ARTICLE. E. O. List & A. J. Palmer & D. E. Berryman & B. Bower & B. Kelder & J. J. Kopchick

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1 Dietologi (2009) 52: DOI /s z ARTICLE Growth hormone improves ody omposition, fsting lood gluose, gluose tolerne nd liver triylglyerol in mouse model of diet-indued oesity nd type 2 dietes E. O. List & A. J. Plmer & D. E. Berrymn & B. Bower & B. Kelder & J. J. Kophik Reeived: 2 Ferury 2009 /Aepted: 5 My 2009 /Pulished online: 26 My 2009 # Springer-Verlg 2009 Astrt Aims/hypothesis Growth hormone hs een used experimentlly in two studies to tret individuls with type 2 dietes, with oth reporting enefiil effets on gluose metolism. However, onerns over potentil dietogeni tions of growth hormone omplite its ntiipted use to tret type 2 dietes. Thus, n niml model of type 2 dietes ould help evlute the effets of growth hormone for treting this ondition. Methods Mle C57BL/6J mie were pled on high-ft diet to indue oesity nd type 2 dietes. Strting t 16 weeks of ge, mie were treted one dily for 6 weeks with one of four different doses of growth hormone. Body weight, ody omposition, fsting lood gluose, insulin, gluose tolerne, liver triylglyerol, tissue weights nd lood hemistries were determined. Results Body omposition mesurements reveled dosedependent derese in ft nd n inrese in len mss. Anlysis of ft loss y depot reveled tht suutneous nd mesenteri ft ws the most sensitive to growth hormone tretment. In ddition, growth hormone tretment E. O. List : D. E. Berrymn : B. Bower : B. Kelder : J. J. Kophik (*) Edison Biotehnology Institute, Ohio University, 101 Konneker Reserh Lortories, 1 Wter Tower Drive, The Ridges, Athens, OH 45701, USA e-mil: kophik@ohio.edu A. J. Plmer : D. E. Berrymn Shool of Humn nd Consumer Sienes, College of Helth nd Humn Servies, Ohio University, Athens, OH, USA J. J. Kophik Deprtment of Biomedil Sienes, College of Osteopthi Mediine, Ohio University, Athens, OH, USA resulted in improvement in gluose metolism, with the highest dose normlising gluose, gluose tolerne nd liver triylglyerol. In ontrst, insulin levels were not ltered y the tretment, nor did orgn weights hnge. However, fsting plsm leptin nd resistin were signifintly deresed fter growth hormone tretment. Conlusions/interprettion Growth hormone therpy improves gluose metolism in this mouse model of oesity nd type 2 dietes, providing mens to explore the moleulr mehnism(s) of this tretment. Keywords Body omposition. Gluose tolerne. Growth hormone. Hyperglyemi. Hyperinsulinemi. Oesity. Triylglyerol. Type 2 dietes Arevition tpai-1 Tissue plsminogen tivtor inhiitor-1 Introdution Reominnt humn growth hormone is drug pproved for vriety of linil onditions. It hs positive effets on ody omposition [1]. Inresed protein retion in musle nd lipolysis in dipose tissue re iologil onsequenes of growth hormone tion nd together promote len phenotype, s reviewed [2]. Despite its ility to improve ody omposition, growth hormone hs lso een desried s dietogeni gent with the ility to inhiit the tion of insulin [1, 3, 4]. Thus, one onern out use of growth hormone for treting type 2 dietes is the possiility of developing or worsening existing insulin resistne. Nevertheless, two humn studies using growth hormone therpy to tret type 2 dietes hve oth, surprisingly, reported improvements in gluose nd insulin levels, nd

2 1648 Dietologi (2009) 52: insulin sensitivity [5, 6]. While growth hormone hs een studied frequently s tretment for non-dieti, oese individuls, with mixed results [5 12], reserh on its use for treting humn type 2 dietes is limited to the two studies mentioned ove. Furthermore, there re no studies using growth hormone in niml models of type 2 dietes, so dditionl insight is lking. Thus, the question of whether growth hormone n e sfely used to tret type 2 dietes remins unresolved, therefore n niml model tht n more fully evlute the impt of growth hormone tretment on physiology nd orgn funtion would e useful. Development of n niml model would e useful for other resons. Studies using humns n e diffiult to interpret, due to heterogeneous genotypes, s well s diffiulty in ontrolling ptient tivities like diet, exerise nd medition [6, 11, 13]. Mouse models, in ontrst, re often used to supplement knowledge of humn diseses nd offer other dvntges. For exmple, mie hve reltively similr physiologies to humns, while pure red stins re genetilly more uniform thn humns nd exogenous ftors, whih re hrd to ontrol in humns, n e esily ontrolled in lortory setting. Therefore, it is impertive to develop mouse model to evlute the sfety nd effetiveness of growth hormone therpy in the tretment of type 2 dietes. In the urrent study, n estlished mouse model of dietindued oesity nd type 2 dietes [14 18] ws used to evlute the impt of growth hormone tretment on type 2 dietes. Type 2 dieti mie (defined here s mie with signifintly elevted lood gluose nd insulin ompred with ontrol mie) were treted with wide rnge of growth hormone doses for 6 weeks while mintined on high-ft diet. Metoli vriles relevnt to oesity (ody weight, ody omposition, dipokine levels, orgn weights nd liver triylglyerol), dietes (gluose, gluose tolerne, insulin levels) nd growth hormone sttus (IGF-1 levels, orgn weights) were monitored. Methods Genertion of mouse model of diet-indued oesity nd type 2 dietes Indution of mouse model of oesity nd type 2 dietes using high-ft diet hs een reported previously [14 18]. We purhsed 49 mle C57Bl/6 J mie (Jkson Lortories, Br Hror, ME, USA) t 21 dys of ge nd divided them into two experimentl groups. The first group (n=42) ws pled on high-ft diet (D12492; Reserh Diets, New Brunswik, NJ, USA), while the seond group (n=7) ws pled on stndrd lortory rodent how (ProL RMH 3000; LDiet, Rihmond, VA, USA). In the high-ft diet, 20% of energy ws from rohydrtes, 20% from protein nd 60% from ft. The stndrd low-ft diet supplied 60% of energy from rohydrtes, 26% from protein nd 14% from ft. Mie were mintined on the respetive diets throughout the study, inluding during the growth hormone tretment phse. Mie were housed two to three per ge, with free ess to food nd wter. The ges were kept in temperture- nd humidity-ontrolled room nd exposed to 14 h light/10 h drk yle. All proedures were pproved y the Ohio University Institutionl Animl Cre nd Use Committee nd fully omplied with ll federl, stte nd lol poliies. Growth hormone tretment Purified reominnt ovine growth hormone ws gift from Monsnto (St Louis, MO, USA). Purified growth hormone powder ws suspended in PBS. All four doses were prepred on the sme dy prior to the study nd stored s dily liquots frozen t 80 C. Individul liquots were thwed just prior to use. The 6- week phse of dily suutneous growth hormone injetions (referred to s growth hormone tretment phse) strted fter 16 weeks on the high-ft diet nd ontinued to week 22. For mie on the high-ft diet, growth hormone ws dministered t four different doses: 0.005, 0.05, 0.5 nd 5 μg growthhormoneg 1 dy 1 (referred to s HF-0.005, HF-0.05, HF-0.5 nd HF-5 in the text). In ddition, low-ft diet group nd high-ft diet group of mie were injeted with n equl volume of PBS nd served s ontrols (referred to s LF-PBS nd HF-PBS, respetively). Seven to nine mie were used per group. Injetion sites were rotted weekly etween the nek nd the lower k to minimise skin irrittions. The injetions ourred t hours±2 h dily. Weight nd ody omposition mesurements Totl ody weight ws ssessed weekly throughout the study. Body omposition ws mesured weekly using Bruker Minispe (The Woodlnds, TX, USA) during the 6 week growth hormone tretment phse. Blood gluose mesurements nd olletion of plsm During the growth hormone tretment phse, fsting lood gluose ws determined nd fsting plsm smples were olleted every 2 weeks. Blood olletion ws performed y utting ~1 mm off the tip of the til nd olleting 200 μl of lood into heprinised pillry tues. Blood olletion ourred etween nd hours following 12 h fst. Plsm ws otined y entrifugtion t 4 C for 10 min t 7,000g nd ws stored t 80 C until further nlysis. Plsm mesurements IGF-1 onentrtions were determined using mouse IGF-1 ELISA kit (DSL ; Dignosti Systems Lortories, Wester, TX, USA) following the mnufturer s instrutions. The sensitivity

3 Dietologi (2009) 52: of the IGF-1 ssy ws 1.3 ng/ml. Plsm dipokines nd ytokines were mesured using Mouse Adipokine Linoplex nd Mouse Cytokine Linoplex pnels (Lino Dignosti Servies, St Chrles, MO, USA). Insulin onentrtions were determined using n ultr-sensitive mouse insulin ELISA kit (ALPCO Dignostis, Windhm, NH, USA) s per the mnufturer s instrutions. Gluose tolerne test Gluose tolerne tests were performed during the finl week of injetions. Mie were fsted for 12 h prior to the mesurements. Eh mouse reeived n intrperitonel injetion of 10% gluose solution t 0.01 ml/g ody weight. Blood gluose mesurements were performed in duplite using gluometer (OneTouh LifeSn, Milpits, CA, USA) efore the injetion nd t 60, 120 nd 180 min fter the injetion. The fsting lood gluose nd gluose tolerne tests were timed to e ssessed in the mornings nd ~18 h fter the lst dily growth hormone injetion. Tissue olletion Animls were killed y ervil dislotion fter 22 weeks of diet tretment nd 6 weeks of dily growth hormone injetions. Liver, kidney, hert, musle nd dipose tissue (inguinl suutneous, epididyml, retroperitonel, mesenteri ft pds) were olleted, weighed, flsh-frozen in liquid nitrogen nd stored t 80 C until further nlysis. Liver triylglyerol ontent Liver tissue ws used for extrtion nd mesurement of triylglyerol levels s desried previously [19]. Briefly, smples were digested in 3 mol/l KOH in 65% (vol./vol.) ethnol solution overnight. A triylglyerol GPO kit (Pointe Sientifi, Cnton, MI, USA) ws used to mesure glyerol ontent of the smples. Clultions were performed to estimte triylglyerol levels, ssuming the verge moleulr mss of triylglyerol is kd (885 g/mol). Sttistil nlysis Dt re presented s men±sem. Sttistis were performed on weights, ody omposition mesurements, plsm mesurements nd orgn weights using SPSS version 14.0 (Chigo, IL, USA). Comprisons were mde using univrite ANOVA with Tukey s honestly signifint differene post ho test. For withinnd etween-group omprison of longitudinl dt, repeted-mesures ANOVA ws used. Differenes were onsidered signifint t p<0.05. Results Genertion of mouse model of oesity nd type 2 dietes mie After 16 weeks, the high-ft group of mie weighed signifintly (42.6±0.7 g) more thn those fed the low-ft diet (28.7±1.0 g). This inrese in weight ws due, in prt, to ft mss umultion (14.4±1.5 g on high-ft diet vs 0.99±0.2 g on low-ft diet). Insulin (483±33 pmol/l) nd gluose (12.7±.043 mmol/l) levels were lso signifintly elevted in the high-ft fed ompred with low-ft fed ontrol nimls (63.9±12 pmol/l nd 7.88±0.63 mmol/l, respetively). These results were similr to those reported previously [14, 16 18] nd show tht the high-ft fed mie used in the growth hormone tretment phse were oese nd dieti efore tretment egn. Growth hormone stimulted IGF-I prodution Plsm IGF- 1 levels were monitored to evlute the poteny of the ovine growth hormone tretment regime. Plsm IGF-1 levels of the LF-PBS injeted group were signifintly lower thn ll high-ft fed groups (F [5, 43]=27.373, p<0.05) where 5 represents the degrees of freedom etween groups nd 43 represents the degrees of freedom within groups (Fig. 1). Of the high-ft fed mie, only the highest dose of growth hormone ws le to signifintly elevte plsm IGF-1 levels ove those of the ontrol group. Body weight nd omposition hnges with growth hormone tretment No signifint differene in ody weight ws oserved etween or mong the groups fed the high-ft diet even with the vrious growth hormone tretments (F [5, 45]=0.502, p=0.78) (Tle 1). However, there were drmti ltertions in ody omposition etween the highft fed groups following growth hormone tretment (Figs 2 nd 3). Speifilly, the highest two doses of growth hormone resulted in dose-dependent derese in ft mss in high-ft fed mie (F [5, 43]=41.1, p<0.001) (Fig. 2). The derese in diposity ws sttistilly signifint y the end of the first week of growth hormone tretment. Interestingly, the pek loss in ft mss ourred fter only 2 weeks, even though the ontrol mie nd those treted IGF-1 (ng/ml) LF Fig. 1 Plsm IGF-1 mesurements (ng/ml) t week6 in mie on the low-ft (LF) diet injeted with PBS (hthed rs), high-ft (HF) diet injeted with PBS (white rs), or in mie on the high-ft diet injeted with inresing doses of growth hormone (GH) s follows: 0.005µg/g (spotted rs), 0.05µg/g (vertil dshed rs), 0.5µg/g (hequered rs) or 5µg/g (lk rs). Dt re expressed s men±sem. Mens shown with ommon supersript letter re not signifintly different, p>0.05 GH HF

4 1650 Dietologi (2009) 52: Tle 1 Whole ody weight, orgn weight nd the weights of four dipose tissue depots following 6 weeks of growth hormone tretment Vriles Study groups LF-PBS HF-PBS HF HF-0.05 HF-0.5 HF-5 n Body weight (g) Pre-tretment 28.7± ± ± ± ± ±1.83 End of tretment 28.8± ± ± ± ± ±1.42 Orgn weight (g) Liver 1.49± ± ± ± ± ±0.08 Hert 0.14± ± ± ± ± ±0.01 Kidney 0.34±0.02, 0.36±0.01, 0.31± ±0.01, 0.33±0.02, 0.38±0.01 Spleen 0.19± ± ± ± ± ±0.02 Adipose depot (g) Suutneous 0.18± ± ± ± ± ±0.13 Epididyml 0.41± ± ± ± ± ±0.10 Retroperitonel 0.20± ± ± ± ±0.05, 0.43±0.02, Mesenteri 0.12± ± ± ± ±0.13, 0.60±0.06, Dt re mens±sem Mens in the sme row without ommon supersript letter re signifintly different (p<0.05) HF, high-ft diet; LF, low-ft diet with lower dose growth hormone ontinued to gin ft mss during the 6 weeks of growth hormone tretment. Thus the totl ft mss hnge fter 6 weeks of injetions ws net gin of 3.7±0.4 g (12.5%) in the HF-PBS group, while the HF-5 group hd net loss of 4.1±0.8 g ( 11.2%) over the sme period; this represents differene of 8 g etween untreted high-ft fed mie nd those mie treted with the Ft mss umultion (g) 6 4,,,, 2,,,,, d d Tretment time (weeks) Fig. 2 Ft mss umultion y week ompred with ft mss determined immeditely prior to tretment of mie on the low-ft (LF) diet injeted with PBS (hthed rs), high-ft (HF) diet injeted with PBS (white rs), or in mie on the high-ft diet injeted with inresing doses of growth hormone (GH) s follows: 0.005µg/g (spotted rs), 0.05µg/g (vertil dshed rs), 0.5µg/g (hequered rs) or 5µg/g (lk rs). Dt re expressed s men±sem. Mens within eh week with ommon supersript letter re not signifintly different (p>0.05) highest level of growth hormone. In terms of per ent ody ft, the high-dose growth hormone tretment ounted for lrge redution when ompred with high-ft fed ontrol mie, with the ltter hving 37.4% men ody ft, while mie reeiving high-dose growth hormone (5μg g 1 dy 1 ) hd 21.4% men ody ft. Len mss differenes were lso seen. With the highest dose of growth hormone, signifint inreses in len mss were oserved (F=[5, 43]=66.1, p<0.001) (Fig. 3). Unlike Len mss umultion (g) 5 4 d ,,, d,, d,, Tretment time (weeks) Fig. 3 Len mss umultion y week ompred with len mss determined immeditely prior to tretment of mie on the low-ft (LF) diet injeted with PBS (hthed rs), high-ft (HF) diet injeted with PBS (white rs), or in mie on the high-ft diet injeted with inresing doses of growth hormone (GH) s follows: 0.005µg/g (spotted rs), 0.05µg/g (vertil dshed rs), 0.5µg/g (hequered rs) or 5µg/g (lk rs). Dt re expressed s men±sem. Mens within eh week with ommon supersript letter re not signifintly (p>0.05),,,,

5 Dietologi (2009) 52: ft mss redution, whih peked fter 2 weeks of tretment, len mss ontinued to inrese over the entire 6 weeks of high-dose growth hormone tretment. At the end of the 6 weeks, the mie in the high-dose growth hormone tretment group hd gined men of 3.9 g of len mss. The mounts of len mss in the HF-PBS, HF , HF-0.05 nd HF-0.5 groups did not differ signifintly (F [5, 43]=46.620, p>0.05) t the end of the 6 week tretment phse. Fluid s determined y nuler mgneti resonne ws mesured, ut did not hnge signifintly with growth hormone tretment regrdless of dose (dt not shown). Gluose, insulin nd gluose tolerne After 6 weeks of growth hormone injetions, lood gluose levels were ltered signifintly mong groups (Fig. 4). The mie with the two highest doses of growth hormone hd signifintly redued lood gluose levels ompred with PBS-treted mie on the high-ft diet. Surprisingly, the highest dosge of growth hormone deresed fsting lood gluose to level similr to those of non-dieti mie fed the low-ft diet (Fig. 4). Gluose tolerne tests reveled similr pttern, with improvements in gluose metolism in mie on higher doses of growth hormone (Fig. 4). However, plsm insulin levels showed different trend (Fig. 4). After 6 weeks of injetions, ll high-ft fed groups hd signifintly elevted plsm insulin levels ompred with low-ft-fed ontrol mie. Growth hormone tretment, regrdless of dosge, hd no effet on plsm insulin levels. Adipokine nd ytokine levels Plsm smples from the LF- PBS, HF-PBS nd the HF-5 growth hormone tretment groups were evluted for ytokine nd dipokine levels (Tle 2). Compred with LF-PBS mie, high-ft feeding resulted in signifint elevtion in insulin, leptin, resistin, tissue plsminogen tivtor inhiitor-1 (tpai-1) nd grnuloyte-mrophge olony-stimulting ftor. In high-ft fed mie treted with growth hormone, signifint redution in plsm levels of leptin nd tpai-1 ws hieved. In the HF-5 group, only tpai-1 ws redued with growth hormone tretment to levels similr to those in LF- PBS mie. Orgn weights Men orgn weights re shown in Tle 1. The weights of hert (F [5, 43]=1.644, p>0.05), liver (F [5, 43] =1.560, p>0.05) nd spleen (F [5, 43] =1.721, p>0.05) were not ltered mong ny of the tretment groups. Kidney weight in the HF group ws signifintly less thn in the HF-5 group; however, these groups were not signifintly different from the remining tretment groups (F [5, 43]=2.914, p>0.05). Compred with the HF- PBS group, the weight of the four dipose depots ws signifintly deresed upon tretment with the highest dose of growth hormone. The suutneous nd mesenteri ft pds showed the gretest per ent derese ( 59% nd 51% respetively) with growth hormone tretment, while the epididyml ft showed the smllest per ent derese ( 36%). Liver triylglyerol Liver triylglyerol levels were signifintly elevted with high-ft feeding (HF-PBS) ompred with LF-PBS ontrols (Fig. 5). Although no effet ws seen with the low growth hormone doses, triylglyerol levels in the livers of HF-0.5 nd HF-5 mie were signifintly lower thn those in HF-PBS ontrols nd, in ft, were sttistilly similr to those in LF-PBS ontrols, suggesting omplete reversl of ftty livers. Gluose (mmol/l) ,000 11, 600 3, , , Insulin (pmol/l) AUC (mmol/l h 1 ) 2, ,500 GH GH GH LF HF LF HF LF HF Fig. 4 Gluose metolism vriles following 6 weeks of growth hormone (GH) tretment. Fsting lood gluose, fsting plsm insulin nd intrperitonel gluose tolerne test in mie on the lowft (LF) diet injeted with PBS (hthed rs), high-ft (HF) diet injeted with PBS (white rs), or in mie on the high-ft diet injeted with inresing doses of GH s follows: 0.005µg/g (spotted rs), 0.05µg/g (vertil dshed rs), 0.5µg/g (hequered rs) or 5µg/g (lk rs). Dt re expressed s men±sem. Mens shown with ommon supersript letter re not signifintly different, p>0.05

6 1652 Dietologi (2009) 52: Tle 2 Fsting plsm mesurements of low-ft diet-fed mie injeted with PBS (LF-PBS), high-ft diet-fed mie injeted with PBS (HF-PBS) nd high-ft diet-mie treted with 5μg growth hormone g 1 dy 1 (HF-5) Vriles LF-PBS HF-PBS HF-5 p vlue p vlue p vlue n Adipokine pnel Leptin (pg/ml) 637±146 16,839±2,213 10,189±1, Resistin (pg/ml) 353±26.2 1,275±92.4 1,099± tpai-1 (pg/ml) 803±265 1,708± ± Cytokine pnel GM-CSF (pg/ml) 89.3± ± ± MCP-1 (pg/ml) 75.0± ± ± TNF-α (pg/ml) 8.75± ± ± Dt re mens±sem For LF-PBS vs HF-PBS; for HF-PBS vs HF-5; for LF-PBS vs HF-5 GM-CSF, grnuloyte mrophge olony-stimulting ftor; MCP-1, monoyte hemotti protein-1 Disussion Growth hormone tretment resulted in signifint improvements in ody omposition with higher doses showing the gretest improvements. Over the 6 week tretment period, signifint redution in ft mss nd signifint inrese in len mss were oserved, even though these mie hd free ess to high-ft diet during the tretment period. Surprisingly, growth hormone tretment signifintly improved severl, ut not ll inditors of dietes. Thus the higher doses of growth hormone drstilly improved fsting lood gluose levels nd gluose tolerne in the high-ft fed mie, reduing them to the level seen in the low-ft fed ontrols. Growth hormone tretment lso redued liver triylglyerol ontent. Despite tretment with inresing doses of growth hormone, hyperinsulinemi did not worsen. Triylglyerol ontent (mg/g) GH LF Fig. 5 Triylglyerol ontent of livers from mie on the low-ft (LF) diet injeted with PBS (hthed rs), high-ft (HF) diet injeted with PBS (white rs), or in mie on the high-ft diet injeted with inresing doses of growth hormone (GH) s follows: 0.005µg/g (spotted rs), 0.05µg/g (vertil dshed rs), 0.5µg/g (hequered rs) or 5µg/g (lk rs). Dt re expressed s men±sem. Mens shown with ommon supersript letter re not signifintly different, p>0.05 HF Severl interesting oservtions regrding ody omposition were oserved. First, improvements in ody omposition were seen t the highest two doses of growth hormone, even though only the highest dose of growth hormone resulted in elevted irulting IGF-1 levels. Thus, the hnges seen in ody omposition ould e due to the tion of growth hormone or to the utorine or prrine tions of IGF-1. The importne of the utorine or prrine prodution of IGF-1 hs een well doumented in the liver insulin-like growth ftor-1-defiient mouse model [20]. Another possiility is tht the level of free, ut not totl IGF-1 ould hve een ltered, sine we only mesured nd reported totl IGF-1 in this study. Seond, not ll dipose depots ppered to e eqully responsive to the growth hormone tretment. The suutneous (inguinl) nd viserl (mesenteri) ft pds were the most impted, while the epididyml ft pd ws lest ltered with growth hormone tretment. Depot-speifi effets of growth hormone hve een reported previously [21, 22]. Thus, these dt further support the notion tht ll ft depots should e evluted independently in studies with growth hormone or other hormonl tretments. Finlly, the timing of the hnge in len mss vs ft mss ws not the sme. The dereses in ft mss oserved for the highest dose of growth hormone were seen s erly s 2 weeks fter initition of growth hormone injetion. In ontrst, the gins in len mss eme more prominent with eh dditionl week of tretment. The ility of growth hormone to protet mie from diet-indued ft umultion nd to rediret nutrient prtitioning wy from dipose tissue nd into len tissue hs een reported previously in growth hormone trnsgeni mie [21, 23]. However, oth of these studies used mie engineered to hve hronilly elevted growth hormone levels, representing more of n romegli stte, nd not dily dosing therpy s performed in this study. Overll,

7 Dietologi (2009) 52: our results show tht ute tretment of growth hormone n rdilly shift ody omposition nd result in signifint len mss gins. It is well known tht growth hormone n exhiit dietogeni or nti-insulin tivities [24]. Indeed, it hs een suspeted to use insulin resistne in tissues ontining oth growth hormone nd insulin reeptors, suh s liver, musle nd ft, ll of whih [25 28] hve een shown to e ffeted y ute growth hormone dministrtion nd to exhiit insulin resistne. The speifi mehnisms responsile for insulin resistne used y growth hormone remin unknown. While some reent findings suggest tht phosphoinositide 3-kinse plys pivotl role [29, 30], others rgue ginst this [27, 28]. Oesity n lso led to n insulin-resistnt stte; thus, one might expet tht growth hormone tretment of oese mie would exerte insulin resistne. While the high-ft diet resulted in oesity nd type 2 dietes, growth hormone tretment with high-ft diet tully improved fsting lood gluose nd gluose tolerne. Numerous linil studies hve reported the impt of growth hormone tretment of oesity on gluose metolism, with mixed results. The findings reported here re not unpreedented, sine improved gluose metolism hs een desried in three of 24 linil trils [5 7]. However, mny studies report tht growth hormone tretment of oesity hs either little to no effet [8, 9, 11, 12] or negtive effets [31 34] on gluose metolism. A reent metnlysis ompring 24 studies in whih growth hormone ws used to tret oesity reveled smll inrese in fsting plsm gluose nd fsting insulin [35]. However, the uthors stte tht the negtive impt on gluose nd insulin ws most notle in studies using interventions of short durtion. Studies with longer tretment regimes showed lesser effet on insulin levels. Furthermore, only few studies inluded dt on overll insulin resistne nd glyemi [35]. Surprisingly, this met-nlysis lso indited tht HOMA-insulin resistne (IR) nd HA 1 levels were improved. It onluded tht the effet of growth hormone therpy on gluose homeostsis in oesity requires further evlution in long-term studies [35]. While severl studies hve reported n inrese in insulin nd gluose levels, studies reporting insulin sensitivity (usully y mesuring gluose disposl rte) generlly gree tht it is either unffeted or improved y growth hormone tretment [5, 7, 36]. One explntion for the disrepnies in the literture my e the inlusion or exlusion riteri of individuls with dietes or the degree of insulin resistne. While more thn two dozen linil trils hve used growth hormone to tret oesity, only two hve reported improvements in gluose nd insulin levels, nd insulin sensitivity [5, 6]. Coinidently, these two studies speifilly trgeted individuls with type 2 dietes. Therefore, possile explntion for our results, with reversl of hyperglyemi nd gluose intolerne with growth hormone tretment, is tht in dieti stte with elevted gluose nd gluose intolerne, the dietogeni tions of growth hormone re overshdowed y its enefiil impt on ody omposition. Sine the mie in our study, s well s humns with type 2 dietes [5, 6], responded well to growth hormone therpy despite hving hyperglyemi nd impired gluose tolerne prior to tretment, suh n explntion is plusile. Future studies in this model, using mie with vrying degrees of insulin resistne nd etter ssessing the degree of insulin resistne with gluose nd insulin lmping, will help ddress this hypothesis. Clinil studies hve used wide rnge of growth hormone doses to tret oesity (5 123 IU/week [~ mg kg 1 dy 1 ]) [11, 37], while doses used for humns with type 2 dietes were 5 11 IU/week (~ mg kg 1 dy 1 )[5, 6]. Unfortuntely, diret omprison of dosges used in this study to those used for humns is not pproprite nd n only e onsidered in terms of reltive dosge for severl resons. There re severl speies differenes s mie were treted insted of humns, ovine growth hormone ws used insted of humn growth hormone nd the system used ws not homogenous (i.e. mie treted with ovine growth hormone s opposed to humns treted with humn growth hormone). This is one of the resons why suh rod rnge of growth hormone levels ws used in this study to ensure we rehed dosge tht ws iologilly relevnt. Importntly, the reominnt ovine growth hormone used in this study ws tive, sine dose-dependent effets were seen in vivo. However, it ppers tht the two lowest doses of growth hormone (0.05 nd 0.005μg/g) provided too little growth hormone tivity to result in ny physiologil hnges mesured. The two highest doses of growth hormone (0.5 nd 5.0μg/g) improved ody omposition, gluose nd liver triylglyerol ontent, while only the highest dose (5.0μg/g) ws high enough to elevte totl IGF-1. Thus these dt suggest tht it is possile to improve ody omposition, gluose nd liver triylglyerol ontent with dosges of growth hormone tht re elow the threshold required to elevte totl IGF-1. For oese dult humns treted with growth hormone, Mekl et l. [35] demonstrted tht doses ove the medin vlue of 31.1 IU/week (10.4 mg/week or mg kg 1 dy 1 ) showed stronger trend towrds inresing serum IGF-1. Bsed on our results nd those of Mekl et l. [35], we would suggest strting dosge pprohing ut not exeeding 30 IU per week, susequently titrting ppropritely on the sis of individul IGF-1 response to growth hormone. In other words, optiml therpy would e the highest level of growth hormone tht does not result in elevtion of IGF-1 ove levels pproprite for the ptient (ge nd sex onsidered).

8 1654 Dietologi (2009) 52: It is diffiult to determine (due to speies differenes) extly how long tretment should e given to humns with type 2 dietes. However, sine hnges in ody omposition ourred well efore the 6-week endpoint in mie, it is likely tht the improvements to ody omposition were responsile for the improvements we oserved in gluose metolism. In the two humn studies using growth hormone to tret ptients with type 2 dietes, only 12 weeks of growth hormone therpy ws needed to improve ody omposition, fsting lood gluose, insulin nd insulin sensitivity [5, 6]. While Ahn et l. [6] did not report progression of their dt during the 12 weeks, Nm et l. [5] reported gluose nd insulin levels t 4-week intervls during the 12-week therpy. They [5] found tht n initil inrese in insulin ourred t 4 weeks, followed y progressive derese in insulin t 8 nd 12 weeks. Similrly, gluose only deresed minimlly t 4 weeks, with steeper delines ourring t 8 nd 12 weeks In humns, therefore, it ppers tht more thn 4 weeks were needed nd 12 weeks were suffiient to see improvements. Tken together, it seems suffiient time should e given for growth hormone to improve ody omposition, s improvements in gluose metolism re likely to e result of ltered ody omposition. In summry, these dt show tht type 2 dietes n e signifintly improved with growth hormone tretment in mouse model of diet-indued oesity nd type 2 dietes. These results re similr to those shown in humns with type 2 dietes [5, 6]. The use of growth hormone to tret humn type 2 dietes remins highly ontroversil, despite two linil trils hving shown positive impt on gluose homeostsis [5, 6]. It would therefore e importnt to develop n niml model, s shown here, to investigte more fully the impt of growth hormone tretment on speifi tissues nd onditions, suh s ftty liver, s well s to etter determine the mehnisms y whih growth hormone impts on type 2 dietes. While our dt do not dvote the use of growth hormone to tret humns with type 2 dietes, they do provide model to etter evlute the effiy nd sfety of its use nd my e used to eluidte the mehnisms involved. Aknowledgements This work ws supported in prt y the Stte of Ohio s Eminent Sholr Progrm, whih inludes gift from lumni of Ohio University (M. nd L. Goll), nd y grnts from the Ohio University Reserh Committee, AMVETS, the Dietes Reserh Inititive nd the BioMoleulr Innovtion nd Tehnology Prtnership t Ohio University, the World Anti-Doping Ageny nd the Ntionl Institutes of Helth (DK nd AG ). The uthors thnk G. Bogosin of the Monsnto Compny for donting the reominnt ovine growth hormone used in this study; thnks lso to R. Mle-Brune for her helpful omments. Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. Referenes 1. Bengtsson BA, Johnnsson G (1998) The use of growth hormone in dults: review of the lst 10 yers, the present nd perspetive for the future. Growth Horm IGF Res 8(Suppl B): Veldhuis JD, Roemmih JN, Rihmond EJ et l (2005) Endorine ontrol of ody omposition in infny, hildhood, nd puerty. Endor Rev 26: Houssy B (1936) The hypophysis nd metolism. N Engl J Med 214: Jorgensen JO, Krg M, Jessen N et l (2004) Growth hormone nd gluose homeostsis. Horm Res 62(Suppl 3): Nm SY, Kim KR, Ch BS et l (2001) Low-dose growth hormone tretment omined with diet restrition dereses insulin resistne y reduing viserl ft nd inresing musle mss in oese type 2 dieti ptients. Int J Oes Relt Met Disord 25: Ahn CW, Kim CS, Nm JH et l (2006) Effets of growth hormone on insulin resistne nd therosleroti risk ftors in oese type 2 dieti ptients with poor glyemi ontrol. Clin Endorinol (Oxf) 64: Johnnsson G, Mrin P, Lonn L et l (1997) Growth hormone tretment of dominlly oese men redues dominl ft mss, improves gluose nd lipoprotein metolism, nd redues distoli lood pressure. J Clin Endorinol Met 82: Clemmons DR, Snyder DK, Willims R, Underwood LE (1987) Growth hormone dministrtion onserves len ody mss during dietry restrition in oese sujets. J Clin Endorinol Met 64: Kmel A, Norgren S, Elimm A, Dnielsson P, Mrus C (2000) Effets of growth hormone tretment in oese prepuertl oys. J Clin Endorinol Met 85: Tomlinson JW, Crtree N, Clrk PM et l (2003) Low-dose growth hormone inhiits 11 et-hydroxysteroid dehydrogense type 1 ut hs no effet upon ft mss in ptients with simple oesity. J Clin Endorinol Met 88: Alert SG, Moordin AD (2004) Low-dose reominnt humn growth hormone s djuvnt therpy to lifestyle modifitions in the mngement of oesity. J Clin Endorinol Met 89: Rihelsen B, Pedersen SB, Kristensen K et l (2000) Regultion of lipoprotein lipse nd hormone-sensitive lipse tivity nd gene expression in dipose nd musle tissue y growth hormone tretment during weight loss in oese ptients. Metolism 49: Thompson JL, Butterfield GE, Gylfdottir UK et l (1998) Effets of humn growth hormone, insulin-like growth ftor I, nd diet nd exerise on ody omposition of oese postmenopusl women. J Clin Endorinol Met 83: Surwit RS, Kuhn CM, Cohrne C, MCuin JA, Feinglos MN (1988) Diet-indued type II dietes in C57BL/6J mie. Dietes 37: Surwit RS, Feinglos MN, Rodin J et l (1995) Differentil effets of ft nd surose on the development of oesity nd dietes in C57BL/6J nd A/J mie. Metolism 44: Qiu L, List EO, Kophik JJ (2005) Differentilly expressed proteins in the pnres of diet-indued dieti mie. Mol Cell Proteomis 4: List EO, Berrymn DE, Plmer AJ et l (2007) Anlysis of mouse skin revels proteins tht re ltered in diet-indued dieti stte: new method for detetion of type 2 dietes. Proteomis 7: List EO, Berrymn DE, Plmer AJ et l (2007) Applition of ioinformtis nd slle omputing to perform proteomi nlysis of stomh tissue from dieti mie. Slle Computing 8:

9 Dietologi (2009) 52: Slmon DM, Fltt JP (1985) Effet of dietry ft ontent on the inidene of oesity mong d liitum fed mie. Int J Oes 9: Ykr S, Liu JL, Stnnrd B et l (1999) Norml growth nd development in the sene of hepti insulin-like growth ftor I. Pro Ntl Ad Si U S A 96: Berrymn DE, List EO, Kohn DT et l (2006) Effet of growth hormone on suseptiility to diet-indued oesity. Endorinology 147: Berrymn DE, List EO, Coshigno KT et l (2004) Compring diposity profiles in three mouse models with ltered GH signling. Growth Horm IGF Res 14: Olsson B, Bohlooly YM, Fitzgerld SM et l (2005) Bovine growth hormone trnsgeni mie re resistnt to diet-indued oesity ut develop hyperphgi, dyslipidemi, nd dietes on high-ft diet. Endorinology 146: Clemmons DR (2002) Roles of insulin-like growth ftor-i nd growth hormone in mediting insulin resistne in romegly. Pituitry 5: Rinowitz D, Klssen GA, Zierler KL (1965) Effet of humn growth hormone on musle nd dipose tissue metolism in the forerm of mn. J Clin Invest 44: Ykr S, Setser J, Zho H et l (2004) Inhiition of growth hormone tion improves insulin sensitivity in liver IGF-1- defiient mie. J Clin Invest 113: Jessen N, Djurhuus CB, Jorgensen JO et l (2005) Evidene ginst role for insulin-signling proteins PI 3-kinse nd Akt in insulin resistne in humn skeletl musle indued y short-term GH infusion. Am J Physiol Endorinol Met 288:E194 E Nielsen C, Gormsen LC, Jessen N et l (2008) Growth hormone signling in vivo in humn musle nd dipose tissue: impt of insulin, sustrte kground, nd growth hormone reeptor lokde. J Clin Endorinol Met 93: del Rinon JP, Iid K, Gylinn BD et l (2007) Growth hormone regultion of p85lph expression nd phosphoinositide 3-kinse tivity in dipose tissue: mehnism for growth hormonemedited insulin resistne. Dietes 56: Brour LA, Miznoor Rhmn S, Gurevih I et l (2005) Inresed P85lph is potent negtive regultor of skeletl musle insulin signling nd indues in vivo insulin resistne ssoited with growth hormone exess. J Biol Chem 280: Snyder DK, Clemmons DR, Underwood LE (1988) Tretment of oese, diet-restrited sujets with growth hormone for 11 weeks: effets on nolism, lipolysis, nd ody omposition. J Clin Endorinol Met 67: Snyder DK, Underwood LE, Clemmons DR (1990) Anoli effets of growth hormone in oese diet-restrited sujets re dose dependent. Am J Clin Nutr 52: Snyder DK, Underwood LE, Clemmons DR (1995) Persistent lipolyti effet of exogenous growth hormone during lori restrition. Am J Med 98: Rihelsen B, Pedersen SB, Borglum JD et l (1994) Growth hormone tretment of oese women for 5 wk: effet on ody omposition nd dipose tissue LPL tivity. Am J Physiol 266: E211 E Mekl KC, Tritos NA (2009) Effets of reominnt humn growth hormone therpy in oesity in dults: met-nlysis. J Clin Endorinol Met 94: Frno C, Brnderg J, Lonn L et l (2005) Growth hormone tretment redues dominl viserl ft in postmenopusl women with dominl oesity: 12-month pleo-ontrolled tril. J Clin Endorinol Met 90: Luidi P, Prlnti N, Piioni F, Snteusnio F, de Feo P (2002) Short-term tretment with low doses of reominnt humn GH stimultes lipolysis in viserl oese men. J Clin Endorinol Met 87: