Dabetologa (1996) 39:758-765 Dabetologa 9 Sprnger-Verlag1996 Orgnals Long-term and rapd regulaton of ob mrna levels n adpose tssue from normal (Sprague Dawley rats) and obese (db/db mce, fa/fa rats) rodents M Igel 1, H Kanulanen 2, A Brauers 1, W Becker 1, L Herberg 1, H-G Joost 1 1Insttute of Pharmacology and Toxcology,RWTH Aachen, Aachen, Germany 2 Insttute of Medcal Technology,Unversty of Tampere, Tampere, Fnland 3Dabetes Research Insttute, Dasseldorf,Germany Summary Increased levels of m R N A transcrbed from the ob gene n adpose tssue of obese/hypernsulnaemc Zucker (fa/fa) rats were detectable as early as 3 weeks after brth and contnued to rse there after n parallel wth body weght and serum nsuln m R N A levels of two other fat-specfc genes (ARL4, FST44) were unaltered In C57BL/KsJ db/db mce, ob m R N A levels also ncreased n parallel wth body weght and serum nsuln, and remaned elevated n older anmals when nsuln levels decreased In heterozygous control anmals (db/+ ; fa/fa), m R N A levels were comparable wth those n the homozygous controls In normal Sprague Dawley rats, the ob m R N A ncreased contnuously, but more slowly than n Zucker rats, n parallel wth body weght and nsuln levels, and reached 15 tmes hgher levels n the heavest rats (400 g) studed In Sprague Dawley rats made dabetc by an njecton of streptozotocn, ob It has long been assumed that food ntake s regulated by the adpose tssue mass through a feed-back mechansm whch nhbts or stmulates hypothalamc nucle [1] Recently, ths hypothess has been proven by postonal clonng of a sngle gene mutaton n the C57BL/6J ob/ob mouse stran [2], the obese mutaton (ob), whch causes a syndrome of hyperphaga and extreme obesty accompaned by numerous metabolc and hormonal alteratons Ths gene encodes a proten, tentatvely desgnated leptn, whch appears Receved: 27 October 1995 and n revsed form: 5 December 1995 Correspondng author: Dr HG Joost, Insttut ftr Pharmakologe und Toxkologe, Medznsche Fakult~t der RWTH Aachen, Wendlngweg2, D-52 057 Aachen, Germany m R N A levels were reduced by approxmately 50 % after 24 h A 24-h fastng perod reduced the ob m R N A by 50 % n lean Sprague Dawley and Fa/Fa, but not n obese Zucker fa/fa rats, although nsuln levels were reduced n both groups These data ndcate that ob m R N A levels ncrease n both normal and obese rodents n parallel wth age, body weght and serum nsuln, reflectng an early (Zucker rats, db-mce) or slowly developng (Sprague Dawley rats) resstance to leptn and nsuln Ths ncrease does not appear to be medated by the recently descrbed rapd regulaton of ob m R N A by nsuln, but seems to be due to a dfferent, long-term control mechansm whch sgnals the sze of the fat depots [Dabetologa (1996) 39: 758-765] Keywords Leptn, ob gene, gene expresson, nsuln resstance, obesty to be secreted from adpose tssue In the obese ltter mates (ob/ob) of the C57BL/6J stran, a nonsense mutaton n codon 105 of the ob gene generates an nactve proten whch s responsble for excessve overeatng, obesty and secondary metabolc alteratons, eg a marked nsuln resstance Furthermore, m R N A levels of the ob gene are 20 tmes hgher n the obese mce than ther lean ltter mates, ndcatng that the lack of actve leptn produces a counterregulatory ncrease n ts gene expresson More recently, t has been shown that wld-type leptn ndeed reduces food ntake n ob/ob mce [3-5] Thus, leptn appears to be the crucal medator n the feed-back control between adpose tssue and feedng behavour Data from obese patents ndcate that ob m R N A levels are ncreased n approxmate proporton wth
M Igel et al: Long-term and rapd regulaton of obmrna levels 759 the body mass ndex [6] So far, n none of these patents has a mutant leptn gene been found, suggestng that the ncrease n ob mrna levels s secondary to a reduced senstvty to leptn Increased levels of leptn mrna have also been found n two strans of obese rats, the Zucker rat fa/fa and the Otsuka Long Evans Tokushma fatty (OLETF) rat [7] The cdna sequence of leptn was normal n these strans, and nether of them carres a mutaton near the ob locus Thus, ther obesty appears to reflect a malfuncton of the adpose tssue/ventromedal hypothalamus (VMH) feed-back regulaton, possbly due to a defectve leptn receptor A smlar concluson has prevously been drawn for the db/db mouse on the bass of paraboss experments whch ndcated that the obese anmals produced an excess of an anorexgenc factor whch could nhbt overeatng of the ob/ob mouse [8] Recently, ths assumpton was supported by the fndng that mrna levels of leptn were markedly elevated n the db/db mouse [9] The dscovery of leptn has rased several mportant questons related to ts functon and also to the regulaton of ts synthess Therefore, we have studed mrna levels of leptn n adpose tssue from morbdly obese (C57BL/KsJ db/db, KK, NZO mce, Zucker fa/fa rats) and normal rodents (Sprague Dawley rats) n order to descrbe both long-term and rapd alteratons Based on the assumpton that a defectve leptn receptor n db/db mce and Zucker rats gves rse to elevated ob mrna levels, we studed the tme course of ths elevaton durng the development of the metabolc syndrome n order to determne ts onset and to correlate t wth other parameters In order to evaluate a possble rapd regulaton of the ob gene expresson, the effects of fastng and streptozotocn dabetes were studed The data ndcate that leptn mrna levels ncrease n obese rodents n parallel wth age, body weght and serum nsuln, reflectng an early (Zucker rats, db-mce) or slowly developng (Sprague Dawley rats) resstance to leptn and nsuln Furthermore, the data suggest that ths ncrease does not appear to be medated by the recently descrbed rapd regulaton of ob mrna by nsuln [10, 11], but seems to be due to a dfferent, long-term control mechansm whch sgnals the sze of the fat depots Materals and methods Anmals Mutant strans (C57BL/KsJHl-db/db, ZuckerHl-fa/ fa, NZO, KK, NON) were bred n the Dabetes Research Insttute, Dsseldorf Sprague Dawley rats were bred n the colony of the Insttut ftr Versuchsterkunde, Aachen All anmals were fed a standard laboratory det and had free access to food and water Homozygous and heterozygous db/db mce and Zucker rats were dentfed by ther breedng record Sprague Dawley rats were rendered dabetc by a sngle njecton of streptozotocn (65 rng/kg) as descrbed prevously [12] The "Prncples of laboratory anmal care" (NIH publcaton No 85-23, revsed 1985) were followed, and approval for the anmal experments, where necessary, was obtaned from the Insttuton of Anmal Care and Use at the Unversty of Tampere, Fnland, and the Regerungspr~sdent Dsseldorf, Germany Preparaton of RNA Anmals were klled by decaptaton, and subcutaneous, perrenal and gonadal fat was dssected and mmedately frozen n lqud ntrogen The samples were homogenzed wth a Polytron homogenzer n 4 mo[/1 guandne thocyanate supplemented wth 7% mercaptoethanol Lysates were layered on a cesum chlorde cushon (588 mol/1) and centrfuged at 28 000 rev/mn (rotor SW40) for 29 h at 20~ Pelleted RNA was dssolved wth 300 ~tl sodum acetate/trs buffer, and was neutralzed by addton of 50 ~tl 2 mol/1 potassum acetate (ph 55) Preparaton of a partal ob cdna Total RNA was solated from 3T3-L1 cells as descrbed prevously [13], and 486 base pars of the codng regon of the ob gene were amplfed by polymerase chan reacton (PCR) wth total RNA from dfferentated 3T3-L1 cells [13] as the template (upstream prmer 5'-CTG TGT CGG TTC CTG TGG-3'; reverse prmer 5'- GCA TI'C AGG GCT AAC ATC-3') The reacton product was separated on agarose and subcloned nto the SmaI ste of puc19 (Sureclone kt: Pharmaca; Freburg, Germany) Plasmd DNA was prepared and dgested wth BamHI/EcoRI n order to solate the ob cdna Northern blot analyss Samples of total RNA (15 ~tg) were separated by electrophoress on 1% agarose gels contanng formaldehyde and transferred onto nylon membranes (Hybond N + ; Amersham-Buchler, Braunschweg, FRG) Before transfer, gels were staned wth ethdum bromde n order to ascertan that equal amounts of total RNA had been separated Probes were generated wth the Klenow fragment of DNA polymerase I and [c~-32p]dctp from the partal ob cdna by random olgonucleotde prmng [14] The nylon membranes were hybrdzed at 42~ and blots were washed twce wth 012mol/1 NaC1/0012mol/1 sodum ctrate/01% SDS and once wth 0015 mol/1 NaC1/00015 mol/l sodum ctrate/01% SDS Autoradographs were analysed wth the LKB2400 laser denstometer and the software GelScan XL 20 from Pharmaca Other assays Serum mmunoreactve nsuln was assayed wth RIA kts from Pharmaca Purfed rat nsuln (Novo Research Insttute, Bagsvaerd, Denmark) was used as standard Blood glucose was determned by an automated glucose oxdase method (Care Dagnostca, Voerde, Germany) Results Elevated levels of mrna of the ob gene n four strans of obese/hypernsulnaemc mce Northern blot hybrdzaton of a mouse ob cdna probe wth total RNA from adpose tssue yelded the antcpated sngle band at 45 kb [2] Very low levels of ob mrna were detected n lean control mce (KsJ +/+, homozygous controls of the C57BL/KsJ stran) As was reported prevously [9], mrna levels were approxmately 10 tmes hgher n the obese ltter mates (db/ db, Fg 1) Hgh levels of leptn mrna were also
760 M Igel et al: Long-term and rapd regulaton of obmrna levels Fg 1 Northern blot analyss of ob gene expresson n adpose tssue from dfferent strans of obese mce Total mrna from adpose tssue of the ndcated mouse strans (C57BL/KsJ, KK, NZO, NON) was prepared as descrbed and separated on agarose Northern blots were probed wth ob and ARL4 cdna, and the ntenstes of the 45 and 12 kb band (upper panel), respectvely, were assayed by laser denstometry of autoradographs (lower panel) The data represent means + SD of samples from three dfferent anmals found n KK and NZO mce, whereas the NON stran showed a less pronounced ncrease (Fg 1) Note that n KK, NZO and NON mce, the obese-hyperglycaemc syndrome s beleved to be polygenc, and that normal congenc lnes are not avalable for comparson Thus, the lean ltter mates of the db-mce (KsJ + / + ) have to be used as controls for the polygenc mutants Snce the data as presented n Fgure 1 are normalzed per total RNA, the possblty has to be consdered that obesty s paralleled by an ncrease of all mrna speces, or of the fat-specfc mrna speces, n relaton to the rbosomal RNA Thus, n order to demonstrate the specfcty of the observed ncrease, we assayed the levels of two other fat-specfc transcrpts whch were recently cloned n our laboratory: the ras-related GTPase ARL4 [13] and a partal cdna sequence desgnated FST44 (Becker et al, unpublshed data) As s llustrated n Fgure 1, levels of ARL4 mrna were somewhat varable, but were not sgnfcantly elevated n any of the obese/hyperglycaemc mce Smlarly (data not shown), FST44 mrna levels were unaltered n the obese anmals Table 1 summarzes the characterstcs of the obese/hypernsulnaemc anmals n order to dentfy parameters whch mght predct, and thus be lnked to, the elevaton n ob mrna levels There was no apparent correlaton between blood glucose or nsuln levels and ob mrna, snce both parameters were normal n the NON mce n spte of a three-fold ncrease n ob mrna In ths expermental seres, the best predctve parameter for the elevaton of the ob gene expresson appeared to be the body weght, e the adpose tssue mass, of the anmals It appears reasonable to assume that the ncrease n the ob gene expresson n the db mce reflects a resstance to ts product, leptn, and s a consequence of the hyperphaga and/or nsuln resstance Based on ths assumpton, we expected to fnd normal levels of leptn mrna n heterozygous controls, snce they exhbt a normal phenotype as far as hyperphaga and nsuln levels are concerned Indeed, as s depcted n Table 2, mrna levels n the heterozygous anmals (db/+ ) were smlar to those n the homozygous contros The small and nsgnfcant dfference was probably due to the greater age and weght of the heterozygous controls Thus, the normal allele can fully compensate for the effect of the defectve gene on obesty and ob gene expresson Furthermore, smlar data were obtaned n the obese/hypernsulnaemc Zucker rat (fa/fa) In these rats, t has been prevously shown that ob mrna levels are ncreased as compared wth lean controls [7] The data depcted n Table 2 extend these fndngs by showng that leptn mrna levels are comparable n homozygous and heterozygous controls Tme course of the ncrease n ob mrna levels n C57BL/KsJdb/db mce In order to further nvestgate the relaton between the ob gene expresson, body weght, blood glucose and serum nsuln levels, these parameters were determned n db/db mce of dfferent ages (3, 4, 6 and 18 weeks) As s shown n Fgure 2, ob mrna levels were sgnfcantly Table 1 Characterzaton of the dfferent strans of obese mce wth regard to age, body weght, blood glucose and nsuln levels KsJ +/+ KsJ db/db KK NZO NON Body weght (g) 275 + 24 524 + 25 361 + 10 566 + 23 349 + 16 Age (weeks) 14 16 14 15 25 Blood glucose (mmol/1) 83 + 10 265 _+ 49 146 + 43 178 + 57 74 + 04 Serum nsuln (pmol/1) 780 + 34 2053 + 71 7600 + 553 4653 + 244 727 + 26 Ob mrna (arbtrary unts) 04 + 01 37 + 05 44 + 06 34 + 02 12 + 05 Data represent means + SD for three anmals
M Igel et al: Long-term and rapd regulaton of obmrna levels 761 Table 2 Levels of ob mrna n obese anmals, homozygous and heterozygous lean ltter mates of db mce and Zucker rats Body weght (g) Age (weeks) Blood glucose (mmol/l) Serum nsuln (pmol/1) Ob m R N A (arbtrary unts) KsJ +/+ KsJ db/+ KsJ db/db Fa/Fa fa/fa fa/]a 313 22 73 693 20 356 _+15 27/32/38 71 _+12 1040 + 26 26 + 20 556 20 255 1680 83 280 19 65 173 16 306 29 48 193 26 463 20 108 3047 64 + 07 + 19 + 25 + 01 _+ 154 _+37 + 76 + 09 _+22 _+ 17 + 27 + 05 +% _31 _+07 _+15 _+22 _+29 _+06 _+94 _+06 The data r e p r e s e n t m e a n s + SD of three anmals 7- {n~ 6-~ ~ A 1000- I / ~- ~<~, 4- _ ~0 t ' 4 W2 + a I_ 800 J ~'~4006004 Te~ + ++ 50 60t oa 401 30 3, 4, 6 18 I t ++ ++ :l n3 +I 4 6 + ' I Weeks ncreased at the earlest tme pont analysed (3 weeks) Thus, the effect appears to represent a very early event n the development of the metabolc dsturbances n the db mouse Smlarly, hypernsulnaema and consequently nsuln resstance were detectable at the earlest tme pont analysed, m R N A levels of leptn contnued to rse thereafter (Fg 2, panel B) and reached a plateau at 18 weeks Insuln levels, n contrast, were markedly decreased due to the exhauston of the slet organ [15] n the older anmals (18 weeks) Blood glucose levels were normal at 3 and 4 weeks, and were sgnfcantly ncreased there after As was antcpated from the prevous experments, levels of A R L 4 were unaltered throughout (data not shown) Tme course o f ob m R N A levels n adpose tssue from Zuckerfa/fa rats Fgure 3 llustrates a tme course for the development of obesty and m R N A levels of leptn n relaton to the hypernsulnaema n Zucker rats As n the db mce, m R N A levels of leptn were sgnfcantly elevated at the earlest tme pont analysed (3 weeks) and contnued to rse thereafter In parallel, hypernsulnaema developed n the obese rats (Panel B) Unlke the db mce, the Zucker rats had normal blood glucose levels throughout (panel D) as has been reported prevously [16] Fg2 A-D Ob mrna-levels n adpose tssue from C57BL/KsJ db/db mce of dfferent ages compared wth the alteratons ~_ z=4ot t +'3+ B 18 n body weght, blood glucose, and serum nsuln levels Total RNA from obese anmals (db/db) 9 and lean ltter mates ( + / + ) [] of the ndcated age was solated and probed as descrbed n the legend of Fgure 1 Each data set are means + SD of samples from three dfferent anmals Weeks Relaton between body weght and leptn m R N A levels n adpose tssue o f Sprague Dawley rats Agng Sprague Dawley rats develop obesty and a moderate hypernsulnaema, and have prevously been used as a model of nsuln resstance [17] Therefore, we studed adpose tssue from normal Sprague Dawley rats of dfferent weghts As s llustrated n Fgure 4, there was an almost proportonal ncrease n ob m R N A levels wth body weght Smlarly, levels of serum nsuln ncreased roughly n parallel wth the body weght (Table 3) Both ob m R N A and nsuln levels n the hghest weght group (400 g) were stll consderably lower than those n 300 g Zucker rats It should be noted, however, that a drect comparson of age and weght between Sprague Dawley and Zucker rats s napproprate because of ther largely dfferent growth rate As s shown by a comparson of the body lengths at 13 weeks, Sprague Dawley rats grow much faster than the Zucker rats (Zucker rats, 175 cm; Sprague Dawley rats, 30 cm) Effects o f fastng on leptn m R N A levels n adpose tssue o f normal and Zucker fa/fa rats Recently, t was descrbed that fastng produces a rapd decrease of leptn m R N A levels n rat adpose tssue whch was probably medated by a decrease n the serum nsuln [10] Confrmng ths fndng, we obtaned smlar
762 M Igel et al: Long-term and rapd regulaton of obmrna levels Fg 3 A_-D Ob mrna-levels n adpose tssue from Zucker (fa/fa) rats of dfferent ages compared wth alteratons n body weght, blood glucose and nsuln levels Total RNA from obese anmals (fa/fa) 9 and lean ltter mates (Fa/Fa) [] of the ndcated age was solated and probed as descrbed n the legend of Fgure 1 Each data set are means + SD of samples from three dfferent anmals Note, however, that the absolute levels n the obese anmals were much hgher than those n the lean controls As antcpated, fastng resulted n a reducton of serum glucose and nsuln levels n both normal and obese anmals Fg 4 Ob mrna levels n adpose tssue from Sprague Dawley rats of dfferent ages and body weghts Total RNA was solated from anmals of the ndcated weght and probed as descrbed n the legend of Fgure 1 Each data set are means + SD of samples from four dfferent anmals data wth the Sprague Dawley rats after a 24-h fastng perod: mrna levels of leptn were decreased by 60 % (15 + 08 vs 399044 n the controls, mean + SD arbtrary denstometry unts), whereas those of ARL4 were unaltered (026 + 002 n both groups) The same experment was performed wth obese Zucker rats and ther lean controls (Fg 5) Surprsngly, fastng faled to sgnfcantly affect leptn mrna levels n the obese anmals, whereas the expected 50 % decrease was seen n the lean controls Effect of streptozotocn-dabetes on ob mrna levels n adpose tssue of normal rats In order to further characterze the relaton between serum nsuln and ob mrna levels, the effect of an expermental reducton of nsuln levels by the dabetogenc agent streptozotocn was studed Streptozotocn reduced ob mrna levels by approxmately 65 % (Fg 6, upper panel), whereas the levels of mrna of ARL4 were unaltered Smlar data demonstratng a marked decrease n leptn mrna levels 5 days after njecton of streptozotocn were very recently publshed by others [11] Extendng these fndngs, the lower panel of Fgure 6 llustrates a tme course of the effect of streptozotocn Rats were rendered dabetc by njecton of the agent, and ob mrna levels were assayed 1, 2, 4, 8 and 16 days after the njecton The ob mrna was reduced as early as 1 day after njecton, n parallel wth the steep ncrease n blood glucose whch reflects the abolton of nsuln secreton from the endocrne pancreas No further decrease n ob mrna was observed after the frst day of dabetes Thus, the experment revealed a relatvely rapd regulaton of ob mrna n response to the streptozotocn dabetes Dscusson The role of leptn as a major regulator of food ntake has recently been establshed convncngly by the fndng that exogenously appled leptn reduces the
M Igel et al: Long-term and rapd regulaton of obmrna levels 763 Table 3 Metabolc characterstcs of Sprague Dawley rats of dfferent age SD SD SD SD fa/fa Body weght (g) 100 200 300 400 300 Age (weeks) 4 6 8 13 13 Blood glucose (mmol/1) 78 + 02 88 _+ 03 77 + 01 86 + 13 66 + 14 Serum nsuln (pmol/1) 100 + 07 320 + 60 280 + 27 613 _+ 167 2300 +_ 121 Ob mrna (arbtrary unts) 04 + 04 09 _+ 01 20 + 01 42 + 06 69 _+ 02 ARL4 mrna (arbtrary unts) 05 + 01 03 _+ 001 03 + 002 05 +_ 003 02 _+ 001 Data are means +_ SD of three anmals Fg5 A-C Effect of fastng on serum nsuln, blood glucose and ob mrna n adpose tssue from obese Zucker rats (fa/fa) and lean ltter mates (Fa/Fa) Total RNA was solated as descrbed n the legend of Fgure 1 and probed wth mouse ob cdna The data represent means _ SD of samples from three dfferent anmals; note the dfferent ordnate scale on the rght sde of panel A Control, 9 ; 24-h fastng, [] food ntake n obese (ob/ob) and normal mce [4] In addton, the fndng that mrna levels of leptn n mce carryng the nonsense mutaton are markedly elevated suggested a counterregulatory control va the ob gene expresson [2] Extendng these earler fndngs, the present data ndcate that mrna levels transcrbed from the ob gene are regulated by longterm adaptaton, n apparent correlaton wth the sze of the fat depots, as well as through rapd control mechansms n normal and obese rodents In proporton to the degree of obesty, marked ncreases n mrna levels were observed n several heredtary forms of early obesty as well as n moderately obese Fg 6 A-C Effect of streptozotocn dabetes on mrna levels of leptn n epddymal adpose tssue Male Sprague Dawley rats were rendered dabetc by a sngle njecton of 65 mg/kg streptozotocn Total RNA was solated 1 week after the njecton (A, B) as descrbed n the legend of Fgure 1, and was probed wth ether mouse ob (m-ob) or rat ARL4 (r-arl4) cdna The data are means + SD from three anmals C" Tme course of the effect of streptozotocn Total RNA was solated at the ndcated tme ponts after njecton of streptozotocn, and was probed wth mouse ob cdna Each data pont was obtaned wth at least two anmals Sprague Dawley rats Thus, an ncrease n the ob gene expresson and consequently resstance to leptn not only occurs n rare mutants of early obesty but also durng the course of agng n 'normal' rodents These fndngs are compatble wth the prevous
764 M Igel et al: Long-term and rapd regulaton of obmrna levels hypothess [2] that leptn, the product of the ob gene, s a crucal component of a feed-back mechansm controllng body weght through feedng behavour, and that t s the sze of the adpose tssue stores that controls the feed-back nhbton of food ntake The present data and two recent studes [10, 11] ndcate that leptn mrna levels are also rapdly regulated as n fastng or streptozotocn-treated anmals, presumably through alteratons of the serum nsuln levels Blood glucose levels can be ruled out as a factor regulatng the expresson of the ob gene, because the tme course of the metabolc syndrome n the db/ db mouse ndcated that hyperglycaema emerged much later than the ncrease n leptn mrna levels In addton, fastng and streptozotocn produced smlar effects on leptn mrna levels, but opposte effects on serum glucose By the same token, we beleve that serum lpds whch are elevated n streptozotocn-dabetc anmals are unlkely canddates for the regulaton of the ob gene expresson Insuln levels, however, were altered n parallel wth mrna levels of leptn n fastng and streptozotocn dabetes Thus, t appears safe to conclude that nsuln, or a parameter of adpocyte metabolsm controlled by nsuln, medates the short-term regulaton of the ob gene expresson Two key fndngs of the present study suggest that the long-term ncrease n ob mrna levels observed n the obese rodents can be dssocated from alteratons of the serum nsuln levels Leptn mrna levels remaned elevated n older db/db mce n spte of a dramatc decrease n serum nsuln Secondly, fastng decreased serum nsuln levels n Zucker rats but faled to produce a reducton n leptn mrna levels proportonal to that seen n the controls Thus, we suggest that the age and weght-dependent ncrease n leptn mrna levels n the obese rodents s unrelated to the rapd regulaton exerted by serum nsuln levels, and reflects an addtonal long-term control mechansm whch sgnals the sze of the fat depots Both db mce and Zucker rats represent models for the study of obesty and nsuln resstance [18-20] It s well establshed that these strans carry mutatons n homologous genes [21] and that dfferences n the severty of the dabetes are due to the dfferent genetc backgrounds of these mutants It appears reasonable to conclude on the bass of the present and prevous data [8] that both strans are resstant to leptn and develop hyperphaga and obesty because of ths defect, and that the contnuous ncrease n the ob gene expresson s secondary to the leptn resstance Consequently, one would have to conclude that all other metabolc alteratons observed n the obese anmals, n partcular nsuln resstance, are secondary to hyperphaga The present data ndcate, however, that the marked elevaton n mrna levels of leptn n the obese ltter mates started at an early age at whch hyperphaga s hardly notceable Hypernsulnaema, e nsuln resstance, appeared to start at a smlar tme pont and roughly paralleled the ob gene expresson thereafter Thus, 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