, Ning Wng, Mei Yng, Zhi-Min Du, Yong-Chun Zhng, o-feng Yng Jing Ai, Ning Wng, Mei Yng, Yong-Chun Zhng, o-feng Yng, Deprtment of Phrmcology, Hrbin Medicl University, io-phrmceuticl Key Lbortory of Heilongjing Province, Hrbin , Heilongjing Province, Chin Zhi-Min Du, Stte se for Drug Clinicl Tril, Hrbin Medicl University, Hrbin , Heilongjing Province, Chin Supported by the Key Found of the Technologicl Office of Heilongjing Province, No , the Ntionl Nturl Science Foundtion of Chin, No , Foundtion of Eductionl Office of Heilongjing Province, No Co-correspondents: Jing Ai Correspondence to: Professor o-feng Yng, Deprtment of Phrmcology, Hrbin Medicl University, Hrbin , Heilongjing Province, Chin. yngbf@ems.hrbmu.edu.cn Telephone: Fx: Received: Accepted: Abstrct AIM: To estblish simplified nd relible niml model of insulin resistnce with low cost in Wistr rts. METHODS: Wistr rts were treted with high ft emulsion by ig for 10 d. Chnges of the diets, drinking nd body weight were monitored every dy nd insulin resistnce ws evluted by hyperinsulinemic-euglycemic clmp techniques nd short insulin tolernce test using cpillry blood glucose. Morphologic chnges of liver, ft, skeletl muscles, nd pncretic islets were ssessed under light microscope. mrna expressions of GLUT2 nd α-glucosidse in smll intestine epithelium, GLUT4 in skeletl muscles nd Kir6.2 in bet cell of islets were determined by in situ hybridiztion. RESULTS: K ITT ws smller in treted nimls (4.5±0.9) thn in untreted control Wistr rts (6.8±1.5), nd so ws glucose injection rte. oth dipocyte hypertrophy nd lrge pncretic islets were seen in high ft fed rts, but no chnges of skeletl muscles nd livers were observed. mrna levels of GLUT2, α-glucosidse in smll intestinl epithelium nd Kir6.2 mrna in bet cells of islets incresed, wheres tht of GLUT4 in skeletl muscles decresed in high ft fed group compred with norml control group. CONCLUSION: An insulin resistnce niml model in Wistr rts is estblished by ig specil ft emulsion The WJG Press nd Elsevier Inc. All rights reserved. Key words: Wistr rt; Insulin resistnce; Model Ai J, Wng N, Yng M, Du ZM, Zhng YC, Yng F. Development of Wistr rt model of insulin resistnce. World J Gstroenterol 2005; 11(24): INTRODUCTION Epidemiologicl studies suggest tht insulin resistnce is not only n independent risk fctor tht induces type 2 dibetic mellitus, but lso the common cuse of hypertension, coronry hert disese, nd cerebrl vessel disese, thus the key to cure nd prevent hert nd cerebrl vessel disese. It is of prmount importnce to estblish n insulin resistnce niml model, in order to hve better understnding of the pthologicl process of insulin resistnce nd to develop therpeutic drugs. Severl insulin resistnce niml models re vilble, including hereditry ob/ob mice nd SD or Wistr rt models developed by either injecting low-dose dexmethsone (2 µg/d) into bdominl cvity or feeding food rich in fructose nd sucrose [1-7]. The mjor disdvntges of ll these niml models re the long experimentl cycles (4-30 wk) nd the less relevnt pthologic sttus of insulin resistnce induced by sole fctor s opposed to the nturl multi-fctorl process. The present study ws to estblish n insulin resistnce niml model using Wistr rts with more cliniclly relevnt pthophysiologicl chrcteristics of insulin resistnce bsed on glucose utility of the body nd ltertions of vrious cellulr nd moleculr events relted to insulin resistnce. MATERIALS AND METHODS Animls nd regents Mle Wistr rts weighing g were obtined from Deprtment of Animls, Hrbin Medicl University. Insulin ws purchsed from the First iochemicl Drug Compny of Shnghi. The kit for in situ hybridiztion of smllintestine GLUT2, α-glucosidse, Kir6.2 in islet bet cells nd GLUT4 in skeletl muscles ws purchsed from OSD iotech, Wuhn, Chin. Preprtion of ft emulsion A constnt volume of 100 ml ft emulsion contining 20 g lrd, 1 g thyreostt, 5 g cholesterol, 1 g sodium glutmte, 5 g sucrose nd 5 g scchrose, 20 ml Tween 80, 30 ml propylene glycol ws prepred by dding distilled wter nd stored t 4. Animl tretment Twenty-four Wistr rts were rndomly divided into norml control group nd high ft emulsion group, 12/group. Rts in norml control group received common wter, rts in high ft emulsion group received ft emulsion for 10 d.

2 3676 ISSN CN / R World J Gstroenterol June 28, 2005 Volume 11 Number 24 Insulin resistnce sensitivity ssy by short insulin tolernce test using cpillry blood glucose [8] Rts were weighed nd plced into mouse cge fter fsting overnight. lood sugr in rts ws detected six times fter ip insulin (0.05 U/kg) using blood sugr detector. Absciss indictes time nd ordinte expresses nture logrithm of blood sugr. Regression coefficient (r) or slope ws determined by liner regression nd K ITT ws clculted by multiplying r by 100. K vlue indictes insulin sensibility with smller K vlues for lower sensibilities. Hyperinsulinemic-euglycemic clmp experiment As described previously [9], food ws withdrwn 12 h before the experiment. The rts were then nesthetized by ip mobrbitl sodium (25 mg/kg) fter they hd been weighed. Rts were cnnulted in the jugulr vein for infusion of glucose nd insulin (dul cnnul) nd in the crotid rtery for smpling. All cnnule were tunneled subcutneously, nd encsed in silstic tubing (0.08 cm) sutured to the skin. After infusion of glucose (10%) nd insulin (1 IU/mL) from dul cnnul (constnt velocity), blood sugr ws mesured. To keep the blood sugr in reltively stedy stte, the rte of glucose infusion ws continuously djusted. Glucose injection rte (GIR) ws mesured under homeostsis six times during the experiment. Morphologicl ltertions of rts with insulin resistnce After rts were fsted for 3 h, perirenl ft, gstrocnemius muscle, liver, nd pncres were dissected. The tissues were then fixed with 4% prformldehyde, embedded in prffin wx, sectioned, nd stined with eosin by sppn wood. Morphologicl ltertions were exmined under light microscope. The procedure for mrna levels ws described previously. The positive expression of α-glucosidse nd Kir6.2 showed brown stining signls in villi of smll intestine nd in pncretic islet β-cells. GLUT2 mrna ws expressed in the incisure between two villi of smll intestine nd in cytoplsm of skeleton muscle. Expression level ws ssyed by the degree of color. Sttisticl nlysis Dt were nlyzed by Student s t-test. The results were expressed s men±sd. RESULTS TS Chnges of diet, drinking, body weight, nd viscerl ft in Wistr rts fed with ft emulsion Compred with norml control group, the drinking, body weight, nd viscerl ft incresed mrkedly 10 d fter ig ft emulsion (Tble 1, P<0.05). In contrst, no chnges were observed in diet group. Tble 1 Chnges of diet, drinking, body weight, nd viscerl ft in Wistr rts fed with ft emulsion (men±sd, n = 12) Groups n W (g) Diet (g) Drink (ml) Viscerl ft (g) NC ± ± ± ±4.1 FD ± ± ±26.5 b 16.3±5.4 P<0.05, b P<0.01 vs control. W: body weight; NC: control group; FD: ft emulsion group. Insulin resistnce sensitivity ssy by short insulin tolernce test using cpillry blood glucose nd hyperinsulinemiceuglycemic clmp experiment in Wistr rts The results of short insulin tolernce test using cpillry blood glucose reveled tht K ITT decresed to 4.5±0.9 in rts treted with ft emulsion (ig, 10 d) (P<0.05, n = 12). Our hyperinsulinemic-euglycemic clmp test indicted tht the GIR for keeping homeostsis of blood sugr in rts of high ft emulsion group ws decresed in ft treted group (Figure 1A). A GIR (ml/kg/min) KITT b NC b NC Figure 1 Results of insulin resistnce sensitivity ssy in hyperinsulinemiceuglycemic clmp test (A) nd chnges of glucose induced by insulin injection (). NC: control diet group; FD: ft emulsion diet group; GIR: glucose injection rte. P<0.05, b P<0.01 vs NC. Morphologicl ltertions of skeletl muscle, liver, ft, nd pncres islets in rts with insulin resistnce No morphologicl chnges were observed in skeletl muscle nd liver, but in lrger dipocytes nd pncres islets when the tissues were stined with HE in high ft emulsion group compred to control group (Figure 2). mrna expression ssy of GLUT2 nd α-glucosidse in smll intestinl epithelium, Kir6.2 mrna in bet cells of islets nd GLUT4 in skeletl muscles of rts with insulin resistnce oth GLUT2 nd α-glucosidse mrnas in smll intestinl epithelium were incresed in ft emulsion group compred to control group (Figures 3A-D). The number of cells expressing α-glucosidse mrna in high ft emulsion group ws 50±6 nd 40±5 in control group (P<0.05, n = 12). GLUT2 mrna ws diffused in the incisure between two villi of smll intestine. In the sight of 100 cm 2, the expression re of GLUT2 mrna in high ft emulsion group ws incresed to 3.8±0.6 cm 2 from 3.0±0.4 cm 2 in control group (P<0.05, n = 12). In cytoplsm of skeleton muscle of ft FD FD

3 Ai J et l. Wistr rt model of insulin resistnce 3677 A C D Figure 2 Morphologicl observtion of ft nd pncres islets stined HE in rts with insulin resistnce (60 ). A: Ft cells in control group; : ft cells in high ft emulsion group; C: pncres islets in control group; D: pncres islets in high ft emulsion group. emulsion group, the expression of GLUT4 mrna ws lower, s indicted by the brighter brown color, thn in control group (Figures 3E nd F). Kir6.2 mrna in β-cells showed tendency to increse in ft emulsion group becuse its positive expression density did not chnge but the volume incresed compred to control group. DISCUSSION In the present study, we developed new formul of ft emulsion bsed on the insulin-resistnt niml models estblished by other lbortories. The formul for prepring ft emulsion is to combine high ft diets with fructose nd sucrose. Moreover, the ft emulsion ws dministered to rts by ig but not by food feeding so s to control the dily ft intke. The possible imblnce of ft intke due to decresed ppetite s result of high ft diets ws voided. Incresed drinking ccompnying n increse in body weight nd viscerl ft ws observed though the rts were dministered high ft emulsion for 10 d. This is typicl of the clinicl phenotype. In order to clrify if the insulin-resistnt niml model ws estblished in our study, we detected the dynmic chrcteristics of blood sugr fter insulin injection by short insulin tolernce test using cpillry blood glucose. The result demonstrted tht the K ITT vlue decresed mrkedly fter ig ft emulsion for 10 d compred to control group, indicting tht the rts re insensitive to exogenous insulin, i.e. insulin resistnce. Insulin resistnce refers to the insensitivity of tissues (such s skeletl muscle, liver, kidney, nd dipose tissue) to insulin ction, i.e. the weker glucose utiliztion of body fter insulin ction tht results in hyperglycemi. The clssicl test used to evlute insulin resistnce is hyperinsulinemiceuglycemic clmp experiment [10,11]. We determined the insulin resistnce using hyperinsulinemic-euglycemic clmp technique tht is known to be the most relible method for verifying whether or not insulin resistnce is chieved in rts fter feeding ft emulsion. According to negtive feedbck mechnism, if we wnt to keep blood sugr in the bsl level by chnging injection rte of exogenous glucose, we should regulte the injection rte of exogenous insulin. The exogenous GIR is equl to the glucose utiliztion rte of peripherl orgns becuse the endogenous glucose production (from liver) could be completely inhibited when the plsm insulin level is excessively high. Hence, GIR cn be considered s n index for evluting the ction of insulin on peripherl orgns. Our dt showed tht the GIR ws indeed decresed in rts treted with high ft emulsion, indicting tht insulin resistnce is chieved in ft emulsion group. Dibetes mellitus is pthologicl process ffecting the whole body system. Skeletl muscle, ft, nd liver re considered s the insulin-sensitive tissues [12-16]. Altertions of the functionl sttus of these tissues my result in insulin resistnce of the body. The min mnifesttion is the dysfunction of glucose bsorption nd utiliztion, metbolism disturbnce of glucose in liver cells nd lipo-

4 3678 ISSN CN / R World J Gstroenterol June 28, 2005 Volume 11 Number 24 A C D E F Figure 3 mrna expression ssy of GLUT2 nd α-glucosidse in smll intestinl epithelium nd GLUT4 in skeletl muscle in rts with insulin resistnce (30 ). A: GLUT2 in control group; : GLUT2 in high ft emulsion group; C: α-glucosidse in control group; D: α-glucosidse in high ft emulsion group; E: GLUT4 in control group; F: GLUT4 in high ft emulsion group. metbolism disturbnce in dipose cells [15,17]. Severl ppers hve reported increses in dipose cell volume nd number ccompnied with redistribution of these cells over the body in insulin-resistnt nimls nd humns. Nevertheless, it is more importnt tht dipose cell volume increses when insulin resistnce occurs [14]. Our experiments demonstrted tht insulin resistnce could be induced in rts fter being treted with high ft emulsion by ig. However, we still do not know if morphologicl chnges occur s result of dysfunction. We therefore crried out histopthologicl ssy by HE stining of insulin-sensitive tissues, such s ft, skeletl muscle, nd liver. We found no morphologicl chnges in both skeletl muscle nd liver, but dipose cell volume ws mrkedly enlrged in rts of ft emulsion group. In high ft emulsion group, pncretic islets enlrged but no chnges in the other prt of pncres were seen. The morphologicl chnges in both ft cells nd pncretic islets in high ft emulsion group confirmed the success of our insulin-resistnt niml model. Glucose is n importnt substnce tht keeps the blnce of energy metbolism nd life. Glucose is polrity molecule tht cnnot pss the lipid bilyer of cell membrne by free diffusion. Smll intestine nd renl tubule cn bsorb glucose nd other tissues must intke glucose by fcilitting diffusion with glucose trnsporter proteins in cell membrne. Among the vrious glucose trnsporter proteins, GLUT2 nd GLUT4 hve been more intensively studied. GLUT2 is n importnt glucose trnsporter protein which distributes in heptocytes, pncretic islet β-cells, smll intestine, nd kidney [18-20]. People py close ttention to GLUT2 becuse it is relted to pthogenesis of dibetes mellitus. GLUT2 expression in pncretic islet β-cells decreses in rts nd Zuker obesity rts with utoimmune dibetes mellitus. Some studies reported tht GLUT2 mrna nd protein expressions re enhnced in the liver of dibetes mellitus rts induced by streptozotocin, but others found no chnges. Studies on expression of GLUT2 in epithelil cells found tht GLUT2 protein expression in srcolemm of smll intestine increses in both STZ-induced dibetes mellitus rts nd high sugr feeding rts, but no chnges occurred in hyperglycemi rts cused by glucose filling, while glucose trnsporter ctivity increses in these conditions [21-23]. Until now, study on mrna expression of GLUT2 in smll intestine of insulin-resistnt rts hs not been reported. GLUT4 is n insulin rection protein trnsporter which minly distributes in dipose cells nd strited muscles [24]. On the one hnd, GLUT4 is the richest protein trnsporter in both dipose cells nd strited muscles. On the other hnd, GLUT4 expression level is coincident with glucose utiliztion bility of vrious types of dipose cells. Almost ll the glucose bsorbed by muscles gets converted into glycogen in high insulin condition. Glucose trnsport of musculr tissues is very importnt for the glucose utility of body becuse glucose trnsport is the rte-limiting step of glucose utility of muscle tissue. To dte, studies hve evidenced tht not only the protein expression of GLUT4 decreses in insulin-resistnt condition, but lso the trnsloction of GLUT4 from cytoplsm to cell membrne is defected [25]. sed on the bove resons, we mesured mrna expression of GLUT2 in smll intestinl epithelil cells nd GLUT4 in gstrocnemius muscle in our insulin-resistnt rt model. On the one hnd, we detected mrna expression of GLUT2 in smll intestinl epithelil cells. On the other hnd, we further verified t molecule level tht we successfully estblished the insulin-resistnt niml model. Our study demonstrted tht mrna expression of GLUT2 in smll

5 Ai J et l. Wistr rt model of insulin resistnce 3679 intestinl epithelil cells incresed in high ft emulsion group compred with norml control group. In contrst, GLUT4 mrna expression decresed. The results revel tht the insulin-resistnt niml model is successfully estblished. In order to ensure tht sugr cn be bsorbed in smll intestine, polyscchride must be divided into monoscchride by α-glucose glycosidse of smll intestine. In our experiments, mrna expression of α-glucose glycosidse in smll intestine incresed in ft emulsion group. The mrna expression of β-cell Kir6.2 in pncretic islets did not chnge in ft emulsion group, but the totl expression level incresed becuse of the enlrgement of pncretic islet volume. In conclusion, n insulin-resistnt rt model is estblished by ig specil ft emulsion. REFERENCES 1 Severino C, rizzi P, Solins A, Secchi G, Mioli M, Tonolo G. Low-dose dexmethsone in the rt: model to study insulin resistnce. 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