Volume 309, number 1, 15%156 8 1992 Fcdcrution of Europcam Uioehcmkrrl
Scptcnbcr 1992
FEDS 11491 Socichs
0014579U9355~~
Insulin receptor ancl glucose transporter mRNA expression in skeletal muscle of genetically obese Backer rats Rslchcl M. Knott and John E. Hcskcth
Rcceivcd 19 July 1992 Insulin rcsistnncc and dsfcctivc gluror uanspon 11rc rssacio~ed with mutic tissue in ~hc &cncrblly obese Zuckcr rat zmd arc vccompunicd by changes in the number of insulin rcscp~~n: and the atuilubility of glurc~c transporlrn. The present study wns carried out to assess whether. in mrlc Eucker rnts 11110 weeks of t~gc. there dcl’ccll; were rcilccicd by chnngcs in lhc lcvcls of mRNAs for lhc insulin rcccplor und for the insulin responsive glucose trrnsporrcr (GLUT-4). To~rrl RNA was cn~rx~cd from plnmvrir and rolcur muscles ;Ind the Icvcls of insulin racptor and GLUT-4 mRNAs and 111S rRNA wcrc dstcrmincd hy Northern hybridtitian and quanlificd by imugc ontllyris of the autoritdiogruphr, A 50% incrcarc in 111~lcvcl of insulin rcccplor mRNh was dcrcctcd in both the plooluris und lhc solcur rnusclc fruln the obese fats compared to lhc lcim mts, No change in the lcvcl of GLUT-4 mRNA tins dctcclrd in lhc pluntnrir muscle ullhou#h inrrcclscs wcrc observed in tllct rolcus mu& from
Zuckcr rats: Insulin rcccplor: Gluross trtmrponcr:
1, INTRODUCTION
of
Skeletal muscle. by virtue its muss, is u mtljor turycl organ for insulin in the body and is the primary tissue responsible for insulin-dcpcndcnc whole-body glucose uptake [I]. Conscqucntly. if glucose utiliztrtion and/or insulin rcsponsivcncas of the muscle urc nltcrcd this may significuntly uffsct the efficiency of whole-body glucose mctabotism. An insulin-resistant state bus been well documented in the genetically obese Zuckcr rat [2] and this is rcflcctcd in defects ot the rcccptor und the post-receptor lcvcl [3] in skclctt\l rnusclc und liver [4j. A reduction in insulin binding and the phosphorylation of rhc insulin receptor [2.5] my contribute Co the insulin-rctiistant state, although this cannot fully explain the condition, Dccrcascd glucose uptake, oxidation and incorporation into glycogcn have been churrrctcristically associrrtcd with skeletal muscle of genetic obcsitics [2], The soncomittlnc incrctrsc in basul and insulin-stimulutcd glucose oxidation in adipose cells [6] arc also important features associ;Wd with the obese syndrome. In addition. mctubolicdcfccts huvc been dctcstcd in the ylucotic transport system in cultured adipoac cells isolated from young (30 day old) Zuckcr rats and thcsc arc associated with a marked incrcusc in the glucose transporters in the intracellular pool [7]. Evaluation of glucose transporter or insulin rcscptor
C~~~~YCI~~~~~~I~~ tt&trc,vx Dcparuncnt of Ophthalmoloyy, University Mcdicul Buildings. Porcc~crhill, Abcrdccn. AD9 ?2D, Scotlund. UK.
mRNA
mRPJA Icvcts tallows usscssmcnt of which mcshnnisms (ix. trrmslutionul or pretranslutional) arc dcfcctivc in tissues from obssr unimals. The mRNA for the insulin. responsive glusosc tmnsportcr. GLUT-4 has been shown to be clcvtltcd in cardiac myocytcs [i3] and in adipocytcs from young obese Zuckcr ruts (91. NOWCYCF, thcrc is no direct rcltttionship bctwscn insulin rcccptor number and the insulin receptor mRNA in the liver of gcncticully obcsc mice (oh/o&)since the number of insulin rcccptors in the liver of such mice [IO] is rcduccd, whereas the tcvcl of insulin receptor mRNA is incrcoscd [I 13. Despite the ubovc studies. the mechanisms which lead
to the insulin rcsistansc of muscle in gcnctMly obcsc animals arc not clearly understood. The aim of the prcscnt study was to invcstigatc if the obese phcnotyp-c is associated with changes in the lcvcl of mRNA for the insulin receptor and the insulin-responsive glucose transporter, GLUT-4, in skeletal muscle of Zuckcr rats. Mu& samples wcrc taken when the animals were IOweeks old, an age at which a state of insulin rcsistansc is known to exist [4.12! Both solcus muscle, consisting predominantly of slow oxidativc fibrcs. and the plantaris muscles, of mixed fibre type, were examined, as insulin sensitivity depends upon muscle fibrc type and inhcrcnt oxidtrtivc capability [13,14]. 2. MATERIALS AND MET#ODS Male lcun (FtiirlFu or Fu/fi) and olxsc Zuckcr rats (r-2@) wcrc proup hourcd und fed ad libhum. Wlvzn the animals wcrc IO-weeks old they
153
Volume 309. number 1
FEBS LETTERS
+vcrc killed by dcc@ta\ion ;md blood %implo; wcrc callrclcd from s~vcrcd blood vasrlr, The plrnk& and lhc rwlrus mu%&% wcrc rc. mavcd and quickly l’razcn in liquid nilropn imd aubqrrrmly rirorcd III -7O.C unril rcyuircd. The inrulin Ic\cI in Ihe plasma of lean and obclrc Zuckcr rats wurdc~crmincd by rudioinrarunpilIrul~~ usinp[‘:‘l]inrulin as a tracer and ral insulin (Nova Uiolubori~kxics. UK) YJLa anndard.
Icst yroup ali rpccilird in the 1~x1.Datn nrc prcrcnicd throuyhoul as means F S.E.M. SIaWcll analysis of the deta *W Lwrricd out ulriny u SludcnCs Wsl.
2.2. A&i ewtrninrr. prrrt$cttriur~tttrtl rlrrrru@iurc?rix RNA was errtruclcd from the muscle tirJuc uriny llw mahod dcrcribcd by Chomcxynrki und Lcchi (151. Urielly. the Gl;wa were homogcnixcd in 11dcnaturiny rolution conhiininr) 4 M guanidinium i~o~hioryanutc. 15 mM lrodiunr citrate (pH 7.0). 0.5% Ji;rrkuql and 0. I M ?-mcralp~otihanol. The RNA was CW:ICIC~ uring phrnol-chloro. fomr-isoamyl alcohol and the nqucouv luger WIS removed. The RNA wax prccipitutcd with i~propanol and further conscntmlcd by :I Mond prcciphation with itipropanol. The RNA @let was then wurhrd with 708 cthlrnol and fm;rlly ruulrpcndcd in 0,5% tiiunr dodccyl sulphatc (SDS). Ths conccntralion af lhc RNA ~115dctsrnrincd by itri ubsorbancc UI 140 nm. The RNA war then rcwlvcd by elcrtrophor& in a I.?% rr~raric ycl canlsining formuldchydc [ t6). Each sample wus anelyrcd in dupli. CYICor in triplicate uriny !Opy of RNA per hms. The ycl was run at 55 V for 3 h ir11drhr RNA WIIIlrubrqucnlly vigualixcd by sulining with cthidiunr bromide. The RNA ~5 trrnrfcrrcd to a nylon numbranc (Gcncxrccn) by overnight capillary blolMy and inrmobiliard by cxporurc to $hort.%%vc uhnviolct light.
Plusmu insulin lcvcls WMCdctcrmincd in 6 animals of both Ican and obcsc Zuckcr rtrts. The obcro rats wcrc clsrrrly hypcrinsulinucmic with rrn insulin lcvcl of 4,70 ngIm1 (2 024) compilrcd to 1.75 @ml (2 0,161 for the lcun Zuckcr rats.
Northern hybridizationr #‘crc carried out on ~otnlRNA cx~racud from rkclcW1 murclc in order 10 dcknninc the lcvcl of mRNh bath lor the insulin rsscplor and GLUT.4 and for the Itl S rRNA: rhc cDNA probe Tar the human insulin rcccplor war II i .5 kb f?r~RI/Pfli fmsrncn~ corrcrponding IO Ihc region l,lOl-2.602 hart pairs which coda for a rcsion in thccxiraccllular domain [ 171.The human GLUT. 4 probe was a 1.1 kb, SIIll frarpllmcmfrom a cDNA (181and the 18 S rRNA probc was a 1.4 kb DUIIIHI fragmcni 1191, All af Ihc hybridizalionr were carried OUI under rimilar conditionr at 41.C. The nylon mcmbra~ were prr-hybridized for 16 h in buffer consisting of 50% formumidc. IO% dcxtrun I;ulphntc. 0.1% paly vinylpyrrolidonc(PVP). 02% bovine Yrum albumin.O.?% Ficoll. 0.05 M TrirmHCI (pH 7.5). I .OM NuCI. 0. I% sodium pyrophorphzlc, 1.0% SDS and denatured rulman ~pcrm DNA (> 1OOjhnl). The nppropriutc probe (25-50 np) was lubrllcd by random priminy with 1%.‘:PJdCTP urins the Multiprime DNA labclling dy~icm (Amcnhsm Interna. tianal. UK) nnd rcparntcd from hbcllrd nuclcohlcr using a Scphdcn 0.50 column. The lubcllcd probe WBI made up in lhc hybridization buffer (cxcludiny dcxtmn rulphau) and added ta the cxining buff.cr. Hybridiwulion was carried out for 16 h at 4X. Nonqccific hybrid. ixation WI: rcmovcd from the fihcn by the folloivinp washing procc. durcr: Z x SSC (0.3 M NuCWJOmM radium citnlc, p1-i 7.0) a~raom tcmpcraturc for 5 min; 0.5 x SSOI% SDS ;II 65’12 for 30 min; 02 x SSC al room tcmpcrclturc for 30 min. Each procedure WP carried out in duplicalc and ~lrcfillerr wcrc then laid down in ;luloradioyr;lphy cmctus wilh HypcrfWMP and stored al -70%‘. The length of exposure WUIarrcrscd using a Gcigcrmunler ulicr Ihc mcmbrancs had been washed, and cxposurctinic varied l’roni 3 h(l8 S rRNA) to 7dayr (insulin rcccpror. GLUTA). The lcvcl of mRNA that was dctcc.vd using ~hcrc praccdur*r was dctcrmincd by quantifying the inrcndty of the signal on the nutoradiograph usins a QUIPS imayc pro-ccssingwork+t;ction. opcra~ing with YCS image proccrrinp rofiwarc. The signals for Ihc mRNAr under scrutiny were calculakd rclativc 10 the lcvcl of It( S rRNA (optical density ratin) in each ample lane. This cnublcd correction for any diffcrcnccs in signal intensity Mt may bc due tn variution in the ini\i;\l loading of the RNA onto the gel. The optical dcnrhy ratio of rhc rcspcctivc ~RNAI in skclctul murclc of obcac Zuckcr ri+lz wcrc lhcn cxprcEccdrclalivc to values for their Icun coumcrparls (lOO%b).Qunn. litalion WY curried out on dupliciilc snmplrr from 5 animulx in each
154
3, RESULTS
Using Northern hybridiztion of total RNA from solcus and pluntfrris muscle of lean ;\nd obese Zuckcr ruts, mRNA species for the insulin rcccptor (72 and 8.5 kb) and For GLUT-4 (2.8 kb) were dctcctcd (Fig. 1). As observed in normal rodent spccics. two mirjsr bnnds wcrc obscrvcd For the insulin receptor mRNA. Insulin receptor mRNA WY qurmtificd by combining rhc opti= cul density of both bilnds. No significant chlmgcs in the lcvcl of GLUT-4 mRNA wcrc abscrvcd bctwccn the km and the obcvc ruts in the pluntnris ~sclc. ulthouyh the insulin receptor mRNA increased significimtly (Fig. 1) to 153%. In the solcus muscle significunt incrcilscs wcrc obscrvcd in the lcvcl of insulin rcccptor (189%) tend GLUT-4 (141%) mRNA spccics in the obcsc rats compared with their lci\n countcrports (Fig, 2), 4. DLSCUWGN The rcsutts demonstrate rrn incrcuscd lcvcl of insulin receptor mRNA in the solcus und the pl;lnt;rris muscles of obese Zuckcr rats when compurcd to lcirn ruts. In the solcus muscle the level of GLUT-4 mRNA WAS ills0 incrctrscd. The alterations in the Icvcl of insulin rcccptor mRNA in the plunttlris muscle appear to be rcltrtivcly specific as the conccntrutions oFGLUT- mRNA were unchungcd. In the light of previous observutions on the low numbcr of insulin receptors in muscle tissue of ycnctically obcsc Zucker milts[3.20]. it ilppcurs that the dccrcax in receptors mily bc due to altcrntions of trunslatian of the mRNA u&or to post-translutionul proscsscs, Although post-rcdeptor defects can ulso occur, the de= creased number of insulin receptors is likely to contribute to the insulin rcsistirncc of the skclcttll muscle tissue. An increase in the lcvcl of insulin receptor mRMA was also observed in the soleus muscle, suggesting thut a similar dcfcctivc translation process may bc operating. Howcscr, unlike the situation in the plantaris muscle. this w~ti arcompanird by nn increase in the conccntration of GLUT-4 mRNA. An incrcasc of similar magnitude has been observed in isolated cardiac myocytes from Zuckcr rats [8], with u dccrcasc in basal end inuulin-atimulitted trunsport. The incrctise in the level of
FEBS LETTERS
Volume 308, number 2
(a)
gcptcmbcr 1992
plentaris
insulin receptor
GLUT-4
(b) solcus
insulin receptor
GLU T-4
L
L
0
0
Fig. I. Northern hybridization of total RNA (40 ~g) extracted from (a) plantark and (b) solcun muscle usin8cDNA probes forthe insulinrcccplor and GLUT4 Results indiclltcd the specific dctcction of the mRNA specicr in both musflc typrs and the rclativc diffcrcnccs bctwc~n the 1-n and the obcr wts. For further technical details. see Materials nnd Methods.
GLUT-4 rnRNA my bc in rcsponsc to chungcs in circufuting insulin concentration. tis GLUT-4 flas been idcntiffcd in insulin rcsponsivc tissues [2fj. TIIC abscncc of any incrwsc in the planturis muscfc impI& the cxistcncc of diffcrcntiuf regulatory mechanisms that arc associated with the varying fibrc type of tfx two muscles [ 131.This observation is consistent with previous obscrvacions [‘,I which demonstrated that in a mixed fibrctype muscle (gastrocnemius), no change in the lcvcl of GLUT-4 mRNA was dctcctcd in the obese phenotype while changes were obssrvcd in udipocytcs from the obese animals. The rcquircmcnt for an incrcascd nurnbcr oi glucose trmsporkrs may nut bc as criticat in th pfuntaris as in the sofcus because the plantaris has a higher proportion of oxidativc fibrcs, Thcrc stiff rcmains a reduction in the ovcrulf glucose utilization of
muscle tissue from genetically obcsc Zuckcr rats [2] whicfz may bc mediated througfl defects in other pathways. Insulin rcgufatcs the levels of insulin receptors [22] and insulin rcccptor mRNA [23]. in vitro. It has also been shown that acute nutritional changss [24-261 significantfy incrcascs the lcvcl of insulin rcccptor mRNA in skcfctaf muscle, possibly through incrcascd transcription of the insulin receptor gene or insrcasc in the stubility of the mRNA itself. Since the abesc Zuckcr rats arc charactcristicalfy hypsr-insufinacmis, the changes in insulin receptor mRNA in their muscle may bc a rcsponsc to incrcascd lcvcfs of circuhfing insdin. In summary, an increased levcf of insulin receptor mRNA has been detected in the plantaris and the solcus muscle of gcncticalfy obcsc Zucke: rats. Thcrcforc. the
FEBS LEfTERS
Volume 309. number 1
kptcmbcr
1992
REFERENCES [I) DcFronxo. R.A..
lnaullt’t rooogtor
BUJTd
Plnntarie
inrulln
QLtJTd
rocqator SQl@tJ~
Fig. 2. Quantitation of insulin rcaptor and GLUT4 mRNA lcvcls in the pluntnrir ttnd the r&w tnurlc of lean(L) und ohc (0) Zuckcr ruts. A rigniliwnt instrasc (‘P c 0.05) in rhc lsvcl of insulin receptor mRNA wus dctatcd in 1hcpltlntarir murclc ofobesc Zuekcr rais when compared lo Ican coumcrpttrts. No ri~oifiwnt chanpr wcrc observed in lhc GLUT.4 mRNA in this muscle. Signitkzanl incrctttir wcrc obrcrvcd in the rolcur muscle for the insulin rcccptor mRNA (“‘PcO.001) and GLUT.4 mRNA (“Pe 0.01). The lcvcl of mRNA in the murclcsamplcs from the lcun rats is shown as 100%and 1hc changer 1hat arc obssrvcd are indicated rclutivc to 1hir.
dccrcuscd number of insulin rcccptors in the muscle tissue of obese 2ucktr rats cnnnot bc simply cxplaincd by reduced amounts of availi\blc mRNA, On the con= trury this suggests both that thcrc is udditional control ut translational and/or post-translational lcvcls and that the increased mRNA lcvcls rcflcct a compcnsutory mechanism which is attempting to ovcrcomc the tissue’s resistance to insulin. Increases in the lcvcl of GLUT-4 mRNA wcrc also evident in the solcus nlusclc but not in the plantnris rnueclc. This may indicate specific diffcrcnccs thnt arc rrssocintcd with the fibre types of the muscles or a gsncralizcd enrichment of the mRNAs in the solcus muscle. Ac~~~o~vkr~~a~tc~trr: WC would like 10 1hank Mr. T. Atkinsott for his 1echnical assistance with the insulin ttrruyr and Dr. A. Travis for the provirion of the compulcr program for the quuntitaticn of mRNA lcvclr an 1hcnuloradiographs. ThccDNA prober for the insulin rcccptor and GLUTS wcrc donated by Drr. Lclrnd Ellis and Gwyn Gould, rcspcstivcly.
156
Fcrranninni, E.. SUIO,Y., FcliB, P. and Wlrhmn. J, (1981) 1. Clin. Invcsl. 61, 14611-1474. I21 C;rcch. M.P., Richardson. D.K.. Becker, S.G., Wnltcn. C.G.. Gitomcr. W. and Hcnrich. J. (1978) Metdbolirm Ciin. Exp. 27. 1967-1972. 131Crc1lnx. M. and Jcanrcnaud. B, (1980) Metabolism 29,467-473. [41 Tcrrcttuz J., Auimacopnulos~Jcttnnct, F. and JcPnrcnaud, B. (1986) Endosrinol. I lb. 674-678. [SlShrrrgil. N.S., Ta1oyan, A., El~Rcfni. M-F,, Plciu. MI. and Chnn, T.M. (1986) Biocltem. Biophys. Rcr. Commun. 137.286-294. I61 Eaaki, 0, (1~19) Diabctologia 32, 290-294. [71 Gucrrc*Millo, AI., Lavnu. M., Hornc, J.S. und Wardmtlu. L-J, (19X5) J. Biol. Chcm. 260.2197-2201. [a] Eckcl, J.. Rub, M.. Pctcrscn, S.. Hcrbcrg. L. and Rcinnucr. H. (1990) Abnruct, IV lntcrnntionttl Symposiumon Insulin Rcscp ior and lnrulin Action: Molcrular and Clinical Aspcetr. Vcronn, Imly. 67-68. [91 Hninnulr, t., GucrreMillo, M., Guichard. C. ttnrl Luv~I~. M. (1991) 1. Chit. Inucsl. &7. 1127-l 131. [IO] SOILA.H.. Kahn, CR.. Ncvillc Jr., D.M. und Roth. J. (1975) J. Clin. Invest. 56, 769-780. [I I] Ludwiy. S.. Mullcr.Wicland, D.. Goldstein. B.J. and Kahn, CR. (1981) Endocrinology 123. 594-6GQ. [12] PPniwud, L.. Rohncr~Jcanrcnaurl. F. und Jcunrcnaud. U. (1916) Am. J. Physiol. 250. E662-E66b. [13] Catnpion, D.R.. Shapira, J.F.. Allen. GE.. Haustnttn. G.J. and Mur1in, R,J, (19lt7) Growth 51, 397410. II41 Sherman. W.M.. Katz. A-L., Cullcr.C.L., Withcn. R.T. and tvy, J.L. (1918) Am. 1. Phydol. 255. E374-E382. [ 151Chomca~nrki. P. and Sacchi. N. (1987) Anal. Biochcm. 162. l56159. (161 Sumbrook. J.. Fritrb. E.F. and Muttia1is. T. (1989) (2nd cdn.) Cold Spring Harbor tboraory Press, New York. 1171 Ebina. Y.. Ellis, L,. Jttrnagin, M.. Edcty, M,, Ciraf. L., Cl;rrrs.sr. E.. Ou. J-H., Masian. F.. Kun, Y.W.. Goldfine, I.D.. Roth. R.A. and Rutrcr. W.J. (1985) Cell 40. 747-758. [la] ptcc DOE., 8rubc. S. and Muccklsr. M. (1989) Nrriurc 338. [I91 Erickion. J.M.. Rushford, CL.. Dorncy. D.J.. Wilson, G.N. and Sshmickcl, R.D. (1985) Gcnc 16, l-9. 1201 Lc Murchund&urrcl, Y., Gordcn. P.. Flier. J.S.. Knhn, CR. nnd Frcyclict, P. (19%) Di&etclogiu 14. 31 l-317. [?I] Gould,G,W. and Ucll, 0. (1990)Trcnds Diochcm. Sci. IS. 1X-13. I221 Gavin, J.R.. Roth J.. Ncvillc, D.M., DC Mcytr. P. and l3usll, D.N. (1974) Proc. Null. Acad. Sci. USA 71. &I-88. [23] Levy, J-R,, Krystal, G., Glickman, P. und Dastvan. F. (1991) Diabetes 40, 58-65. [24] Knoll, R-M.. Hnkcth. 1-E. und Trayburn, P. (1990) Biochcm. Soc, Trans. 18, 1263. 1251Knott. R.M.. Gran1. G.. Bardoca, S,, Puaetui, A., DC Curvulho, A.F.F.U. and Hrrkc~h. J.E. (1992) ht. J. Biochcm. 24.897-902. [26] W~~lliicc,S., Cutnpbcll, G., Knott. R.M., Gould, G.W. and Hcs. kcth, I.E. (1992) FEDS 301, 69-72.
Scptcnbcr 1992
FEDS 11491 Socichs
0014579U9355~~
Insulin receptor ancl glucose transporter mRNA expression in skeletal muscle of genetically obese Backer rats Rslchcl M. Knott and John E. Hcskcth
Rcceivcd 19 July 1992 Insulin rcsistnncc and dsfcctivc gluror uanspon 11rc rssacio~ed with mutic tissue in ~hc &cncrblly obese Zuckcr rat zmd arc vccompunicd by changes in the number of insulin rcscp~~n: and the atuilubility of glurc~c transporlrn. The present study wns carried out to assess whether. in mrlc Eucker rnts 11110 weeks of t~gc. there dcl’ccll; were rcilccicd by chnngcs in lhc lcvcls of mRNAs for lhc insulin rcccplor und for the insulin responsive glucose trrnsporrcr (GLUT-4). To~rrl RNA was cn~rx~cd from plnmvrir and rolcur muscles ;Ind the Icvcls of insulin racptor and GLUT-4 mRNAs and 111S rRNA wcrc dstcrmincd hy Northern hybridtitian and quanlificd by imugc ontllyris of the autoritdiogruphr, A 50% incrcarc in 111~lcvcl of insulin rcccplor mRNh was dcrcctcd in both the plooluris und lhc solcur rnusclc fruln the obese fats compared to lhc lcim mts, No change in the lcvcl of GLUT-4 mRNA tins dctcclrd in lhc pluntnrir muscle ullhou#h inrrcclscs wcrc observed in tllct rolcus mu& from
Zuckcr rats: Insulin rcccplor: Gluross trtmrponcr:
1, INTRODUCTION
of
Skeletal muscle. by virtue its muss, is u mtljor turycl organ for insulin in the body and is the primary tissue responsible for insulin-dcpcndcnc whole-body glucose uptake [I]. Conscqucntly. if glucose utiliztrtion and/or insulin rcsponsivcncas of the muscle urc nltcrcd this may significuntly uffsct the efficiency of whole-body glucose mctabotism. An insulin-resistant state bus been well documented in the genetically obese Zuckcr rat [2] and this is rcflcctcd in defects ot the rcccptor und the post-receptor lcvcl [3] in skclctt\l rnusclc und liver [4j. A reduction in insulin binding and the phosphorylation of rhc insulin receptor [2.5] my contribute Co the insulin-rctiistant state, although this cannot fully explain the condition, Dccrcascd glucose uptake, oxidation and incorporation into glycogcn have been churrrctcristically associrrtcd with skeletal muscle of genetic obcsitics [2], The soncomittlnc incrctrsc in basul and insulin-stimulutcd glucose oxidation in adipose cells [6] arc also important features associ;Wd with the obese syndrome. In addition. mctubolicdcfccts huvc been dctcstcd in the ylucotic transport system in cultured adipoac cells isolated from young (30 day old) Zuckcr rats and thcsc arc associated with a marked incrcusc in the glucose transporters in the intracellular pool [7]. Evaluation of glucose transporter or insulin rcscptor
C~~~~YCI~~~~~~I~~ tt&trc,vx Dcparuncnt of Ophthalmoloyy, University Mcdicul Buildings. Porcc~crhill, Abcrdccn. AD9 ?2D, Scotlund. UK.
mRNA
mRPJA Icvcts tallows usscssmcnt of which mcshnnisms (ix. trrmslutionul or pretranslutional) arc dcfcctivc in tissues from obssr unimals. The mRNA for the insulin. responsive glusosc tmnsportcr. GLUT-4 has been shown to be clcvtltcd in cardiac myocytcs [i3] and in adipocytcs from young obese Zuckcr ruts (91. NOWCYCF, thcrc is no direct rcltttionship bctwscn insulin rcccptor number and the insulin receptor mRNA in the liver of gcncticully obcsc mice (oh/o&)since the number of insulin rcccptors in the liver of such mice [IO] is rcduccd, whereas the tcvcl of insulin receptor mRNA is incrcoscd [I 13. Despite the ubovc studies. the mechanisms which lead
to the insulin rcsistansc of muscle in gcnctMly obcsc animals arc not clearly understood. The aim of the prcscnt study was to invcstigatc if the obese phcnotyp-c is associated with changes in the lcvcl of mRNA for the insulin receptor and the insulin-responsive glucose transporter, GLUT-4, in skeletal muscle of Zuckcr rats. Mu& samples wcrc taken when the animals were IOweeks old, an age at which a state of insulin rcsistansc is known to exist [4.12! Both solcus muscle, consisting predominantly of slow oxidativc fibrcs. and the plantaris muscles, of mixed fibre type, were examined, as insulin sensitivity depends upon muscle fibrc type and inhcrcnt oxidtrtivc capability [13,14]. 2. MATERIALS AND MET#ODS Male lcun (FtiirlFu or Fu/fi) and olxsc Zuckcr rats (r-2@) wcrc proup hourcd und fed ad libhum. Wlvzn the animals wcrc IO-weeks old they
153
Volume 309. number 1
FEBS LETTERS
+vcrc killed by dcc@ta\ion ;md blood %implo; wcrc callrclcd from s~vcrcd blood vasrlr, The plrnk& and lhc rwlrus mu%&% wcrc rc. mavcd and quickly l’razcn in liquid nilropn imd aubqrrrmly rirorcd III -7O.C unril rcyuircd. The inrulin Ic\cI in Ihe plasma of lean and obclrc Zuckcr rats wurdc~crmincd by rudioinrarunpilIrul~~ usinp[‘:‘l]inrulin as a tracer and ral insulin (Nova Uiolubori~kxics. UK) YJLa anndard.
Icst yroup ali rpccilird in the 1~x1.Datn nrc prcrcnicd throuyhoul as means F S.E.M. SIaWcll analysis of the deta *W Lwrricd out ulriny u SludcnCs Wsl.
2.2. A&i ewtrninrr. prrrt$cttriur~tttrtl rlrrrru@iurc?rix RNA was errtruclcd from the muscle tirJuc uriny llw mahod dcrcribcd by Chomcxynrki und Lcchi (151. Urielly. the Gl;wa were homogcnixcd in 11dcnaturiny rolution conhiininr) 4 M guanidinium i~o~hioryanutc. 15 mM lrodiunr citrate (pH 7.0). 0.5% Ji;rrkuql and 0. I M ?-mcralp~otihanol. The RNA was CW:ICIC~ uring phrnol-chloro. fomr-isoamyl alcohol and the nqucouv luger WIS removed. The RNA wax prccipitutcd with i~propanol and further conscntmlcd by :I Mond prcciphation with itipropanol. The RNA @let was then wurhrd with 708 cthlrnol and fm;rlly ruulrpcndcd in 0,5% tiiunr dodccyl sulphatc (SDS). Ths conccntralion af lhc RNA ~115dctsrnrincd by itri ubsorbancc UI 140 nm. The RNA war then rcwlvcd by elcrtrophor& in a I.?% rr~raric ycl canlsining formuldchydc [ t6). Each sample wus anelyrcd in dupli. CYICor in triplicate uriny !Opy of RNA per hms. The ycl was run at 55 V for 3 h ir11drhr RNA WIIIlrubrqucnlly vigualixcd by sulining with cthidiunr bromide. The RNA ~5 trrnrfcrrcd to a nylon numbranc (Gcncxrccn) by overnight capillary blolMy and inrmobiliard by cxporurc to $hort.%%vc uhnviolct light.
Plusmu insulin lcvcls WMCdctcrmincd in 6 animals of both Ican and obcsc Zuckcr rtrts. The obcro rats wcrc clsrrrly hypcrinsulinucmic with rrn insulin lcvcl of 4,70 ngIm1 (2 024) compilrcd to 1.75 @ml (2 0,161 for the lcun Zuckcr rats.
Northern hybridizationr #‘crc carried out on ~otnlRNA cx~racud from rkclcW1 murclc in order 10 dcknninc the lcvcl of mRNh bath lor the insulin rsscplor and GLUT.4 and for the Itl S rRNA: rhc cDNA probe Tar the human insulin rcccplor war II i .5 kb f?r~RI/Pfli fmsrncn~ corrcrponding IO Ihc region l,lOl-2.602 hart pairs which coda for a rcsion in thccxiraccllular domain [ 171.The human GLUT. 4 probe was a 1.1 kb, SIIll frarpllmcmfrom a cDNA (181and the 18 S rRNA probc was a 1.4 kb DUIIIHI fragmcni 1191, All af Ihc hybridizalionr were carried OUI under rimilar conditionr at 41.C. The nylon mcmbra~ were prr-hybridized for 16 h in buffer consisting of 50% formumidc. IO% dcxtrun I;ulphntc. 0.1% paly vinylpyrrolidonc(PVP). 02% bovine Yrum albumin.O.?% Ficoll. 0.05 M TrirmHCI (pH 7.5). I .OM NuCI. 0. I% sodium pyrophorphzlc, 1.0% SDS and denatured rulman ~pcrm DNA (> 1OOjhnl). The nppropriutc probe (25-50 np) was lubrllcd by random priminy with 1%.‘:PJdCTP urins the Multiprime DNA labclling dy~icm (Amcnhsm Interna. tianal. UK) nnd rcparntcd from hbcllrd nuclcohlcr using a Scphdcn 0.50 column. The lubcllcd probe WBI made up in lhc hybridization buffer (cxcludiny dcxtmn rulphau) and added ta the cxining buff.cr. Hybridiwulion was carried out for 16 h at 4X. Nonqccific hybrid. ixation WI: rcmovcd from the fihcn by the folloivinp washing procc. durcr: Z x SSC (0.3 M NuCWJOmM radium citnlc, p1-i 7.0) a~raom tcmpcraturc for 5 min; 0.5 x SSOI% SDS ;II 65’12 for 30 min; 02 x SSC al room tcmpcrclturc for 30 min. Each procedure WP carried out in duplicalc and ~lrcfillerr wcrc then laid down in ;luloradioyr;lphy cmctus wilh HypcrfWMP and stored al -70%‘. The length of exposure WUIarrcrscd using a Gcigcrmunler ulicr Ihc mcmbrancs had been washed, and cxposurctinic varied l’roni 3 h(l8 S rRNA) to 7dayr (insulin rcccpror. GLUTA). The lcvcl of mRNA that was dctcc.vd using ~hcrc praccdur*r was dctcrmincd by quantifying the inrcndty of the signal on the nutoradiograph usins a QUIPS imayc pro-ccssingwork+t;ction. opcra~ing with YCS image proccrrinp rofiwarc. The signals for Ihc mRNAr under scrutiny were calculakd rclativc 10 the lcvcl of It( S rRNA (optical density ratin) in each ample lane. This cnublcd correction for any diffcrcnccs in signal intensity Mt may bc due tn variution in the ini\i;\l loading of the RNA onto the gel. The optical dcnrhy ratio of rhc rcspcctivc ~RNAI in skclctul murclc of obcac Zuckcr ri+lz wcrc lhcn cxprcEccdrclalivc to values for their Icun coumcrparls (lOO%b).Qunn. litalion WY curried out on dupliciilc snmplrr from 5 animulx in each
154
3, RESULTS
Using Northern hybridiztion of total RNA from solcus and pluntfrris muscle of lean ;\nd obese Zuckcr ruts, mRNA species for the insulin rcccptor (72 and 8.5 kb) and For GLUT-4 (2.8 kb) were dctcctcd (Fig. 1). As observed in normal rodent spccics. two mirjsr bnnds wcrc obscrvcd For the insulin receptor mRNA. Insulin receptor mRNA WY qurmtificd by combining rhc opti= cul density of both bilnds. No significant chlmgcs in the lcvcl of GLUT-4 mRNA wcrc abscrvcd bctwccn the km and the obcvc ruts in the pluntnris ~sclc. ulthouyh the insulin receptor mRNA increased significimtly (Fig. 1) to 153%. In the solcus muscle significunt incrcilscs wcrc obscrvcd in the lcvcl of insulin rcccptor (189%) tend GLUT-4 (141%) mRNA spccics in the obcsc rats compared with their lci\n countcrports (Fig, 2), 4. DLSCUWGN The rcsutts demonstrate rrn incrcuscd lcvcl of insulin receptor mRNA in the solcus und the pl;lnt;rris muscles of obese Zuckcr rats when compurcd to lcirn ruts. In the solcus muscle the level of GLUT-4 mRNA WAS ills0 incrctrscd. The alterations in the Icvcl of insulin rcccptor mRNA in the plunttlris muscle appear to be rcltrtivcly specific as the conccntrutions oFGLUT- mRNA were unchungcd. In the light of previous observutions on the low numbcr of insulin receptors in muscle tissue of ycnctically obcsc Zucker milts[3.20]. it ilppcurs that the dccrcax in receptors mily bc due to altcrntions of trunslatian of the mRNA u&or to post-translutionul proscsscs, Although post-rcdeptor defects can ulso occur, the de= creased number of insulin receptors is likely to contribute to the insulin rcsistirncc of the skclcttll muscle tissue. An increase in the lcvcl of insulin receptor mRMA was also observed in the soleus muscle, suggesting thut a similar dcfcctivc translation process may bc operating. Howcscr, unlike the situation in the plantaris muscle. this w~ti arcompanird by nn increase in the conccntration of GLUT-4 mRNA. An incrcasc of similar magnitude has been observed in isolated cardiac myocytes from Zuckcr rats [8], with u dccrcasc in basal end inuulin-atimulitted trunsport. The incrctise in the level of
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Volume 308, number 2
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gcptcmbcr 1992
plentaris
insulin receptor
GLUT-4
(b) solcus
insulin receptor
GLU T-4
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L
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Fig. I. Northern hybridization of total RNA (40 ~g) extracted from (a) plantark and (b) solcun muscle usin8cDNA probes forthe insulinrcccplor and GLUT4 Results indiclltcd the specific dctcction of the mRNA specicr in both musflc typrs and the rclativc diffcrcnccs bctwc~n the 1-n and the obcr wts. For further technical details. see Materials nnd Methods.
GLUT-4 rnRNA my bc in rcsponsc to chungcs in circufuting insulin concentration. tis GLUT-4 flas been idcntiffcd in insulin rcsponsivc tissues [2fj. TIIC abscncc of any incrwsc in the planturis muscfc impI& the cxistcncc of diffcrcntiuf regulatory mechanisms that arc associated with the varying fibrc type of tfx two muscles [ 131.This observation is consistent with previous obscrvacions [‘,I which demonstrated that in a mixed fibrctype muscle (gastrocnemius), no change in the lcvcl of GLUT-4 mRNA was dctcctcd in the obese phenotype while changes were obssrvcd in udipocytcs from the obese animals. The rcquircmcnt for an incrcascd nurnbcr oi glucose trmsporkrs may nut bc as criticat in th pfuntaris as in the sofcus because the plantaris has a higher proportion of oxidativc fibrcs, Thcrc stiff rcmains a reduction in the ovcrulf glucose utilization of
muscle tissue from genetically obcsc Zuckcr rats [2] whicfz may bc mediated througfl defects in other pathways. Insulin rcgufatcs the levels of insulin receptors [22] and insulin rcccptor mRNA [23]. in vitro. It has also been shown that acute nutritional changss [24-261 significantfy incrcascs the lcvcl of insulin rcccptor mRNA in skcfctaf muscle, possibly through incrcascd transcription of the insulin receptor gene or insrcasc in the stubility of the mRNA itself. Since the abesc Zuckcr rats arc charactcristicalfy hypsr-insufinacmis, the changes in insulin receptor mRNA in their muscle may bc a rcsponsc to incrcascd lcvcfs of circuhfing insdin. In summary, an increased levcf of insulin receptor mRNA has been detected in the plantaris and the solcus muscle of gcncticalfy obcsc Zucke: rats. Thcrcforc. the
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Volume 309. number 1
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1992
REFERENCES [I) DcFronxo. R.A..
lnaullt’t rooogtor
BUJTd
Plnntarie
inrulln
QLtJTd
rocqator SQl@tJ~
Fig. 2. Quantitation of insulin rcaptor and GLUT4 mRNA lcvcls in the pluntnrir ttnd the r&w tnurlc of lean(L) und ohc (0) Zuckcr ruts. A rigniliwnt instrasc (‘P c 0.05) in rhc lsvcl of insulin receptor mRNA wus dctatcd in 1hcpltlntarir murclc ofobesc Zuekcr rais when compared lo Ican coumcrpttrts. No ri~oifiwnt chanpr wcrc observed in lhc GLUT.4 mRNA in this muscle. Signitkzanl incrctttir wcrc obrcrvcd in the rolcur muscle for the insulin rcccptor mRNA (“‘PcO.001) and GLUT.4 mRNA (“Pe 0.01). The lcvcl of mRNA in the murclcsamplcs from the lcun rats is shown as 100%and 1hc changer 1hat arc obssrvcd are indicated rclutivc to 1hir.
dccrcuscd number of insulin rcccptors in the muscle tissue of obese 2ucktr rats cnnnot bc simply cxplaincd by reduced amounts of availi\blc mRNA, On the con= trury this suggests both that thcrc is udditional control ut translational and/or post-translational lcvcls and that the increased mRNA lcvcls rcflcct a compcnsutory mechanism which is attempting to ovcrcomc the tissue’s resistance to insulin. Increases in the lcvcl of GLUT-4 mRNA wcrc also evident in the solcus nlusclc but not in the plantnris rnueclc. This may indicate specific diffcrcnccs thnt arc rrssocintcd with the fibre types of the muscles or a gsncralizcd enrichment of the mRNAs in the solcus muscle. Ac~~~o~vkr~~a~tc~trr: WC would like 10 1hank Mr. T. Atkinsott for his 1echnical assistance with the insulin ttrruyr and Dr. A. Travis for the provirion of the compulcr program for the quuntitaticn of mRNA lcvclr an 1hcnuloradiographs. ThccDNA prober for the insulin rcccptor and GLUTS wcrc donated by Drr. Lclrnd Ellis and Gwyn Gould, rcspcstivcly.
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Fcrranninni, E.. SUIO,Y., FcliB, P. and Wlrhmn. J, (1981) 1. Clin. Invcsl. 61, 14611-1474. I21 C;rcch. M.P., Richardson. D.K.. Becker, S.G., Wnltcn. C.G.. Gitomcr. W. and Hcnrich. J. (1978) Metdbolirm Ciin. Exp. 27. 1967-1972. 131Crc1lnx. M. and Jcanrcnaud. B, (1980) Metabolism 29,467-473. [41 Tcrrcttuz J., Auimacopnulos~Jcttnnct, F. and JcPnrcnaud, B. (1986) Endosrinol. I lb. 674-678. [SlShrrrgil. N.S., Ta1oyan, A., El~Rcfni. M-F,, Plciu. MI. and Chnn, T.M. (1986) Biocltem. Biophys. Rcr. Commun. 137.286-294. I61 Eaaki, 0, (1~19) Diabctologia 32, 290-294. [71 Gucrrc*Millo, AI., Lavnu. M., Hornc, J.S. und Wardmtlu. L-J, (19X5) J. Biol. Chcm. 260.2197-2201. [a] Eckcl, J.. Rub, M.. Pctcrscn, S.. Hcrbcrg. L. and Rcinnucr. H. (1990) Abnruct, IV lntcrnntionttl Symposiumon Insulin Rcscp ior and lnrulin Action: Molcrular and Clinical Aspcetr. Vcronn, Imly. 67-68. [91 Hninnulr, t., GucrreMillo, M., Guichard. C. ttnrl Luv~I~. M. (1991) 1. Chit. Inucsl. &7. 1127-l 131. [IO] SOILA.H.. Kahn, CR.. Ncvillc Jr., D.M. und Roth. J. (1975) J. Clin. Invest. 56, 769-780. [I I] Ludwiy. S.. Mullcr.Wicland, D.. Goldstein. B.J. and Kahn, CR. (1981) Endocrinology 123. 594-6GQ. [12] PPniwud, L.. Rohncr~Jcanrcnaurl. F. und Jcunrcnaud. U. (1916) Am. J. Physiol. 250. E662-E66b. [13] Catnpion, D.R.. Shapira, J.F.. Allen. GE.. Haustnttn. G.J. and Mur1in, R,J, (19lt7) Growth 51, 397410. II41 Sherman. W.M.. Katz. A-L., Cullcr.C.L., Withcn. R.T. and tvy, J.L. (1918) Am. 1. Phydol. 255. E374-E382. [ 151Chomca~nrki. P. and Sacchi. N. (1987) Anal. Biochcm. 162. l56159. (161 Sumbrook. J.. Fritrb. E.F. and Muttia1is. T. (1989) (2nd cdn.) Cold Spring Harbor tboraory Press, New York. 1171 Ebina. Y.. Ellis, L,. Jttrnagin, M.. Edcty, M,, Ciraf. L., Cl;rrrs.sr. E.. Ou. J-H., Masian. F.. Kun, Y.W.. Goldfine, I.D.. Roth. R.A. and Rutrcr. W.J. (1985) Cell 40. 747-758. [la] ptcc DOE., 8rubc. S. and Muccklsr. M. (1989) Nrriurc 338. [I91 Erickion. J.M.. Rushford, CL.. Dorncy. D.J.. Wilson, G.N. and Sshmickcl, R.D. (1985) Gcnc 16, l-9. 1201 Lc Murchund&urrcl, Y., Gordcn. P.. Flier. J.S.. Knhn, CR. nnd Frcyclict, P. (19%) Di&etclogiu 14. 31 l-317. [?I] Gould,G,W. and Ucll, 0. (1990)Trcnds Diochcm. Sci. IS. 1X-13. I221 Gavin, J.R.. Roth J.. Ncvillc, D.M., DC Mcytr. P. and l3usll, D.N. (1974) Proc. Null. Acad. Sci. USA 71. &I-88. [23] Levy, J-R,, Krystal, G., Glickman, P. und Dastvan. F. (1991) Diabetes 40, 58-65. [24] Knoll, R-M.. Hnkcth. 1-E. und Trayburn, P. (1990) Biochcm. Soc, Trans. 18, 1263. 1251Knott. R.M.. Gran1. G.. Bardoca, S,, Puaetui, A., DC Curvulho, A.F.F.U. and Hrrkc~h. J.E. (1992) ht. J. Biochcm. 24.897-902. [26] W~~lliicc,S., Cutnpbcll, G., Knott. R.M., Gould, G.W. and Hcs. kcth, I.E. (1992) FEDS 301, 69-72.
