Effect of age and moderate food restriction on insulin sensitivity in Wistar rats: role of adiposity

Transcription

1 131 Effet of ge nd moderte food restrition on insulin sensitivity in Wistr rts: role of diposity Fernndo Esrivá, M Luí Gvete, Ysmín Fermín 1, Corli Pérez 1, Nild Gllrdo 2, Crmen Alvrez, Antonio Andrés 2, Mnuel Ros 3 nd José M Crrsos 1 Deprtmento de Bioquími, Fultd de Frmi, Universidd Complutense, Mdrid, Spin 1 Centro de Biologí Moleulr Severo Oho, Fultd de Cienis, Universidd Autónom, Cmpus de Cntolno, Mdrid, Spin 2 Are de Bioquími, Fultd de Químis, Centro Regionl de Investigiones Biomédis (CRIB), Universidd de Cstill L Mnh, Ciudd Rel, Spin 3 Fultd de Cienis de l Slud, Universidd Rey Jun Crlos, Alorón, Mdrid, Spin (Requests for offprints should e ddressed to J M Crrsos; Emil: jmrrsos@m.um.es) Astrt Insulin resistne develops with geing in humns nd rodents. Here, we hve studied the evolution of insulin sensitivity with geing trying to disriminte the role of diposity from tht of geing in this proess. We performed orl gluose tolerne tests nd determined overll nd tissue-speifi gluose utiliztion under euglyemi-hyperinsulinemi onditions in 3-, 8-, nd 24-month-old rts fed d liitum, nd in 8- nd 24-month-old rts fter 3 months of lorie restrition. Body omposition nd dipoytederived ytokines suh s leptin, resistin, nd diponetin were nlyzed. Overll insulin sensitivity dereses with geing. Clorie restrition improves glol insulin sensitivity in 8- ut not in 24-month-old rts. Insulin-stimulted gluose utiliztion in dipose tissues dereses in 8 months, while in oxidtive musles it rehes signifine only in older rts. Clorie restrition restores dipose tissue insulin sensitivity only in 8-month-old rts nd no hnges re oserved in musles of 24-month-old rts. Resistin nd leptin inrese with geing. Food restrition lowers resistin nd inreses diponetin in 8-month-old rts nd dereses leptin in oth ges. Viserl nd totl ft inrese with geing nd derese fter lorie restrition. We onlude tht retion of viserl ft plys key role in the development of insulin resistne fter sexul mturity, whih is reversile y lorie restrition. With ging, umultion of retroperitonel nd totl ody ft leds to impired musle gluose uptke nd to stte of insulin resistne tht is diffiult to reverse. Journl of Endorinology (2007) 194, Introdution Ageing is ssoited with moderte derese in peripherl insulin sensitivity in humns (De Fronzo 1981, Fink et l. 1983, Rowe et l. 1983) nd rodents (Goodmn et l. 1983, Nrimiy et l. 1984, Nishimur et l. 1988, Esrivá et l. 1997). It is lso ssoited with hnges in ody omposition, espeilly in dipose tissue, mking it diffiult to evlute the role of geing y itself in the impirment of insulin tion. Thus, while severl reports hve shown tht deresed insulin sensitivity ours in non-oese elderly humns (Fink et l. 1983, 1986), some hve onluded tht insulin resistne in the elderly derives from hnges in ody omposition due to ft umultion (Ferrnnini et l. 1996, Bsu et l. 2003). In this sense, it hs een desried tht speifi removl of epididyml nd perirenl ft in 15-month-old F344/Brown Norwy rts prevents the development of geing-ssoited insulin resistne (Griely et l. 2002). On the other hnd, dipose tissue is known to serve s n endorine orgn tht liertes severl dipokines whih ould modulte insulin tion on trget tissues (Ahim & Flier 2000, Frühek et l. 2001). Therefore, we suggest tht with geing, the evolution of diposity nd different types of dipose tissue nd their insulin sensitivity proly ply key role in the development of insulin resistne in different tissues nd in the whole niml. Chnges in diposity y lorie restrition should modulte this insulin resistne. Aged Wistr rts present lower gluose disposl rte during euglyemi-hyperinsulinemi lmp thn mture young rts, without hnges in fsting plsm gluose nd insulin onentrtions (Nishimur et l. 1988, Esrivá et l. 1997), demonstrting stte of insulin resistne. However, this derese in insulin sensitivity, fr from eing homogeneous, is tissue speifi. Thus, while white dipose tissue, diphrgm, nd soleus musle present ler derese in gluose metoli index under hyperinsulinemi onditions, other skeletl musles like qudrieps remin insulin sensitive Journl of Endorinology (2007) 194, DOI: /joe /07/ q 2007 Soiety for Endorinology Printed in Gret Britin Online version vi Downloded from Biosientifi.om t 11/17/ :37:46PM vi free ess

2 132 F ESCRIVÁ nd others. Role of diposity on insulin tion in geing with geing (Esrivá et l. 1997). In greement with the resistne oserved in dipose tissue, isolted dipoytes from ged rts show deresed insulin response (Crrsos et l. 1989, Molero et l. 1998, 2002, Villr et l. 2006). Here, we hve investigted the evolution of insulin sensitivity with geing in dipose tissue nd musle nd lso the influene of diposity on this proess. In order to do this, we ompred the overll nd tissue-speifi insulin sensitivity of 3-, 8-, nd 24-month-old Wistr rts, s well s 8- nd 24-month-old rts fter 3 months of moderte food restrition whih is suffiient to lower viserl diposity to vlues even elow those of mture 3-month-old rts fed d liitum. The effets of geing nd food restrition on dipoyte-derived ftors suh s resistin, diponetin, nd leptin hve lso een nlyzed. Mterils nd Methods Animls Mle Wistr rts of 3, 8, nd 24 months old from our in-house olony (Centre of Moleulr Biology, Mdrid, Spin) were housed in limte-ontrolled qurters with 12 h light yle nd fed stndrd lortory how nd wter ville d liitum. They were hndled s per the Europen Union lws nd Ntionl Institutes of Helth (NIH) guidelines for niml re. Experimentl proedures were pproved y the Institutionl Committee of Reserh Ethis. Food restrition Five- nd 21-month-old rts were rndomly seleted to undergo food restrition protool s desried erlier (Pérez et l. 2004). They were pled in individul ges nd fed dily 18 nd 21 g of how respetively (equivlent to z80% of norml food intke). After 2 months of nutritionl restrition, the rts showed ody weight equivlent to z85% of d liitum fed ged-mtes. They were weighed weekly nd the mount of food provided ws djusted individully in order to mintin their ody weight for one dditionl month. Food restrited rts were used t the ge of 8 nd 24 months respetively. Orl gluose tolerne test (OGTT) Overnight fsted rts were dministered 30% gluose solution intrgstrilly (2 g/kg of ody weight) nd lood smples were tken from the til vein efore the gluose lod (tz0) nd 15, 30, 60, nd 120 min fter gluose dministrtion. Blood gluose ws determined immeditely using n Autrend Gluose Anlyser (Rohe). Blood smples were entrifuged nd plsm ws frozen t K70 8C until insulin estimtion. Overll hnges in gluose nd insulin during OGTT were lulted s the re under the urve ove the sl level (DGluose nd DInsulin res respetively). Journl of Endorinology (2007) 194, The rtio of DGluose re to DInsulin re ws used s n index of whole ody insulin sensitivity (Levy et l. 2002). Euglyemi-hyperinsulinemi lmp Overnight fsted rts were nesthetized with pentoritl (4 mg/100 g of ody weight) nd trheotomized to void respirtory prolems. Blood smples were withdrwn from til nd glyemi ws determined s indited ove. Insulin (Atrpid, Novo, Copenhgen, Denmrk) ws infused through sphenous vein t onstnt rte, without priming dose, to reh vlues of 2 or 4 nmol/h per kg respetively, nd solution of 30% gluose ws infused through the other sphenous vein t vrile rte to lmp lood gluose t the level present t the strt of the experiment (Esrivá et l. 1997). This ws hieved tking lood smples every 5 min nd determining gluose onentrtion s indited. Within 40 min of strting the lmp, plsm gluose nd insulin remined onstnt without further djusting the infusion rte. At this stedy stte, the overll gluose utiliztion rehes onstnt vlue, whih ws further monitored for 60 min. Gluose disposl rte (M) ws determined from the rte of gluose infusion normlized to ody weight nd ws used s n index of insulin sensitivity. Gluose utiliztion y individul tissues A olus of 80 mci of 2-deoxy-D-[1-3 H]gluose (Amershm) ws injeted intrvenously into rts either under stedy-stte onditions or not infused with insulin, to estimte sl gluose utiliztion. The onentrtion of gluose nd rdiotivity of 2-deoxy-D-[1-3 H]gluose were determined in lood smples tken every 5 min. Mesurements of plsm gluose nd insulin efore the injetion of the rdiotive olus nd t the end of the experiment (60 min) onfirmed tht stedy-stte onditions were mintined throughout the test. Rts were killed y ervil dislotion nd piees of the different tissues were rpidly removed nd frozen until proessing. Tissue digestion, determintion of 2-deoxy-D-[1-3 H]gluose-6-phosphte, nd estimtion of the rte of gluose utiliztion ws performed s reported previously (Ferré et l. 1985, Esrivá et l. 1992, 1997). Dt re referred s the gluose metoli index nd n e onsidered s n index of gluose utiliztion t different insulin levels (Esrivá et l. 1997). Isoltion of ft ells nd determintion of lipogenesis Rts were killed under CO 2 tmosphere nd viserl epididyml nd retroperitonel ft pds were removed nd weighed to ssess diposity index (Li et l. 1997). Adipoytes were prepred y digestion with ollgense s desried y Molero et l. (1998). Isolted ft ells were suspended in Kres-Ringer phosphte medium ontining 3% BSA, 2 mm gluose, nd mci/ml of (U- 14 C)-gluose, in Downloded from Biosientifi.om t 11/17/ :37:46PM vi free ess

3 Role of diposity on insulin tion in geing. F ESCRIVÁ nd others 133 proportion of 1 ml ells per 3 ml medium. The ells were inuted for 1 h t 37 8C in the presene or sene of 20 nm insulin. Inorportion of gluose into triglyeride nd ftty ids ws determined s desried previously (Fin et l. 1967). Expression of hepti PEPCK Totl liver RNA ws isolted using n RNse kit (Qigen). The RNA (500 ng) ws reverse trnsried nd rel-time quntittive PCR ws performed on TqMn 7000 sequene detetion system (Applied Biosystems, Foster City, CA, USA) using SYBR Geen. Speifi primers for PEPCK gene were designed with primer express 2.0 softwre (F, CGCTA- TGCGGCCCTT; R, AGCCAGTGCGCCAGGTACT) nd 18S rrna ws used s ontrol to normlize gene expression. Other determintions Plsm insulin, leptin, nd diponetin were determined using rt insulin, rt leptin, nd mouse diponetin RIA kits (Lino Reserh, St Chrles, MO, USA) respetively. Resistin ws ssessed using rt resistin ELISA kit (BioVendor, Brno, Czeh Repuli). Oesity Lee index ws lulted s 10 4!ody weight (g)!nso-nl length (mm K1 ), s desried previously (Li et l. 1997). Totl ody ft nd len ody mss were mesured y dulenergy X-ry sorptiometry (DEXA; Norlnd XR-26, Venie, FL, USA). For determining the triglyeride ontent of musles, totl lipids were extrted from 100 mg of tissue (Cohen et l. 2002) nd triglyeride ontent ws mesured using n enzymti kit (Stnio lortory, Boerne, TX, USA). Sttistil nlysis Sttistil omprisons to determine the effet of ge nd insulin tretment were done y one-wy ANOVA using the Prophet softwre (BNN Systems nd Tehnologies, Cmridge, MA, USA). When signifint effet of ge or insulin ws oserved, the Dunn post ho test ws used to nlyze differenes etween mens. To ompre food-restrited nd d liitum fed ged mtes, the unpired Student s t-test ws used. Results Chrteristis of the nimls Tle 1 summrizes the hrteristis of the five groups of rts used. In oth 8- nd 24-month-old rts, ody weight inreses progressively with geing nd the lorie restrition used in this study rings out w15% derese in ody weight. No signifint effet of ge on fsting plsm gluose nd insulin ws oserved nd lorie restrition deresed only the insulin onentrtion in 8-month-old rts. Plsm diponetin remined onstnt with geing. Interestingly, food restrition eliited signifint inrese in plsm diponetin in 8-month ut not in 24-month-old rts. Plsm leptin onentrtion inresed progressively with geing rehing vlues sixfold higher in 24-month-old rts thn in 3-month-old rts. In 8-month-old rts, food restrition eliited mrked derese of plsm leptin up to vlues signifintly lower thn those oserved in younger rts. In 24-month-old rts, more moderte derese in leptin level ws oserved under food restrition. Plsm resistin inresed signifintly in 8-month-old rts; however, it is lower in 24-month-old rts. Food restrition signifintly deresed irulting resistin t the ge of 8 months ut hs no effet on older rts. Chnges in diposity nd ody omposition re shown in Tle 2. One-wy ANOVA indites signifint effet of ge on oesity Lee index (PZ ). Nevertheless, Lee index t the ge of 24 months ws well under tht of young oese Wistr dieti ftty rts (C Pérez nd JM Crrsos unpulished oservtions). Food restrition eliited signifint derese of Lee index in oth 8- nd 24-month-old rts. The perent of viserl ft inresed signifintly up to the ge of 8 months nd remined onstnt therefter. The weight of the two ft pds nlyzed inresed up to the ge of 8 months, ut only the retroperitonel ft ontinued to inrese up to 24 months. Clorie restrition deresed the diposity index eqully in 8- nd 24-month-old rts. A similr derese ws oserved in epididyml ft t oth ges wheres the derese in retroperitonel ft ws lower in 24-month-old rts. Len ody mss did not undergo signifint hnges either with geing or with lorie restrition. In ontrst, signifint inrese in totl nd Tle 1 Chrteristis of the rts. Dt re the mengs.e.m. of seprte determintions 3 months 8 months 8 months-fr 24 months 24 months-fr Body weight (g) 383G8 511G6 431G4 700G12 603G15 Fsting plsm gluos (mmol/l) 4. 5G G G G G0. 2 Fsting plsm insulin (nmol/l) 0. 19G G G G G0. 04 Fsting plsm diponetin (mg/ml) 3. 1G G G G G0. 2 Fsting plsm leptin (ng/ml) 4. 6G G G G G1. 2 Fsting plsm resistin (ng/ml) 18. 0G G G G1. 7 d 24. 7G1. 6 One-wy ANOVA indites signifint effet of ge on ody weight (PZ0. 001), plsm leptin (PZ0. 001) nd resistin (PZ ), nd no signifint effet on plsm diponetin, gluose nd insulin levels (PO0. 05). P!0. 05 versus 3-month-old rts; P!0. 05 versus 3- nd 8-month-old rts; P!0. 05 versus sme ge fed d liitum; d P!0. 05 versus 8-month-old rts. FR, food restrited. Journl of Endorinology (2007) 194, Downloded from Biosientifi.om t 11/17/ :37:46PM vi free ess

4 134 F ESCRIVÁ nd others. Role of diposity on insulin tion in geing Tle 2 Chnges in diposity with ge nd lorie restrition. Dt re the mengs.e.m. of 4 10 seprte determintions 3 months 8 months 8 months-fr 24 months 24 months-fr Oesity Lee index 301G1 304G1 289G1 307G1 298G3 Adiposity index (%) 3. 2G G G G G0. 1 Epididyml ft (g) 4. 5G G G G G0. 6 Retroperitonel ft (g) 5. 2G G G G G0. 6 Totl ft mss (%) 7. 1G G G G G1. 9 Totl ft mss (g) 26. 5G G G G G16. 9 Len ody mss (g) 340G15 375G23 332G5 405G9 388G9 Triglyeride ontent in soleus (mg/g of tissue) 6. 9G G G G G2. 9 Triglyeride ontent in qudrieps (mg/g of tissue) 2. 0G G G G G0. 8 One-wy ANOVA indites signifint effet of ge on oesity Lee index (PZ ), diposity index (PZ0. 001), epididyml (PZ0. 001) nd retroperitonel (PZ ) ft, perent (PZ0. 001) nd totl ft mss (PZ0. 001), nd triglyeride ontent in qudrieps (PZ0. 008). No signifint effet on len ody mss, nd triglyeride ontent in soleus musle ws oserved (PO0. 05). P!0. 05 versus 3-month-old rts; P!0. 05 versus 3- nd 8-month-old rts; P!0. 05 versus sme ge fed d liitum. FR, food restrited. perent ft ws oserved with geing. Clorie restrition deresed totl ody ft in 8- nd 24-month-old rts. However, when expressed in perent, this derese ws not signifint in older rts. Triglyeride ontent in soleus remined unhnged with geing nd food restrition. In qudrieps, signifint inrese ws oserved in 24-month-old rts, tht ws reverted y lorie restrition (Tle 2) Orl gluose tolerne Chnges in gluose nd insulin during OGTT re shown in Tle 3. The inrement of gluose ws similr for the three ge groups inditing tht gluose tolerne ws not modified during geing. In ontrst, the re under the urve for insulin in response to the gluose lod inresed progressively during geing suggesting the development of peripherl insulin resistne. In food restrited rts, hnges in lood gluose during OGTT were similr to tht oserved in their respetive ged mtes fed d liitum, inditing tht it did not lter the gluose tolerne in spite of the mrked derese in diposity (Tle 2). Nevertheless, the insulin needed to ope with the gluose lod ws signifintly lower in 8-month-old rts nd in 24-month-old rts, the inrement in insulin during OGTT eing similr to tht of d liitum fed ged mtes. Insulin sensitivity index, lulted s the rtio of DGluose re to DInsulin re, deresed etween 3 nd 8 months, nd further signifint derese ws oserved up to the ge of 24 months. Clorie restrition indued mrked improvement of insulin sensitivity in 8-month-old rts ut not in 24-monthold rts in spite of similr hnge of diposity in oth groups. Euglyemi-hyperinsulinemi lmp Plsm gluose nd insulin onentrtions t stedy stte, 45 min fter strting the lmp, re shown in Tle 4. Gluose onentrtions were lmped t similr levels in ll groups within the physiologil rnge. Plsm insulin onentrtions t stedy stte were signifintly elevted in oth 8- nd 24-month-old rts t the highest insulin infusion rte nd were not ltered y lorie restrition. The rte of gluose infusion per kilogrm of ody weight neessry to mintin lood gluose onentrtions during the lmp deresed signifintly with ge t oth insulin infusion rtes (Fig. 1 nd Tle 4). In 8-month-old rts, this derese ws signifint only t the highest insulin infusion rte, wheres in 24-month-old rts, signifintly lower gluose infusion ws required for oth insulin doses. In 8-month-old rts, lorie restrition signifintly inresed the gluose disposl rte t oth insulin infusion rtes. In ontrst, in 24-month-old rts, signifint inrese ws oserved only t the lower insulin dose, wheres the gluose utiliztion t the highest dose of insulin ws similr to tht oserved in d liitum fed ged mtes (Fig. 1 nd Tle 4). Tle 3 Vritions of gluose nd insulin onentrtions in lood during orl gluose tolerne test. Dt re mengs.e.m. of 6 7 rts per group DGluose re (mm!min) DInsulin re (nm!min) Insulin sensitivity index Age (months) 3 429G G G G G G2 8-FR 363G G G G G G FR 500G G G3. 3 DGluose re, DInsulin re nd Insulin sensitivity index were determined s indited in Mterils nd Methods. P!0. 05 signifintly different from 3- month-old rts; P!0. 05 signifintly different from 3- nd 8-month-old rts; P!0. 05 signifintly different from sme ge fed d liitum. FR, food restrited. Journl of Endorinology (2007) 194, Downloded from Biosientifi.om t 11/17/ :37:46PM vi free ess

5 Role of diposity on insulin tion in geing. F ESCRIVÁ nd others 135 Tle 4 Metoli hrteristis of the rts during euglyemi hyperinsulinemi lmp. Dt re the mengs.e.m. of 5 6 different lmps performed with eh group of rts Insulin infusion rte (nmol/h per kg) Age (months) 2 4 Stedy stte plsm gluose (mmol/l) G G G G FR 5. 7G G G G FR 5. 2G G0. 2 Stedy stte plsm insulin (nmol/l) G G G G FR 1. 1G G G G FR 2. 1G G1. 0 M(mmol/min/kg) G G G G FR G G G G FR 88. 3G G5. 0 One-wy ANOVA indites signifint effet of ge on stedy stte plsm insulin t the highest infusion rte (PZ0. 049), nd on (M) t oth insulin doses (PZ nd for 2 nd 4 nmol/h per kg of infused insulin respetively). M, gluose disposl rte, determined from the rte of gluose infusion normlized to ody weight. P!0. 05 versus 3-month-old rts; P!0. 05 versus 3- nd 8-month-old rts; P!0. 05 versus sme ge fed d liitum. FR, food restrited. Expression of hepti PEPCK Chnges in gluose utiliztion ould e influened y ltertions in the hepti gluoneogeni funtion. Vritions in liver gluoneogenesis depend minly on the gluoneogeni enzyme PEPCK whose expression hs eome n importnt mrker for hepti gluoneogenesis (Hnson & Reshef 1997, Hkimi et l. 2005). Determintion of PEPCK expression y rel-time PCR indited tht neither ge nor lorie restrition signifintly ltered the level of expression of the enzyme (dt not shown). Gluose utiliztion in speifi tissues Figure 2 shows tht in oth retroperitonel nd epididyml white dipose tissue, insulin stimultes the gluose uptke in 3-month-old rts pproximtely six nd threefold respetively. Insulin lso stimultes the gluose utiliztion (PZ0. 023) signifintly in retroperitonel dipose tissue of 8-month-old rts However, the stimultory effet of the hormone nd the mximl gluose uptke were signifintly lower in 8-month old thn in 3-month-old rts. In 24-month-old rts, lower insulin-stimulted gluose uptke ws oserved. Figure 1 Gluose infusion rte during euglyemi-hyperinsulinemi lmp. Anesthetized overnight fsted rts were infused with insulin s indited in Mterils nd Methods, to reh vlues of 2 ( ) or 4 (.) nmol/h per kg respetively nd solution of 30% gluose ws infused through the other sphenous vein t vrile rte to lmp lood gluose t the level present t the strt of the experiment (see Mterils nd Methods). Gluose infusion rte ws djusted every 5 min fter determining lood gluose onentrtion s indited in Mterils nd Methods. Within 40 min of strt of the lmp, gluose onentrtion remined onstnt without further djusting the infusion rte. Blood gluose ws monitored for further 60 min. Dt t plteu represent the overll gluose disposl rte (M), re the mengs.e.m. of 5 6 different lmps performed with eh group of rts, nd re expressed s mmol of gluose!min K1!kg K1 of ody weight. Sttistil omprisons re shown in Tle 4. FR, food restrited. Journl of Endorinology (2007) 194, Downloded from Biosientifi.om t 11/17/ :37:46PM vi free ess

6 136 F ESCRIVÁ nd others. Role of diposity on insulin tion in geing Figure 2 Gluose metoli index of retroperitonel nd epididyml dipose tissues during euglyemihyperinsulinemi lmp. Gluose utiliztion ws estimted under euglyemi onditions in 3-, 8-, nd 24-month-old rts fed d liitum nd in 8- nd 24-month-old rts fter 3 months of moderte lorie restrition, s indited in Mterils nd Methods. Dt re expressed s mmol of gluose!min K1!kg K1 of tissue nd re the mengs.e.m. of 5 8 independent determintions in eh se. White rs, rts fed d liitum; Blk rs, rts under lorie restrition. (A), Retroperitonel white dipose tissue; (B), Epididyml white dipose tissue. P!0. 05 versus 3-month-old rts; P!0. 05 versus 3- nd 8-month-old rts; P!0. 05 versus sme ge fed d liitum; d P!0. 05 versus sl; e P!0. 05 versus sl nd 2 nmol/h per kg. The stimultory effet of insulin on gluose utiliztion ws lso lower in epididyml white dipose tissue of 8- nd 24-month-old rts (2- nd 1. 4-fold respetively) thn in 3-month-old rts (3-fold). Thus, oth dipose tissues develop mrked insulin resistne t 8 months nd remin poorly sensitive in 24-month-old rts. Journl of Endorinology (2007) 194, In 8-month-old rts, lorie restrition inreses the insulin stimultion of gluose uptke in oth dipose tissues nd insulin infusion rtes up to vlues even higher thn those oserved in young rts. In ontrst, no improvement of insulin tion ws oserved in dipose tissues of 24-month-old rts under lorie restrition when ompred with d liitum fed ge-mthed rts. Downloded from Biosientifi.om t 11/17/ :37:46PM vi free ess

7 Role of diposity on insulin tion in geing. F ESCRIVÁ nd others 137 In soleus nd diphrgm (Fig. 3), signifint gedependent derese of gluose uptke t oth insulin infusion rtes ws oserved (in soleus, PZ nd , nd in the diphrgm, PZ nd , for 2 nd 4 nmol/h per kg insulin infusion rte respetively). Nevertheless, the post ho test used indites tht this derese is only signifint for 24-month-old rts wheres gluose uptke in rts ged 8 months is not signifintly different from tht of 3-month-old rts. Clorie restrition improved the insulin effet on gluose utiliztion in 8- nd 24-monthold rts t the lower insulin infusion rte, ut did not lter the gluose utiliztion of oth tissues t the highest insulin level. Thus, lorie restrition did not inrese the ontriution to overll gluose disposl of soleus nd diphrgm of 24-month-old rts under hyperinsulinemi onditions. Conerning qudrieps musle, dt in Fig. 3 show tht gluose uptke is not signifintly modified during geing t ny of the insulin infusion rtes used, inditing tht this tissue does not ontriute to the deresed overll gluose utiliztion of 8- nd 24-month-old rts. In food restrited 8-month-old rts, lower gluose uptke is oserved only in the sene of insulin infusion, wheres in food-restrited 24-month-old rts, signifint inrese of gluose uptke ours t sumximl insulin onentrtions, suggesting n improvement of insulin sensitivity in this tissue. However, mximl gluose uptke ws not ltered y food restrition in oth 8- nd 24-month-old rts. In greement with previous reports (Esrivá et l. 1997), gluose utiliztion y rin nd lung of 3-month-old rts ws not stimulted under hyperinsulinemi onditions (dt not shown) onfirming the speifiity of the former insulin effets. Insulin tion on isolted dipoytes We studied the insulin responsiveness of isolted dipoytes from rts of different groups (Tle 5). The rte of inorportion of gluose into triglyeride in the sene of insulin remins unhnged with ge. In ontrst, inorportion of gluose into triglyeride-ftty ids is signifintly lower in 8- nd 24-month-old rts. Food restrition eliited mrked inrese in sl lipogeni tivity in oth 8- nd 24-month-old rts. The stimultion of lipogenesis y insulin delined in ft ells of 8-monthold rts when ompred with 3-month-old rts. However, no further deline ws oserved up to the ge of 24 months. Clorie restrition indued mrked inrese in the lipogeni tion of insulin on dipoytes of 8-monthold rts tht eome even more sensitive thn ft ells of 3-month-old rts. Clorie restrition lso improved insulin sensitivity in dipoytes of 24-month-old rts ut to lower extent thn in food restrited 8-month-old rts. Disussion Using two different in vivo pprohes, we show here tht overll insulin sensitivity dereses during geing in the Wistr rt. This ours in spite of norml fsting lood gluose nd insulin onentrtions nd without developing gluose intolerne s oserved in OGTT. The dt in this work indite tht the min derese in insulin sensitivity ours etween 3 nd 8 months of ge ut delines further up to the ge of 24 months. Studies foused to exmine the effets of geing on insulin tion re frught with the diffiulty of disriminting the effet of ge y itself from tht of the ge-ssoited hnges in ody omposition. Although there is signifint inrese in ody weight during geing, oesity Lee index of old rts is mrkedly lower thn tht oserved in morid oese Wistr Dieti Ftty rts. Our dt show tht the derese of insulin sensitivity up to 8 months of ge is prlleled y signifint inrese in viserl nd totl ft mss. Between 8 nd 24 months of ge insulin sensitivity dereses further without signifint hnges in perent viserl diposity. However, it should e noted tht rts grow throughout their life spn. Thus, even though no hnges in diposity index re oserved fter 8 months, there is signifint inrese in retroperitonel nd totl ft with geing, whih my e relted to the deline of insulin sensitivity fter 8 months of ge. In ontrst, len ody mss does not hnge signifintly with geing. The dt otined from food-restrited rts lso point to the relevne of retroperitonel ft in the development of insulin resistne in older rts. Thus, under moderte lorie restrition, oth 8- nd 24-month-old rts exhiit similr nd signifint derese in perent viserl diposity nd totl ft mss, rehing vlues of the former even lower thn those of ontrol young rts. However, only in the se of 8-month-old rts, mrked improvement of whole ody insulin sensitivity index ws oserved. Nevertheless, the dt lerly shows tht there is mjor differene in the redution of retroperitonel ft, whih remins mrkedly higher in 24-month-old rts. This suggests tht the deline in insulin sensitivity in erly dulthood ould e due to ft umultion nd n e reversed y lowering overll diposity. However, eyond this ge limit, the sustined inrese in retroperitonel ft nd its refrtoriness to derese even under food restrition might ring out dmges in some tissues, leding to stte of irreversile insulin resistne. Other uthors hve reported tht surgil removl of viserl ft prevents the ge-ssoited deline in insulin sensitivity (Griely et l. 2002). However, one report (Ctlno et l. 2005) suggests tht intr-dominl diposity ounts for only 30 40% of the hnges in hepti nd peripherl insulin resistne ssoited with geing in BN/ F344 rts. Our dt here indites tht oth retroperitonel nd non-viserl ft ply role in the hnges of insulin sensitivity with ge nd food restrition. Journl of Endorinology (2007) 194, Downloded from Biosientifi.om t 11/17/ :37:46PM vi free ess

8 138 F ESCRIVÁ nd others. Role of diposity on insulin tion in geing Journl of Endorinology (2007) 194, Downloded from Biosientifi.om t 11/17/ :37:46PM vi free ess

9 Role of diposity on insulin tion in geing. F ESCRIVÁ nd others 139 Tle 5 Insulin responsiveness of isolted dipoytes: effet of ge nd food restrition. Dt re mengs.e.m. of 6 8 rts per group Gluose inorportion into Triglyeride Bsl (nmol/10 5 ells/h) Ftty ids Insulin (nmol/10 5 ells/h) % Bsl (nmol/10 5 ells/h) Insulin (nmol/10 5 ells/h) % 3-months 2. 05G G G G G G23 8-months 2. 16G G G G G G9 8-months FR 8. 01G G G G G G82 24-months 2. 21G G G G G G12 24-months 8. 30G G G G G G57 FR Adipoytes were inuted s indited in Mterils nd Methods. P!0. 05, signifintly different from 3-month-old rts; P!0. 05, signifintly different from sme ge fed d liitum. One-wy ANOVA indites signifint effet of ge on gluose inorportion into triglyeride in presene of insulin (PZ0. 033). FR, food restrited. The dt in this work, with respet to the insulin trget tissues, demonstrte tht the gluose metoli index of oth dipose tissues nlyzed is lower in 8- nd 24-month-old rts thn in younger rts. These dt gree with the deresed insulin effet on lipogenesis oserved in isolted dipoytes nd indites tht this insulin-resistnt stte develops erly fter sexul mturtion. Impired insulin tion in dipose tissue ould represent n erly step leding to the overll insulin resistne hrteristi of ged nd/or oese rts (Smith 2002). Moreover, reent in vitro studies hve demonstrted tht dipoyte-onditioned medium impirs insulin signling in skeletl musle ells (Dietze et l. 2004) nd heptoytes (Wng et l. 2006). The dt presented herein indites tht insulin resistne in oxidtive musles suh s soleus nd diphrgm develops lter thn in dipose tissue, suggesting tht it ould e seondry effet used y the prolonged ltertion of dipose tissue insulin sensitivity. Interestingly, hnges in musle insulin sensitivity with geing nd lorie restrition seem to e unrelted to the mount of musle triglyeride. Thus, triglyeride ontent is similr in the soleus of the five groups of rts tested tht show differenes in insulin response, wheres in qudrieps no insulin resistne is oserved, in spite of n inrese of triglyeride ontent with geing. Qudrieps musle is minly onstituted y type 2 nd 2 fiers, whih re minly glyolyti nd hve low pity for using ftty ids s fuel, ft tht might explin the umultion of triglyerides in this tissue in 24-month-old rts, whih re hrterized y inresed serum triglyeride onentrtion (Esrivá et l. 1997). As reported y others (Kiens 2006, Kregen et l. 2006), musle insulin resistne is rther ssoited with ellulr levels of mlonyl-coa, ftty yl CoA, diylglyerol, or ermide, thn with triglyeride umultion. Thus, more experimentl work is needed to lrify the differenes in mehnism underlying the differenes in insulin sensitivity etween oxidtive nd glyolyti musles with geing. The dt in this work revel tht lorie restrition indues mrked inrese in gluose utiliztion under hyperinsulinemi onditions in white dipose tissue of 8-month-old rts ut not in 24-month-old rts. Beyond the quntittive ontriution of dipose tissue to overll gluose disposl, this seems to ontrst with the prtil improvement of insulindependent lipogeni tivity oserved in isolted dipoytes from food restrited 24-month-old rts. However, gluose metoli index is n in vivo estimtion nd some irulting dipokines ould e influening dipose tissue insulin sensitivity in vivo (see elow). In soleus nd diphrgm of 24-month-old rts, lorie restrition inreses the gluose utiliztion t sumximl insulin levels ut not t sturting insulin onentrtions inditing tht insulin resistne still persists in these tissues fter lorie restrition. These dt re lso onsistent with the sene of effet of lorie restrition on mximl overll gluose disposl rte, ut signifint inrese t sumximl insulin levels is oserved in 24-month-old rts. On the other hnd, lthough the dt of PEPCK expression do not suggest signifint hnges in liver gluoneogenesis, it nnot e ruled out tht ltertion in liver insulin sensitivity results in hnges in hepti gluose output tht my ontriute to lter the whole ody gluose disposl rte during geing nd lorie restrition. Ageing is ssoited with ft mss retion (Nishimur et l. 1988) nd long-term hnges in irulting dipokines ould modulte insulin sensitivity. High levels of diponetin hve een ssoited with inresed insulin sensitivity nd, in situtions of insulin resistne low onentrtion of diponetin hs een oserved (Chndrn et l. 2003). Figure 3 Gluose metoli index of musle tissue during euglyemi-hyperinsulinemi lmp. Gluose utiliztion ws estimted s indited in Fig. 1. Dt re expressed s mmol of gluose!min K1!kg K1 of tissue nd re the mengs.e.m. of 5 8 independent determintions in eh se. White rs, rts fed d liitum; Blk rs, rts under lorie restrition. Top, Soleus; Middle, Diphrgm; ottom, Qudrieps. P!0. 05 versus 3-month-old rts; P!0. 05 versus 3- nd 8-month-old rts; P!0. 05 versus sme ge fed d liitum; d P!0. 05 versus sl; e P!0. 05 versus sl nd 2 nmol/h per kg. P!0. 01 signifintly different from 3-month-old rts; P!0. 05 signifintly different from 8-month-old rts; P!0. 01 signifintly different from sme ge fed d liitum. Journl of Endorinology (2007) 194, Downloded from Biosientifi.om t 11/17/ :37:46PM vi free ess

10 140 F ESCRIVÁ nd others. Role of diposity on insulin tion in geing The dt in this work show tht plsm diponetin remins unhnged with geing suggesting tht it does not ply role in the development of ge-ssoited insulin resistne. Reent dt hve indited tht it is the high moleulr weight diponetin multimer, whih is minly ssoited with inresed insulin sensitivity (Lr-Cstro et l. 2006) nd its formtion depends on hydroxyltion nd glyosyltion of four onserved lysine within the ollgenous domin (Wng et l. 2006). Thus, it remins possile tht despite unhnged totl mount of plsm diponetin with geing, n impired lysine hydroxyltion nd/or glyosyltion leds to lower levels of the insulin sensitizing diponetin multimer in ged rts. Interestingly, signifint inrese of diponetin is oserved only in 8-month-old rts fter lorie restrition, ft tht might explin the differentil effet of lorie restrition on insulin sensitivity t the two different ges. Resistin uses insulin resistne in norml mie (Steppn et l. 2001) nd hroni hyperresistinemi leds to insulin resistne in musle, liver, nd dipose tissue (Muse et l. 2004, Rngwl et l. 2004, Sth et l. 2004). The oserved inrese in plsm resistin in 8-month-old rts prllels the development of insulin resistne nd its derese in food restrited rts is ssoited with mrked improvement in insulin sensitivity. Thus, it ould e speulted tht resistin plys role in the development of insulin resistne t erly geing. In ontrst, in 24-month-old rts irulting resistin deresed nd food restrition did not modify insulin onentrtion in plsm, in greement with the sene of insulin sensitivity improvement fter lorie restrition in these rts. The inrese in serum leptin with geing oserved here ould explin the derese in insulin sensitivity of dipose tissue in ged rts (Pérez et l. 2004). Although leptin is known to promote insulin sensitivity in musle, ged rts show entrl leptin resistne (Fernández-Glz et l. 2002), whih ould redue the effiy of leptin tion on musle through the entrl nervous system. After lorie restrition plsm leptin prtilly dereses in 24-month-old rts. However, the reovery of entrl leptin tion ould prevent the improvement of dipose tissue insulin sensitivity nd might led to persistent stte of overll insulin resistne. To summrize, our dt demonstrtes tht there is n erly development of overll insulin resistne up to the ge of 8 months in Wistr rts, the dipose tissue eing the first of the tissues studied herein in developing insulin resistne. This ould e medited y inresed plsm leptin nd resistin onentrtions, s well s overll ft retion. Moderte lorie restrition redues the mount of ft nd improves insulin sensitivity in ssoition with inresed plsm diponetin nd lowered leptin nd resistin levels. At more dvned ge, insulin resistne lso develops in some musles nd lorie restrition is unle to restore neither overll insulin sensitivity nor the insulin effets on dipose tissue nd insulin-resistnt musles. It n thus e postulted tht the persistent hnges in diposity, espeilly in retroperitonel ft, ssoited with geing ould led to irreversile hrm in Journl of Endorinology (2007) 194, insulin trget tissues resulting in insulin resistne. Although dipokines re ndidtes to medite some of these effets, this issue requires further study. The dt presented herein point to the relevne of dipose tissue s primry site of impirment of insulin sensitivity during geing in the rt nd suggest tht n erly reovery of dipose tissue insulin sensitivity is neessry for the effetiveness of moderte lorie restrition on the insulin tion on the whole. Aknowledgements This work ws supported y grnts BFI , BFI nd BFU (Ministerio de Cieni y Tenologí, Spin), 08.6/0009/2001 (Comunidd de Mdrid, Spin), nd ISCIII-RETIC RD06 (Ministerio de Snidd y Consumo, Spin). The Centro de Biologí Moleulr is the reipient of institutionl id from the Rmón Arees Foundtion. C P ws reipient of fellowship from the Mutis Progrm (Spin). N G ws supported y EI grnt from Consejerí de Snidd ( JCCM). Thnks re due to Dr L Gómez-Pellio nd Dr R Rodríguez (Universidd de Allá de Henres, Mdrid, Spin) for their help in determintion of ody omposition y DEXA. The uthors delre tht there is no onflit of interest tht would prejudie the imprtility of this sientifi work. Referenes Ahim RS & Flier JS 2000 Adipose tissue s n endorine orgn. Trends in Endorinology nd Metolism Bsu R, Bred E, Oerg AL, Powell CC, Dll Mn C, Bsu A, Vittone JL, Klee GG, Aror P, Jensen MD et l Mehnisms of the ge-ssoited deteriortion in gluose tolerne. 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Biohemil nd Biophysil Reserh Communitions Wng Y, Lm KSL, Chn L, Chn KW, Lm JBB, Lm MC, Hoo RCL, Mk WWN, Cooper GJS & Xu A 2006 Post-trnsltionl modifitions of the four onserved lysine residues within the ollgenous domin of diponetin re required for the formtion of its high moleulr weight oligomeri omplex. Journl of Biologil Chemistry Reeived in finl form 2 April 2007 Aepted 13 April 2007 Mde ville online s n Aepted Preprint 19 April Journl of Endorinology (2007) 194, Downloded from Biosientifi.om t 11/17/ :37:46PM vi free ess