Regulating Hypothalamus Gene Expression in Food Intake: Dietary Composition or Calorie Density?

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1 Originl rticle Obesity nd Metbolic Syndrome Dibetes Metb J 7;4:-7 pissn eissn DIETES & METOLISM JOURNL Regulting Hypothlmus Gene Expression in Food Intke: Dietry Composition or Clorie Density? Mi Jng, So-Young Prk, Yong-Woon Kim, Seung-Pil Jung, Jong-Yeon Kim Obesity-Dibetes dvnced Reserch Center, Yeungnm University College of Medicine, Degu, Kore ckground: The proportion of sturted ftty cids/unsturted ftty cids in the diet seems to ct s physiologicl regultion on obesity, crdiovsculr diseses, nd dibetes. Differently composed ftty cid diets my induce stiety of the hypothlmus in different wys. However, the direct effect of the different ftty cid diets on stiety in the hypothlmus is not cler. Methods: Three experiments in mice were conducted to determine whether: different compositions of ftty cids ffects gene mrn expression of the hypothlmus over time; different types of ftty cids dministered into the stomch directly ffect gene mrn expression of the hypothlmus; nd ft composition chnges in the diet ffects gene mrn expression of the hypothlmus. Results: The type of ft in cses of purified ftty cid dministrtion directly into the stomch my cuse chnges of gene expressions in the hypothlmus. Gene expression by dietry ft my be regulted by clorie mount ingested rther thn weight mount or type of ft. Conclusion: Therefore, the clorie density fctor of the diet in regulting hypothlmic gene in food intke my be detrimentl, lthough the possibility of type of ft cnnot be ruled out. Keywords: Dietry fts; Eting; Gene expression; Hypothlmus INTRODUCTION Obesity is globl helth epidemic. high-ft/cloric diet leds to overweight nd obesity, which re strongly ssocited with the incresing prevlence of more severe chronic diseses []. In simple terms, obesity occurs when energy intke exceeds expenditure []. The centrl nervous system hs mechnisms to mintin homeostsis of body weight, which is tightly regulted by centrl nd peripherl signls reflecting the vilbility of body energy []. Despite these mechnisms, when humns or rodents re exposed to clorie-rich diets, these mechnisms fil nd they become obese [3]. In both mle nd femle rodents, high ft diet compred to low-ft diet stimultes weight gin, lrgely due to incresed cloric intke [4]. Diets tht re rich in clories re considered to be n importnt cuse of viscerl obesity nd insulin resistnce [4], but the effect of dietry ft on disese risk is controversil. Most studies hve found tht diet low in sturted ft or diets in which sturted ft is prtilly replced with polyunsturted ft decreses the risks of crdiovsculr diseses, dibetes, or deth [5-7]. However, some reserch indictes tht sturted ft is not ssocited with those diseses [8,9]. The proportion of sturted ftty cids (SFs)/unsturted ftty cids in the diet seems to ct s kind of physiologicl regultion on obesity, crdiovsculr diseses, nd dibetes. Sturted ft is relted to decresed insulin signling t the level of centrl nervous system, cusing insulin resistnce in the brin nd overeting; thus, leding to obesity [], which rises the question of whether n-3 polyunsturted ftty cids (n-3 PUFs) hve different regultory roles from other ftty cids [,]. Intke of fish nd long-chin n-3 PU- Corresponding uthor: Jong-Yeon Kim Obesity-Dibetes dvnced Reserch Center, Yeungnm University College of Medicine, 7 Hyeonchung-ro, Nm-gu, Degu 445, Kore E-mil: jykim@ynu.c.kr Received: Jul. 8, 6; ccepted: Sep. 3, 6 This is n Open ccess rticle distributed under the terms of the Cretive Commons ttribution Non-Commercil License ( which permits unrestricted non-commercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Copyright 7 Koren Dibetes ssocition

2 Jng M, et l. Fs in young dulthood is inversely ssocited with metbolic syndrome lter in life []. The n-3 PUFs re beneficil in the secondry prevention of coronry hert disese, hypertension, nd type dibetes mellitus [3], re negtively ssocited with the plsm leptin level [4], nd reduce the expression of leptin in vivo [5,6]. Leptin regultes food intke nd energy expenditure through neurl nd endocrine mechnisms medited by the leptin receptors nd their downstrem stiety regultors, such s orexigenic neuropeptide Y (NPY) nd norexigenic pro-opiomelnocortin (POMC) [7]. Differently composed ftty cid diets my induce stiety of the brin in different wys. However, the direct effect of the different ftty cid diets on stiety in the hypothlmus is not cler. In this study, gene expressions ssocited with the food intke regultion of the hypothlmus were nlyzed using reltime polymerse chin rection (PCR) to exmine the effect of different types of purified ftty cid nd dietry ft composition on hypothlmic gene expressions. Thus, the objective of this study ws to ssess whether the ft composition chnge in the diet differentilly ffects hypothlmic gene expressions. METHODS nimls Mle C57L/6N mice were purchsed from the Kotech Experimentl niml Center (Pyeongtek, Kore), weighing 5 to 3 g ( weeks of ge). nimls were mintined t C± C, humidity of 55%±5%, with -hour:-hour light-drk cycle (7: M to 7: PM). ll pplicble institutionl nd governmentl regultions concerning the ethicl use of nimls were certified before nd during this reserch nd pproved by the Institutionl niml Cre nd Use Committee of Yeungnm University. Tble. Experimentl diet compositions Ingredient Group CHO SF PUF Unit, g/kg Chow 7 Sucrose utter b Fish oil c Unit, % kcl Crbohydrte Ft Protein Cloric vlue, kcl/g CHO, high crbohydrte diet group; SF, high sturted ftty cid diet group; PUF, high n-3 polyunsturted ftty cid diet group. Chow, mouse chow from Hrln Lbortories, b utter, Seoulmilk, c Fish oil, Member s Mrk. Experimentl designs Experiments were designed to evlute the effect of ftty cid profiles on hypothlmic gene expressions in food intke regultion of mice. For this gol, three experiments were conducted to determine whether: () ftty cid ingestion ffects gene mrn expression of the hypothlmus over time; () different types of ftty cids dministered into the stomch directly ffect gene mrn expression of the hypothlmus; nd (3) ft composition chnges in the diet ffect gene mrn expression of the hypothlmus. Ftty cids (.4 mg/g body weight) dissolved in dimethyl sulfoxide (DMSO, 5 μl/g body weight; Sigm, St. Louis, MO, US) in experiments nd were dministered into the stomch directly fter overnight fsting. DMSO ws used becuse of poor ftty cid solubility in other solvents, such s ethnol nd wter. Mice were divided into control (DMSO) nd SF dministrtion groups for experiment, nd gene expressions were mesured t (n=4),.5 (n=3), (n=3), (n=3), nd 5 hours (n=3) fter dministrtion of ftty cid; control (DMSO, n=), SF (n=9), nd n-3 PUF (n=9) groups for experiment ; nd high crbohydrtes (CHO; n=9) s control, high SF (n=6), nd high n-3 PUF (n=6) diets groups for experiment 3. In experiment, SF ws steric cid nd n-3 PUF ws linolenic cid. In experiment 3, diets were provided from 6: PM to : M the next dy fter 9 hours of fsting prior to the diet. The detils of the composition of diets re presented in Tble. nimls hd free ccess to food nd wter for 5 hours. Mice were nesthetized by intrperitonel injections of pentobrbitl sodium (5 mg/kg body weight) immeditely fter the diet, nd the hypothlmus ws excised crefully. Quntittive rel-time PCR The hypothlmus ws homogenized in TRI regent (Sigm- ldrich, St. Louis, MO, US) using sonictor (rnson Sonifier 45; rnson Ultrsonics Corp., Dnbury, CT, US). RN underwent reverse trnscription to cdn from μg of totl RN using High-Cpcity cdn Reverse Trnscription Kit (pplied iosystems, Foster City, C, US). Quntittive rel- Dibetes Metb J 7;4:-7

3 Hypothlmus gene expression by dietry ft composition time PCR ws performed using the rel-time PCR 75 system nd Power SYR Green PCR Mster Mix (pplied iosystems) ccording to the mnufcturer s instructions. The expression level of β-ctin ws used s n internl control. The rections were incubted t 95 C for minutes, followed by 45 cycles of 95 C for 5 seconds, 55 C for seconds, nd 7 C for 35 seconds. The primers were: POMC sense GG CCT GC CG TGG G T; nti-sense GG CC CG CTC CC C T; NPY sense TCC GCT CTG CG CC TC T; nti-sense TGC TTT CCT TC TT G GGT CTG ; gouti-relted protein (grp) sense TGC TC TGC CGC TTC TTC nti-sense CTT TGC CC C C TC; nd β-ctin sense TC TGC CCT GGC TCC TG C; nti-sense TGG C GTG GG CC GG TG (ioneer, Dejeon, Kore). POMC NPY 6 4 b Sttisticl nlysis Vlues re expressed s men±stndrd error. The significnce of the differences mong groups ws evluted by one-wy nlysis of vrince (NOV). When NOV reveled significnt differences, post hoc nlysis ws conducted vi the Newmn-Keuls multiple rnge test. Null hypotheses of no difference were rejected if P vlues were less thn.5. RESULTS Experiment. time series of hypothlmic gene expressions by dministrtion of sturted ftty cid dissolved in DMSO directly into the stomch To the best of our knowledge, there re no publictions exmining hypothlmic gene mrn expressions fter dministrtion of ftty cids in time series. Therefore, experiment ws conducted to ssess the pek time of gene expressions fter the dministrtion of ftty cid into the stomch directly. The norexigenic POMC mrn expression incresed grdully up to hours nd decresed t 5 hours fter dministrtion of SF into the stomch (Fig. ). There ws no significnt difference between orexigenic NPY nd grp genes expressions from.5 to 5 hours fter ftty cid dministrtion (Fig. ). grp.5 5 Time (hr) Fig.. Time series of () pro-opiomelnocortin (POMC), () neuropeptide Y (NPY), nd (C) gouti-relted protein (grp) mrn expressions fter dministrtion of sturted ftty cid into the stomch. Vlues re men±stndrd error for three to four mice per group. P<. vs. other groups, b P<. vs. other groups. Experiment. Hypothlmic gene expressions by different ftty cids dministrtion dissolved in DMSO directly into the stomch The POMC, NPY, nd grp mrn expressions were mesured hours fter dministrtion of two types of ft dissolved in DMSO directly into the stomch. The POMC mrn expressions of the hypothlmus in the group given SF significntly incresed to 38% (P<.) nd n-3 PUF to 5% (P<.5), compred with the control group. The gene expression in the n-3 PUF group ws lower (P<.5) thn in the SF group. The NPY mrn expression of the hypothlmus in the SF group did not differ significntly from the control, wheres gene expressions decresed to 59% in the n-3 PUF group. The gene expression in the n-3 PUF group ws lower (P<.) thn in the SF group (Fig. ). The grp mrn expression of the hypothlmus in the SF group did not differ significntly from the control, wheres the gene expressions decresed to 63% in the n-3 PUF group. The gene expression in the n-3 PUF group ws lower (P<.) thn in the SF group (Fig. ). Experiment 3. Hypothlmic gene expressions nd food consumptions by ft composition chnge in the diet over 5 hours The food intke ws mesured. The mount of food ingested in C Dibetes Metb J 7;4:-7 3

4 Jng M, et l. 3 POMC b POMC b,c Control SF PUF CHO SF PUF NPY c NPY Control SF PUF CHO SF PUF grp c grp Control SF PUF Group C CHO SF PUF Group C Fig.. Reltive () pro-opiomelnocortin (POMC), () neuropeptide Y (NPY), nd (C) gouti-relted protein (grp) mrn expressions hours fter dministrtion of sturted (high sturted ftty cid [SF]) or n-3 polyunsturted ftty cid (PUF) into the stomch. Vlues re men±stndrd error for nine to mice per group. P<. vs. control, b P<.5 vs. control or SF, c P<. vs. control or SF. the CHO group ws higher (P<.) thn in high-ft diet groups, but the clories ingested were lower. There were no differences of food intke nd plsm free ftty cid levels (dt not shown) between SF nd n-3 PUF groups (Tble ). The POMC mrn expressions of the hypothlmus in the SF group significntly decresed to 83% (P<.5) nd n-3 PUF group to 7% (P<.) compred with the CHO group. The gene expression in the n-3 PUF group ws lower (P<.5) thn in the SF group (Fig. 3). The NPY mrn expression of the hypothlmus in the SF nd the n-3 PUF groups were higher (P<.5) compred with the CHO group (3% in SF Fig. 3. Reltive () pro-opiomelnocortin (POMC), () neuropeptide Y (NPY), nd (C) gouti-relted protein (grp) mrn expressions in vrious diet fed mice. Vlues re men± stndrd error for six to nine mice per group. CHO, high crbohydrtes diet group; SF, high sturted ft diet group; PUF, high n-3 polyunsturted ftty cid diet group. P<.5 vs. CHO, b P<.5 vs. SF, c P<. vs. CHO. Tble. Food intke in CHO, SF, nd PUF diet fed mice Vrible CHO SF PUF Weight mount, g 5.3±. 4.±.6 4.±.7 Clorie intke, kcl 7.7±.7.3±.79 b 9.8±.84 b Vlues re men±stndrd error for six to nine mice per group. CHO, high crbohydrte diet group; SF, high sturted ftty cid diet group; PUF, high n-3 polyunsturted ftty cid diet group. P<., b P<.5 vs. CHO group. nd 3% in the n-3 PUF compred with CHO). There were no significnt differences between SF nd n-3 PUF groups. 4 Dibetes Metb J 7;4:-7

5 Hypothlmus gene expression by dietry ft composition Food intke (g) y=.6355x r =.849 Food intke (kcl) NPY/POMC NPY/POMC 5 y=3.685x r =.433 Fig. 4. Correltions between the rtio of neuropeptide Y (NPY)/pro-opiomelnocortin (POMC) gene expressions nd () food intke (g) or () food intke (kcl) in vrious diet fed mice. The grp mrn expression of the CHO group did not differ from other groups (99% in SF nd 94% in n-3 PUF compred with CHO) (Fig. 3). There ws strong correltion (r =.433, P<.) between the rtio of NPY/POMC gene expressions nd food intke in clorie mount, but not with food intke in weight mount (Fig. 4). DISCUSSION The present study demonstrted tht the purified different ftty cids hve differentil effects on hypothlmic gene expressions in mice, but the type of dietry ft do not. ody weight homeostsis is mintined through the blnce between energy expenditure nd energy intke. The hypothlmus in the centrl nervous system is the mster regultor of energy intke, energy expenditure, nd body weight blnce [8-]. Hormones regulte food intke nd energy expenditure through neurl nd endocrine mechnisms by the stiety regultors, such s NPY nd POMC []. In ddition to the hormonl signls entering the hypothlmus, the hypothlmic neurons lso directly sense the nutrient signls in the blood, such s hyperglycemi nd hyperlipidemi, nd ctivte the regultory processes of energy blnce in the hypothlmus [-4]. ccording to experiment, the norexigenic POMC gene expression peked hours fter the dministrtion of steric cid, nd orexigenic NPY nd grp gene expressions were lowest hour fter dministrtion of ftty cid, lthough not sttisticlly significnt. Therefore, gene expressions of experiment were mesured hours fter dministrtion of sturted nd n-3 PUF dissolved in DMSO into stomch. The norexigenic POMC gene expression of the hypothlmus in the n-3 PUF group were significntly lower thn in the SF group in experiment. The orexigenic NPY nd grp gene expressions of the hypothlmus in the n-3 PUF group were lso lower thn in the SF group. These dt seemed logiclly untenble becuse both of norexigenic nd orexigenic genes expressions in the n-3 PUF group were lower thn in the SF group. This discordnce supports the hypothesis tht hypothlmic gene expression in food regultion would be decisive by regulting which orexigenic or norexigenic pthwy is dominnt []. Therefore, this result suggests tht SF my be more norexigenic thn n-3 PUF when the norexigenic POMC gene pthwy is dominnt, nd if the orexigenic NPY nd grp gene pthwys re dominnt, SF my be more orexigenic thn n-3 PUF, lthough we do not yet know which pthwy is dominnt. The results from experiment 3 show tht POMC gene expression is higher with the SF diet thn the n-3 PUF diet, nd higher in the CHO group thn in SF nd PUF diet groups. The NPY gene expressions in the SF nd n-3 PUF groups were high compred with the CHO group. On the other hnd, the NPY gene expression in the SF group did not differ significntly from the n-3 PUF group. The grp gene expressions did not differ mong groups. Lower expression of POMC nd higher expression of NPY in the ftty cid diet groups were expected to cuse incresed food intke, but the food intke in weight mount ws not consistent with gene expression. If we use clorie mount insted of weight mount in food intke, higher orexigenic nd lower norexigenic gene expressions in SF nd n-3 PUF groups cused more clorie intke in spite of lower weight mount intke compred with the CHO group. There ws strong correltion (r =.433, P< Dibetes Metb J 7;4:-7 5

6 Jng M, et l..) between the rtio of NPY/POMC gene expressions nd food intke in clorie mount, but not with food intke in weight mount. These results suggested tht POMC nd NPY expressions re regulted by clorie mount ingested rther thn weight mount in food intke, nd grp gene would be less importnt to regulte dietry ft intke. Therefore, hypothlmus gene expression by dietry ft would be dependent on clorie density rther thn type of ft. recent report lso suggested tht consumption of hypercloric high sturted ft diet cused higher clorie intke thn normocloric sturted ft diet [5]. In conclusion, our results suggest tht the type of ft in cses of purified ftty cid dministrtion directly into the stomch my cuse chnges of gene expression in the hypothlmus, tht gene expression by dietry ft my be regulted by clorie mount ingested rther thn weight mount or type of ft. Therefore, the clorie density of the diet my be detrimentl fctor in regulting hypothlmic genes in food intke, lthough the possibility of type of ft cnnot be ruled out. CONFLICTS OF INTEREST No potentil conflict of interest relevnt to this rticle ws reported. CKNOWLEDGMENTS This reserch ws supported by grnt from the Yeungnm University Medicl Center. REFERENCES. Must, Spdno J, Cokley EH, Field E, Colditz G, Dietz WH. The disese burden ssocited with overweight nd obesity. JM 999;8: ouret SG. Erly life origins of obesity: role of hypothlmic progrmming. J Peditr Gstroenterol Nutr 9;48 Suppl :S Woods SC, D lessio D, Tso P, Rushing P, Clegg DJ, enoit SC, Gotoh K, Liu M, Seeley RJ. Consumption of high-ft diet lters the homeosttic regultion of energy blnce. Physiol ehv 4;83: Kim JY, Nolte L, Hnsen P, Hn DH, Ferguson K, Thompson P, Holloszy JO. High-ft diet-induced muscle insulin resistnce: reltionship to viscerl ft mss. m J Physiol Regul Integr Comp Physiol ;79:R Hooper L, Mrtin N, bdelhmid, Dvey Smith G. Reduction in sturted ft intke for crdiovsculr disese. Cochrne Dtbse Syst Rev 5;(6):CD Schwb U, Luritzen L, Tholstrup T, Hldorssoni T, Riserus U, Uusitup M, ecker W. Effect of the mount nd type of dietry ft on crdiometbolic risk fctors nd risk of developing type dibetes, crdiovsculr diseses, nd cncer: systemtic review. Food Nutr Res 4;58: Mozffrin D, Mich R, Wllce S. Effects on coronry hert disese of incresing polyunsturted ft in plce of sturted ft: systemtic review nd met-nlysis of rndomized controlled trils. PLoS Med ;7:e5. 8. de Souz RJ, Mente, Mrolenu, Cozm I, H V, Kishibe T, Uleryk E, udylowski P, Schunemnn H, eyene J, nnd SS. Intke of sturted nd trns unsturted ftty cids nd risk of ll cuse mortlity, crdiovsculr disese, nd type dibetes: systemtic review nd met-nlysis of observtionl studies. MJ 5;35:h Siri-Trino PW, Sun Q, Hu F, Kruss RM. Met-nlysis of prospective cohort studies evluting the ssocition of sturted ft with crdiovsculr disese. m J Clin Nutr ;9: Dvidson MH. Mechnisms for the hypotriglyceridemic effect of mrine omeg-3 ftty cids. m J Crdiol 6;98:7i-33i.. Deckelbum RJ, Worgll TS, Seo T. n-3 ftty cids nd gene expression. m J Clin Nutr 6;83(6 Suppl):5S-5S.. Kim YS, Xun P, Iribrren C, Vn Horn L, Steffen L, Dviglus ML, Siscovick D, Liu K, He K. Intke of fish nd long-chin omeg-3 polyunsturted ftty cids nd incidence of metbolic syndrome mong mericn young dults: 5-yer follow-up study. Eur J Nutr 6;55: Simopoulos P. Essentil ftty cids in helth nd chronic disese. m J Clin Nutr 999;7(3 Suppl):56S-9S. 4. Winnicki M, Somers VK, ccurso V, Phillips G, Puto M, Pltini P, Puletto P. Fish-rich diet, leptin, nd body mss. Circultion ;6: Ukropec J, Reselnd JE, Gsperikov D, Demckov E, Mdsen L, erge RK, Rustn C, Klimes I, Drevon C, Sebokov E. The hypotriglyceridemic effect of dietry n-3 F is ssocited with incresed bet-oxidtion nd reduced leptin expression. Lipids 3;38: Fn C, Liu X, Shen W, Deckelbum RJ, Qi K. The regultion of leptin, leptin receptor nd pro-opiomelnocortin expression by N-3 PUFs in diet-induced obese mice is not relted to the 6 Dibetes Metb J 7;4:-7

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