Bohobe bo phahameng bo tloaelehileng ba ho ja li-Fat boa eketseha ka li-rats (2013)

Dianne P. Figlewicz,^1,2 Jennifer L. Jay,² Molly A. Acheson, Irwin J. Magrisso,⁴ Constance H. Bophirimela,¹ Aryana Zavosh,² Stephen C. Benoit,³ 'me Jon F. Davis³

Re kile ra tlaleha hore ho ja lijo tse nang le mafura a mangata ka tsela e itekanetseng ho eketsa tšusumetso ea ho ikemela ho likhoto tse kholo. Thutong ena, re lekile litlamorao tsa phepo e matla, ea methapo ea kutlo le ea metabolism ea lijo tse nang le mafura a mangata ho liphetoho tsa likhoto tsa banna nakong ea bokhachane, nakong ea libeke tse 5-8. Re hlokometse hore phepo e phahameng ea mafura e ekelitse ho arabela ka sucrose, e neng e ikemetse ka liphetoho tsa metabolic kapa liphetoho tsa catecholamine neurotransmitter metabolites ho li-nucleus accumbens. Leha ho le joalo, maemo a AGRP mRNA ho hypothalamus a ile a phahamisoa haholo. Re bontšitse hore ts'ebetso e eketsehang ea li-neuron tsa AGRP e amana le boits'oaro bo khothalletsoang, le hore exo native (third cerebroventricular) Tsamaiso ea AGRP e hlahisitse khothaletso e kholo ea sucrose. Litlhokomeliso tsena li fana ka maikutlo a hore tlhahiso le ts'ebetso ea AGRP e atileng morothong oa hypothalamus e ka baka karabelo e eketsehang ea ho itšunya-tšunya ho bakiloeng ke ho kenella ha mafura a mangata. Kamora nako, re ile ra bapisa tšusumetso ea sucrose ho li-rats tsa batho ba baholo mme ra bona hore na ho na le sepheo se matla sa ho khetha li-rats, "se lumellanang le litlaleho tse fetileng tsa hore liphoofolo le batho ba na le khetho e batsi bakeng sa tatso e monate, ha ba bapisoa le batho ba baholo. Ka bobeli, lithuto tsa rona li fana ka maikutlo a hore lijo tsa morao-rao li bapala karolo e matla ea ho susumetsa tatso e monate ho liphoofolo tsa bongoaneng.

Lihlooho

Likarolo e ne e le likhoto tsa Albino tsa banna tse tsoang Simonsen (Gilroy, CA). Likhoto li ne li bolokoa ho chow (Laborator Rodent Diet 5001, LabDiet) kapa lijo tse nang le phepo e phahameng (31.8%; Research Diets Inc) ad libitum. Lijo tse nang le lijo li tsamaisoa ka bongata bakeng sa dikahare tsa carbohydrate (58% kcal vs. 51% kcal bakeng sa mafura a tlase le mafura a phahameng, ka ho latellana). Mofuta o tlase oa mafura o na le tsoekere ea mahala ea 6.23 gm% mme phepo e phahameng ea mafura e na le 29 gm% sucrose. Ba ne ba bolokiloe ka potoloho e lefifi ea lefifi la khanya ea khanya ka hora ea 12 hoseng. Ntle le ha ho bonts'itsoe ka tsela e 'ngoe, likhoto li tlisoa ha li le libeke tse 12, hang-hang li tloheloa,' me li beoa kamore ea ho rorisoa ho fihlela libeke tse 6 li le teng. Lekhetlong lena, ho ne ho qalile ho ja le ho ikoetlisa ka boitšoaro le liteko. Liprotheine tse ikhethang li hlalositsoe ka botlalo ka tlase, mme li khutsufatsoe ho Lethathamo 1. Hobane likhoto tse tona li feta nakong ea bokhachane ho 6^th-7^th beke ea lilemo, nako ea lithuto e ne e reretsoe ho ithuta likhoto ha li ntse li haola sethaleng sena sa nts'etsopele. Ts'ebetso tsohle tse entsoeng ho likhoto li ile tsa latela tataiso ea NIH bakeng sa tlhokomelo ea liphoofolo, 'me ea amoheloa ke Komiti ea Tlhokomelo ea Liphoofolo le Ts'ebeliso ea Komiti ea Lipatlisiso le Ntshetsopele ho VA Puget Sound Health Care System.

  

Lethathamo 1

Protocol ea liteko

Itokolle boithapollo

Protocol e akaretsang. Mekhoa e ne e ipapisitse le mekhoa ea rona e phatlalalitsoeng (Figlewicz et al., 2008; Figlewicz et al., 2006). Ts'ebetso tsohle tsa koetliso le liteko li entsoe pakeng tsa 0700 le 1200 hr. Teko eo e kenyelelitse mekhahlelo e meraro ea 2-3: ho ikoetlisa le ho ikoetlisa ka mokhoa o tsitsitseng (FR); ho buoa le ho hlaphoheloa maemong a boletsoeng (bona Lethathamo 1); le boithuto bo tsoetseng pele (PR) bo sebelisang PR algorithm ea Richardson le Roberts (Richardson & Roberts, 1996). Algorithm ea PR e hloka 1, 2, 4, 6, 9, 12, 16, 20, 28, 36, 48, 63, 83, 110, 145, 191, 251, 331, 437, 575, 759, 999, 999 ( joalo-joalo. Mechine ea khatiso ea lever bakeng sa ho tlisa meputso nakong ea thuto, 'me ke teko e matla ea ho khothaletsa le ho fumana moputso (27). Likhoto li ile tsa koetlisetsoa ho itaola 5% sucrose (0.5 ml) e isitsoeng ka pokellong ea metsi a metsi. Mabokose a sebetsang, a laoloang ke sistimi ea Med Associates (Georgia, VT), a ne a e-na le levers tse peli, empa lever e le 'ngoe feela (lever e sebetsang, e khutšoang) e ile ea kenya pampiri ea infusion. Mechine ea khatiso ho lever e ngoe (ea sa sebetseng, ea litheko) le eona e tlalehiloe. Tharollo ea sucrose e ile ea kenngoa ka setsing sa pokello ea metsi bakeng sa ts'ebeliso ea molomo (Med Associates). Koetliso ea mantlha e ile ea etsoa nakong ea mananeo a h bakeng sa matsatsi a 10 tlasa kemiso e tsoelang pele ea ho matlafatsa (FR1: mochini o mong le o mong oa khatiso o ile oa matlafatsoa), ka meputso e ka bang 50 ea sucrose e fanoeng ka thuto e ngoe le e ngoe. Seboka se seng le se seng se ne se qala ka ho kenngoa ha serethe se sebetsang le khanya ea ntlo e tšoeu e lutseng thutong eohle. Molumo oa li-5-s (2900 Hz, 20 dB kaholimo ho morao) + (7.5 W e tšoeu e kaholimo ho lever e sebetsang) e neheletsanang le pente e 'ngoe le e' ngoe, e lateloe ke nako ea mashome a 20 ka mor'a ho fana ka puo e 'ngoe le e' ngoe. Koetliso ea PR e ile ea etsoa bakeng sa kholo e ka khonehang ea 3 h / letsatsi ka matsatsi a leshome. Likopano tsa letsatsi le letsatsi li ile tsa fela kamora metsotso e 30 ea ho se na mochine oa khatiso o sebetsang o neng o arabela, ka nako eo lebone la ntlo le ileng la tima 'me' mokelli ea sebetsang a huloa.

Kameho ea AGRP ho itaola

Ha liphetho tsa rona li bonts'a keketseho ea polelo ea AGRP mRNA ka har'a likhoto tsa pubertal e fepa lijo tse nang le mafura a mangata, re ne re batla ho netefatsa hore AGRP e ka eketsa ho itaola. Likhoto tse 5-wk tsa khale-tse fepiloeng li ile tsa nkuoa ka thupelo ea FR, ea ntan'o amohela li-cannulas ka har'a k'holeseterole ea boraro ea likhoerekhoere (ICV). Kamora beke ea ho hlaphoheloa, netefatso ea ho beoa ka teko ea karabo ea ho noa ea angiotensin II (bona Figlewicz et al., [2006]), le thuto e le 'ngoe ea koetliso ea FR, koetliso e ile ea qalisoa ho PR ea tsamaiso ea tsamaiso. Kamora letsatsi la PR 1, likhoto li ile tsa abeloa se seng sa lihlopha tse peli tse bolelang hore ts'ebetso ea PR Day 1 e ne e sa fapana lipakeng tsa lihlopha tsena tse peli (koloi ea maiketsetso ea CSF, aCSF; kapa AGRP, 2 μl ea 0.01 nmol). Ba fumane liente tsa ACSF (n = 8) kapa AGRP (n = 7) ka matsatsi a PR, 2, le 5. Kakaretso ea lijo tsa letsatsi le letsatsi e ile ea eketsoa nakong ea thupelo ea PR.

Phello ea botsofaling molemong oa ho itaola

Re bapisa boitšoaro ba ho itaola lipakeng tsa likhoto tsa pubertal le batho ba baholo ba banyenyane, fepa chow kapa lijo tse mafura a 31.8%. Litšila li bile le libeke tse peli tsa ho rorisoa ho VAPSHCS vivarium (3-5wk kapa 8-10 wk). Ba fumane lijo ka nako eohle ea tlhahlobo / koetliso (4 wk). Kahoo, joalo ka boitekong ba pele, likhoto tsa pubertal li ile tsa ithutoa ka lilemo tse 5-8 wk. Bacha ba baholo ba ne ba ithutoa ba le lilemo li 10-13 wk.

Boikemisetso ba sebopeho sa 'mele

Sebopeho sa 'mele se ile sa lekanngoa ho sebelisoa lipalo tsa matla a bonoang a boholo ba' mele (QMR [Nixon et al., 2010]) ho fumana se lekanang le metsi a 'mele a likhoto ka bomong, moo ho baloang mafura a mmele a amanang. Liphoofolo tsena li ne li beoa ka mekhukhung e se nang anthime, ebe li ts'oaroa ka mochini oa QMR bakeng sa tlhahlobo ea metsotso e 2, e etsang likhato tse tharo. Lintlha li bolokiloe ho komporo e kopaneng (EchoMRI, Echo Medical Systems, Houston, TX) bakeng sa lipalo-hang tsa metsi a 'mele o felletseng, mafura le boima bo boima.

Teko ea mamello ea tsoekere ea intravenous (IVGTT)

IVGTTs e hlokometseng e ne e etsoa ka litoeba tse nang le cannulas ea IV e neng e kentsoe ka nako e telele bosiu bo bong pele ho thuto, ho sebelisoa mokhoa o thehiloeng ho tsona Frangioudakis, Gyte, Loxham, & Poucher (2008). Li-cannulas tse kenang kahare ho pelo li ile tsa kenngoa libeke tse peli pele ho thuto, joalo ka mokhoa o thehiloeng oa rona (Figlewicz et al., 2009). Mehlala ea motheo e thehiloe ho t-10 min (0.5 ml bakeng sa boikemisetso ba insulin le tsoekere, ka linako tsohle) le metsotso ea t0. Likhoto li fumane infusion ea 1 gm glucose / 2ml / kg tse fetang metsotsoana e 15-20 e lateloe ke moriana oa 0.5 ml oa saline. Mehlala ea mali e nkuoe ho 5, 15, 30, 60, 90, le 120 min. Ka lebaka la ho kotula ha catheter nakong ea ts'ebetso (ka hona, ho sitoa ho fumana disampole tsa mali), li-'m's tsa data tsa mantlha / IVGTT tse hlahisitsoeng ke li-7-8 bakeng sa likhoto tse feptjoang le tse 8 bakeng sa likhoto tse fepiloeng ka 31.8% lijo tsa mafura (Lethathamo 3). Insulin ea plasma e ne e ikemiselitse ho sebelisa li-insulin RIA kits (# RI-13K le SRI-13K, Linco) mme glucose ea plasma e ne e behiloe ho YSI Glucose Analzer). Sebaka se ka tlas'a curve (AUC) bakeng sa karabo e tsoang ho motheo se ne se baloa ho 5 min le 120 min. Lenane la HOMA le ne le baloa e le ho itima lijo (glucose [mM] × insulinm [U / L]) / 22.5 mme e baliloe ho sebelisa mehlala ea ho itima lijo e lekantsoeng bakeng sa insulin le tsoekere.

  

Lethathamo 3

Paramethara ea Metabolic¹

Ho potlakisa methapo ea metabolic

Likhoto tse tsoang ho Teko ea 1 li ile tsa potlakisoa bosiu pele ho euthanasia, matsatsi a 'maloa ka mor'a ho qeta IVGTT. Likhoto li ile tsa khahloa haholo ke ho hema moea o mongata oa isoflurane le ho holisoa. Mabili a ile a tlosoa ka potlako 'me a hoamisoa ka har'a naetrojene e nang le metsi bakeng sa ho lekanya hypothalamic peptide mRNA le li-catecholamine tsa nucleus. Plasma ea 'mele kapa serum e ne e sebelisetsoa ho lekanya insulin, tsoekere, leptin le triglycerides. Bakeng sa li-triglycerides, Point Saense ea Triglyceride GPO Kit # T7531-400 (Fisher # 23-666-418) le litekanyetso KIT # 7531-STD (Fisher # 23-666-422) li sebelisitsoe, 'me 3 μl ea serum e ile ea kenngoa ka makhetlo a mabeli. Plasma leptin e lekantsoe le Millipore Linco RIA Kit # RL 83K.

Catecholamine HPLC Mekhoa ea [Davis et al., 2008]

Likhoto li ne li khothatsoa ke anesthesia ea isoflurane, 'me masapo a kelello a ile a tlosoa kapele, a hoamisoa lehe mme a bolokoa ho −80 ° C. Li-puncheal tse nyane tsa "li-" punctal punches "tsa li-nucleus kukusanya" li ne li arotsoe ho phoofolo e 'ngoe le e' ngoe. Le ha tlhokomelo e kholo e ile ea nkuoa ho fokotsa tšilafalo ke libaka tse haufi tsa boko, ka lebaka la sebopeho le boholo ba mofuta o mong le o mong oa "mini-punch" mokhoa oa rona ha oa ka oa re lumella ho khetholla lits'oants'o (ke hore NAcc msingi le khetla) ka hara NAcc. Bakeng sa tlhahlobo e phahameng ea ts'ebetso ea metsi chromatography (HPLC), tharollo ea antioxidant (0.4 N perchlorate, 1.343 mM ethylenediaminetetraacetic acid (EDTA) le 0.526 mM sodium metabisulfite e ile ea kenyelletsoa ho mehlala e lateloang ke homogenization e sebelisang homogenizer ea ultrason tis (Biologics; Gainesville, VA ) Karolo e nyane ea tishu homogenate e qhibilihile ho 2% sodium dodecyl sulfate (SDS) (w / v) bakeng sa boikemisetso ba liprotheine (Pierce BCA Protein Reagent Kit; Rockford, IL) Ho emisoa ho setseng ho ile ha butsoa ka 14,000 g bakeng sa 20 metsing ka sehatsetsing sa sehatsetsi. supernatant e ne e boloketsoe HPLC.

Mehlala e arotsoe kh'orong ea Microsorb MV C-18 (5 Am, 4.6_250 mm, Varian; Walnut Creek, CA) mme ka nako e le ngoe ba hlahlobe DA, 3,4-dihydroxyphenylacetic acid (DOPAC) le Homovanillic acid (HVA), ka bobeli ke matšoao a bosholu ba dopamine, 5-HT le 5-HIAA. Likhomphutha li fumanoe li sebelisa detector ea li-coulometric array detector tse 12 (CoulArray 5200, ESA; Chelmsford, MA) e hokahantsoeng le Waters 2695 Solvent Delivery System (Waters; Milford, MA) tlasa maemo a latelang: sekhahla sa phallo ea 1 ml / min; ho hlwaya matla a ka bang 50, 175, 350, 400 le 525 mV, le; ho hlafuna monyetla oa 650 mV. Mokhahlelo oa mobile o ne o e-na le tharollo ea 10% ea methanol ho dist H₂O na le 21 g / l (0.1 M) citric acid, 10.65g / l (0.075 M) Na2HPO4, 176 mg / l (0.8 M) heptanesulfonic acid le 36 mg / l (0.097 mM) EDTA ka pH ya 4.1. Mehlala e sa tsejoeng e ne e arotsoe khahlano le khoro e tloaelehileng ea lintlha tse 6 le bonyane R² ea 0.97. Sampole ea taolo ea boleng e ile ea kenelletsoa ka sekhahla se seng le se seng ho netefatsa tlhahlobo ea HPLC.

Li-peptide tsa Orexigenic mRNA qPCR

Re lekantse polelo ea "peptides" ea hypothalamic e khothalletsang ho fepa mme e entsoe ka mokhoa o susumetsang le boits'oaro ba moputso (Figlewicz & Sipols, 2010): neuropeptide Y (NPY [ Hahn, Breininger, Baskin, & Schwartz, 1998; Jewett et al., 1995; Kelley, Nannini, Bratt, & Hodge, 2001]); peptide e amanang le agouti (AGRP [Aponte, Atasoy, & Sternson, 2011; Barnes, Argyropoulos, le Bray, 2010; Broberger et al., 1998; Hagan et al., 2000; Hahn, Breininger, Baskin, & Schwartz, 1998; Kaushik et al., 2011; Krashes et al., 2011; Rossi et al., 1998; Tracy et al., 2008]); le orexin (Cason et al., 2010; Choi, Davis, Fitzgerald, & Benoit, 2010). Likhoto li ile tsa ntlafatsoa ka isoflurane anesthesia, 'me boko ba tlosoa ka potlako, ba hoamisoa le ho bolokoa ho -80 ° C ho fihlela ho sebetsoa. Hypothalamus ea mahareng le ea morao-rao e ile ea khethoa ka microdissect e le block e le 'ngoe e sebelisang sefofane se batang sa AHP-1200CPV (Thermoelectric Cooling America, Chicago, Il) se neng se boloka mocheso o sa feleng oa 12 ° C nakong eohle ea ts'ebetso ea ho seha. Kakaretso ea RNA e tsoang liseleng tse nyane tse arotsoeng ka microdissected e ile ea aroloa ke Trizol reagent (Invitrogen, Carlsbad, CA) mme ea hloekisoa ho sebelisoa RNeasy Mini Kit (Qiagen, Valencia, CA) ho latela litaelo tsa moetsi. RNA e felletseng e ile ea alafshoa ho tlosa tšilafalo efe kapa efe e ka bang teng ka har'a genomic e sebelisa RNase mahala DNase (Promega, Madison, WI), mme ea lekantsoe ka ts'ebeliso ea NanoVue spectrophotometer (GE Healthcare, Cambridge, UK). Boleng ba RNA bo netefalitsoe ke standard agarose gel electrophoresis. Tlatsetso ea DNA (cDNA) e ile ea fetoloa hape (RT) ho tloha ho 1-2 μg ea RNA e felletseng ka motsoako oa li-hexamers tse sa reroang le oligo DT e sebelisang iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA). Likarabo tse seng tsa retrotranscribed (no RT) le tsona li hlophisitsoe ho tsoa sampoleng ka 'ngoe ho laola tšilafalo e ka bang teng ea genomic DNA. Tsamaiso ea cDNA le no-RT e ile ea hlapolloa, 'me 5-10 ng ea template cDNA ho tsoa sampoleng ka' ngoe e sebelisitsoe ho lekanya polelo ea mRNA ea liphatsa tsa lefutso tse khethiloeng ka nako ea nnete ea PCR e sebelisang MyIQ Real-Time PCR Detection System (Bio-Rad, Hercules Litekanyo tse phetoang tsa sampole ka 'ngoe li ne li sebelisoa ka lipoleiti tse tloaelehileng tsa iCycler 96, hammoho le li-template (NTC) ho fumana tšilafalo e ka bang teng sefapanong, ka bongata ba 20 μl reaction reaction e nang le 10 μl 2 × iQ Sybr Green Supermix (Bio- Rad, Hercules, CA), 2 μl ea 0.2-0.5 μM primer ka 'ngoe, 3 μl metsi a DEPC, le 5 μl ea template. Liketso tsohle tsa qPCR li kenyelelitse tlhahlobo ea melt e netefatsang hore na lets'oao le ikhethang ke lefe. Polelo e amanang le lefutso le leng le le leng la thahasello e baliloe ka ho ikaparella ho mothinya o tloaelehileng o tsamaeang ka poleiti ka 'ngoe' me o nkuoe ka li-dilution tsa serial tsa sampole e kopantsoeng ea cDNA, 'me e tloaetsoe ho hlahisa liphatsa tsa lefutso (acidic ribosomal phosphoprotein 36B4 bakeng sa polelo ea lefutso ho mesifa ea hypothalamic, le protheine ea mitochondrial ribosomal L32 bakeng sa polelo ho bokellaneng ba li-accumbens). Litsela tse latelang tsa primer (IDT, San Diego, CA) li sebelisitsoe ho holisa rat prero-orexin, NPY, le AGRP: Prepro-orexin, Pele: 5'-TTCCTTCTACAAAGGTTCCCT-3 ', 5'-GCAACAGTTCGTAGAGACGGAG-3'; NPY: Tsamaea, 5- TACTCCGCTCTGCGACACTACATC-3 ′; Khutlela morao: 5'-CACATGGAAGGGTCTTCAAGCC-3 '; AGRP, Tsamaea: 5'-GCAGAAGGCAGAAGCTTTGG-3 '; Khutlisa: 5'-CCCAAGCAGGACTCGTGCAG-3 '.

cFos Immunocytochemistry (ICC) le Bohlokoa

Fluorescence ICC e ne e sebelisetsoa ho supa 'mele ea lisele tse nang le tšoaetso ea "Fos-positive" le "AGRP" ho medial hypothalamus, ho latela mokhoa o thehiloeng oa rona (Figlewicz et al., 2011). Letsatsing la ho qetela (PR Day 10), likhoto li ile tsa beoa ka likamoreng tsa bona tsa boipheliso joalo ka tloaelo, bakeng sa 90 min. Hang kamora seboka sa ho qetela sa 90 min, likhoto li ile tsa thethesoa haholo ke isoflurane inhalation mme ea tšeloa ka 0.9% NaCl e lateloa ke tharollo ea serame ea 4% paraformaldehyde. Nako ea ho thethefatsa bohloko le euthanasia e ne e ipapisitse le nako e tsebahalang ea polelo ea tlhoro ea protheine ea cFos ho 90-120 min-post ea ketsahalo. Kahoo polelo ea cFos e tla bonts'a ts'ebetso ea CNS qalong ea mosebetsi oa boits'oaro, ho fapana le ho ba sephetho sa liphoofolo tse nang le mosebetsi oo. Boko bo ile ba tlosoa 'me ba hlophisoa kamora' paraformaldehyde matsatsi a 'maloa, ba ntan'o beoa 20% sucrose-PBS, ebe tharollo ea 30% ea sucrose-PBS. Boko bo ne bo arotsoe ka cryostat (Leica CM 3050S cryostat) bakeng sa immunohistochemistry. Re sebelisitse mokhoa oa rona o hlophisitsoeng oa ho lekanya protheine ea cFos e itšireletsang mafung likarolong tsa boko (Figlewicz et al., 2011). Likarolo tsa "corone" tse nang le li-slide tse kentsoeng li-12 μm li ile tsa hlatsuoa makhetlo a mararo ho saline ea phosphate (PBS, OXOID, Hampshire, England). Likarolo li ile tsa hlatsuoa metsotso e 20 ka metsi a 100% ethanol / DI (50%, v / v) a lateloa ke hlatsoetsi ea PBS, ebe e koaloa hora e le ngoe ka mocheso oa kamore ho PBS e nang le 1% e tloaelehileng ea poli kapa serum ea esele. Likarolo li ne li hlatsuoa makhetlo a mangata ho PBS ebe li kenella bosiu bo le bong ho 5 ° C ho tharollo ea mantlha ea antibody e entsoeng ka PBS. Likarolo li ne li hlatsuoa makhetlo a mararo ho PBS ebe li kenngoa ka lefifi mocheso oa kamore ho tharollo ea antibody ea bobeli e entsoeng ka PBS bakeng sa hora e le ngoe. Likarolo li ile tsa hlatsuoa kamora nako PBS, 'me ea beoa le ho koaheloa ka sekhukhu ka Vectashield e behang bohareng (Vector; Burlingame, CA). Litšoantšo tsa dijithale tsa likarolo li fumanoe ho sebelisoa microscope ea Nikon Eclipse E-4 fluorescence e hokahantsoeng le kh'amera e ts'oaroang ea Qimaging Retiga e sebelisang software ea NIS Elements (Nikon).

Re ipapisitse le lithuto tsa PCR tse bonts'ang maemo a eketsehang a AGRP mRNA, re tsepamisitse maikutlo libakeng tse amang methapo ea methapo ea kutlo, haholo-holo leqhubu la moea le arcuate nucleus (ARC). Likarolo tsa Atlas-12 μm li ile tsa hlahlojoa bakeng sa polelo ea cFos le palo ea likarolo le libaka tse tšoanang, ho ipapisitsoe le atlas ea Paxinos le Watson [2005]. Bakeng sa lipalo-palo (ka 40 × ho holisa), libaka tse tšoanang le tsa atlas li ile tsa khethoa. Software ea NIS Elements (Nikon) e ile ea sebelisoa ho nka setšoantšo sa sebaka seo u se batlang. Sebaka se ne se reretsoe ho bala le ho ba monyako oa lipalo tse ntle tsa sele. Sebaka se ts'oanang le semelo (monyako) li ile tsa sebelisoa bakeng sa likarolo tse tsoang lihlopheng tse fapaneng tsa liteko, 'me palo ea software e baloang ka lisele tse ntle (palo ea tsona) e ile ea etsoa sehlopheng se le seng bakeng sa lihlopha tsohle tsa liteko, ho thibela liphetoho lipakeng tsa seboka ka morao. Bakeng sa tlhahlobo ea lipalo-palo, lipalo li nkuoe ho tsoa ho rat e le 'ngoe feela haeba likarolo tse lumellanang kapa tse felletseng kahara sebaka ka seng li fumaneha; Lintlha tse mabapi le sebaka se itseng li ne li sa nkuoe khoebong haeba ho ne ho se na moemeli oa lefatše ka botlalo ea sebaka seo.

Ntle le palo ea cFos, immunohistochemistry ea cFos le AGRP e entsoe habeli. Hobane re ne re sa batle ho sitisa ts'ebetso ea liphoofolo, li ne li sa alafshoa pele ka colchicine ho ntlafatsa pono ea AGRP. Ka hona, pono ea li-neuron tse ntle tsa AGRP e kanna ea nyenyefatsoa. Tsamaiso e habeli ea litšila bakeng sa AGRP e ne e ts'oana le tlhahlobo ea cFos-immunoreactivity ka bo eona, ntle le hore likarolo li ne li koetsoe hora e le 'ngoe mochesong oa kamore ho PBS-5% serum ea esele. Joale, motsoako oa li-antibodies tsa mantlha tsa fos-Ab le AGRP e sebelisitsoe bakeng sa ho koahela bosiu bo le bong ho 4 ° C; Ka mokhoa o ts'oanang li-antibodies tsa bobeli li ne li le ka tharolong e le 'ngoe' me li koahetsoe hora e le 'ngoe lefifing mocheso oa kamore. Liteko tsa pele tsa ho ntlafatsa li ne li etsoa ho fumana hore na li-antibodies tsa mantlha li hlapolisoa ka nepo. Masole a mmele a mantlha a sebelisitsoeng e ne e le anti-cFos ea mmutla (1: 500) (sc-52) le anti-AGRP (1: 100) (18634) ea poli (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Li-antibodies tsa bobeli tse sebelisitsoeng e ne e le anti-mmutla oa tonki ea Cy3-conjugated (Jackson Immunoresearch; West Grove, PA), le Alexa fluor 488 esele e khahlanong le poli IgG (Molecular Probes, Eugene, OR); masole a mmele ohle a mahareng a hlapollotswe ho 1: 500.

Lipatlisiso tsa sethathamo

Lintlha tsa sehlopha li hlahisoa e le liphoso ± phoso e tloaelehileng ea se boleloang (SEM) ho sengoloa, Litafole le Lipalo. Bohlokoa bo hlalosoa e le p ≤ 0.05. Papiso ea lipalo-palo e etsoa lipakeng tsa lihlopha tsa liteko, joalo ka ha ho hlahisitsoe tlasa "Liphetho" ho sebelisoa 'testest (mohlala, lijo, lilemo, kapa papiso ea kalafo ea baithuti). 'Normalization' ea data e hlalosoa ka tsela eo e sebelisoang ka eona.

Tšusumetso ea lijo tse nang le mafura a mangata ka lebaka la tšusumetso ea peri-pubertal bakeng sa sucrose

Likhoto li fepa mafura a 31.8% nakong ea li-wks 5-8, ha li ntse li le maemong a boithatelo, li ne li e-na le tšusumetso e phahameng bakeng sa sucrose, ha e bapisoa le likhoto tse fepetsoeng chowon. Joalokaha ho bontšitsoe Setšoantšo sa 1a, ho ne ho se na phapang ea ts'ebetso nakong ea thupelo ea pele ea FR (li-frequency FRDays 1-10 mafura a sebetsang a lever, 38 ± 5 vs 39 ± 2 bakeng sa chow vs. 31.8% ja lijo, ka ho latellana). Leha ho le joalo, ha likhoto li ne li fetoleloa mesebetsing e thata ea PR, ho bile le keketseho e kholo ea palo ea mechine e hatisang ea lever, le palo ea meputso ea sucrose e nkiloeng, empa eseng ka bolelele ba thuto e akaretsang (Setšoantšo sa 1b). Ha ho na phello ea kalafo e sa foleng ea lijo ka palo ea mechine e sa sebetseng ea lever. Ha likhoto li ne li feptjoa lijo tse phahameng tsa mafura nakong ea wks 5-8 empa ka mor'a moo li khutlisetsa lijong tsa 'm'e tse nkiloeng ka koetliso ea FR le PR nakong ea libeke tsa 9-12, ho ne ho e-na le tšusumetso empa ho se na phapang ea bohlokoa mehateng ea khatiso ea lever. Kahoo, ha ho bonahale ho ka ba le tšekamelo ea boikoetliso bo fetang tekano ba lijo tse nang le phepo e phahameng tse sebelisoang nakong ea nako ea peri-pubertal. Lintlha tsa paramente ea PR tsa li-cohorts tsena li khutsufalitsoe ho Lethathamo 2. Bakeng sa ho qala ho hlakisa mochine o kenyang letsoho (s) ho keketseho e susumetsoang ke ho ja ea khothatso ea sucrose, re entse litekanyo tse ngata tsa metabolic le CNS.

  

Setšoantšo sa 1

PR e khothalletsoang ho arabela bakeng sa meputso ea sucrose e eketseha ho likhoto tsa peripubertal li fepetsoe ka 31.8% mafura a lijo (n = 8). 1a. Hohle mananeong a FR, ho ne ho se na phello ea lijo, empa phello ea lijo e bonahala ha likhoto li fetisetsoa ho paradigm ea PR. 1b. Lintlha li teng ...

  

Lethathamo 2

Kameho ea Peri-Pubertal High Fat Lijo mabapi le Ts'ebetso ea Ratios ea Ts'ebetso ea Ts'ebetso ea Ts'ebetso ea Ts'ebetso ea Ts'ebetso ea Ts'ebetso ea Ts'ebetso ea Ts'ebeliso ea Ts'ireletso

Phello ea phepelo e phahameng ea mafura a lekaneng litekanyetsong tsa metabolic

Hang hoba kamora tlhahlobo ea boits'oaro, sebopeho sa mafura a 'mele se ne se khethiloe ho likhoto tse nang le ts'ebetso ea phepo le boits'oaro bo botle nakong ea wks 5-8. Likhoto li ile tsa fumana li-cannulas tse sa tsitsang tsa 'mele bakeng sa liteko tsa (IV) tsa mamello ea tsoekere (IVGTTs). Kamora nako, ho ile ha fumanoa plasma ea sejoale-butle le serum bakeng sa mehato e meng ea metabolism. Joalokaha ho bontšitsoe Lethathamo 3, ho ne ho se na phapang ea sebopeho sa 'mele, boima ba' mele, insulin ea lijo tse potlakileng kapa mehato ea tsoekere, kutloisiso ea insulin (lipalo tsa HOMA) kapa likarabo ho IVGTT, lipakeng tsa likhoto tse matlafatsoang ke mafura. Mehato ea ho itima lijo ea leptin le litekanyo tsa triglyceride li ne li sa fapana lipakeng tsa lihlopha tsena tse peli. Kahoo, leha kalafo ea ho ja e bile le phello e kholo molemong oa ho ikemela, e bonts'a karabelo ea boitšoaro ho likhoto tse mafura tse phetseng pele ho nako.

Kameho ea phepelo e phahameng ea mafura ho CNS homeostatic le moputso oa neurochemistry

Ntle le litekanyo tsa "metabolic metabolic" tsa kelello tse tsoang ho cohort tse neng li e-na le tšebeliso ea lijo le boikoetliso nakong ea libeke tsa 5-8 li ne li lekanngoa bakeng sa li-profiles tsa amine (n = 4 ka sehlopha sa lijo) kapa litekanyetso tsa mRNA tsa li-peptides tsa hypothalamic orexigenic. Joalokaha ho bontšitsoe Lethathamo 4, ha ho na phello e kholo ea lijo tse phahameng tsa mafura ho dopamine, norepinephrine, kapa serotonin metabolites ho li-nucleus accumbens, sebaka sa bohareng sa moputso le ts'ebetso ea susumetsang (Ikemoto le Panksepp, 1996; Kelley le Berridge, 2002) moo e 'ngoe le e' ngoe ea li-neurotransmitter e bapalang karolo ea bohlokoa ea taolo. Ka har'a lisebelisoa tsa hypothalamic, litekanyetso tsa mRNA tsa li-peptide tsa orexigenic, NPY, AGRP le orexin li ile tsa lekanngoa. Tloaelo e matla empa e se ea bohlokoa ea keketseho ea AGRP ka likhoto tse fepehileng ka mafura e ile ea bonoa sehlopheng sena (n = 8 bakeng sa lijo tse ling); ka hona re phetile paradigm ea koetliso ea boits'oaro / boits'oaro sehlopheng se eketsehileng mme ra lekanya NPY, AGRP, le orexin mRNA ho hypothalamus. Lihlopheng tse kopaneng, re bone keketseho ea bohlokoa (p <0.05) ho AGRP mRNA ho likhoto tse fepang phepo e phahameng ea mafura vs.Setšoantšo sa 2), empa ha ho phetoho e kholo ho polelo ea NPY kapa orexin. Ho lekola likamano tse ka bang teng lipakeng tsa polelo ea AGRP le boits'oaro ba ho itaola, ra lekanya li-cFos le li-neurons tsa AGRP tsa "immobopositive" ho "mediobasal hypothalamus". Lihlopha tsa likhoto li ne li fepeloa li-chow kapa mafura a 31.8%; tse ling li nkuoe ka har'a protocol ea boits'ebetso (libeke tsa 5-8) mme tse ling li ne li sebetsoa e le taolo ea boitšoaro. Setšoantšo sa 3a e bontša mohlala oa ho sebelisana 'moho oa li-cFos le AGRP ka har'a arcuate nucleus neuron. Joalokaha ho akaretsoa ka Lethathamo 5, ts'ebetso ea li-neurons tsa AGRP (tšebelisano-hammoho ea cFos-ICC le AGRP-ICC ka har'a lisele tse tšoanang) e ne e amana le ts'ebetso ea ho itaola. Sena se bontšoa ka Setšoantšo sa 3b, moo palo ea li-neurons tse sebetsang (cFos-positive) e bonts'itsoeng e le palo ea lisele tsa methapo, kapa joalo ka liperesente tsa li-neurons tse nang le AGRP e phethahetseng: ho na le ts'ebetso ea bohlokoa ea li-neuron tsa AGRP mehatong e ikemetseng e sebetsanang le maemo , ka lihlopha tse kopaneng tsa lijo. Papiso ea phepo ea lijo tse ka hare ho lijo bakeng sa palo ea li-neurons tse sebetsang tsa AGRP sehlopheng sa boits'ebetso sa sehlopha sa taolo ea ho itšireletsa e bonts'itse mokhoa o sa fihlelleng bohlokoa ba lipalo (chow, p = .078; 31.8% lijo tsa mafura, p = .073) . Habohlokoa, ha se feela hore lintlha tsena li hokahanya ts'ebetso ea neuronal ea AGRP le boits'oaro, empa ka lebaka la nako ea tekanyo ea cFos (metsotso e 90 kamora hore likhoto li kentsoe likamoreng tsa bona tsa tsamaiso), polelo ea cFos e bonts'a tšebetso ea li-neurons tsa AGRP ho litebello tsa, kapa tsa pele tsa ts'ebetso ea ho itaola. Ho ne ho na le mokhoa o seng oa bohlokoa bakeng sa li-neurons tse ntlafalitsoeng ka botlalo tsa AGRP sehlopheng sa tsamaiso ea boits'oaro (vs. handling management, p = 0.16). Likhotong tseo, moo ho toants'ang lever ho neng ho kopantsoe lipakeng tsa lihlopha tsa lijo, palo ea li-neuron tse ntle tsa AGRP le eona e ile ea bapisoa. Ha ho na phello ea kalafo ea ho ja u le mong ka palo ea li-neuron tse ntle tsa AGRP litekong tsa taolo ea boitšoaro.

  

Setšoantšo sa 2

Phello ea phepo e mafura ea 31.8% mafapheng a medial hypothalamic peptide mRNA expression. Lintlha li tloaelehile bakeng sa likhoto tse nang le mafura a mangata (n = 17) khahlanong le tsa chow controls (n = 16). AGRR mRNA e phahame haholo (p <0.05).

  

Setšoantšo sa 3

Ho kenya ts'ebetso ea li-neuron tsa AGRP qalong ea ho itaola. 3a. Co-localization of cFos and AGRP in arcuate nucleus neuron, 60x kholo. 3b. Palo ea li-neurons tse sebetsang (cFos-immunopositive) AGRP-immunopositive neurons ho Mediobasal hypothalamus ...

  

Lethathamo 4

Nucleus Accumbens Amine Metabolites

  

Lethathamo 5

Ts'ebetso ea Agrp Neuron: Pheko ea Lithethefatsi le Boitšoaro

Kameho ea tsamaiso ea AGRP ka tšusumetso ea sucrose

Tlhaloso ea rona ea ho fumana sena ke hore polelo ea AGRP ka har'a likhoto tsa pubertal ke mokhoa oa mantlha tlasa ts'ebetso e ikhethileng ea boits'oaro ba likhoto tse phahameng tse fepisitsoeng ke lijo. Ho netefatsa katleho ea AGRP ho eketsa tšusumetso ea ho ikemela, AGRP e ile ea tsamaisoa ka ts'ebetso ea boraro ho ea ho likhoto tse matlafatsoang tsa peri-pubertal nakong ea karolo ea PR ea paradigm ea boits'oaro. Tekanyetso ena ea AGRP e ne e le sebopeho se tlase sa ho khothaletsa ts'ebetso ea ts'ebeliso ea libeke tse peli tsa PR paradigm, empa e felletse ka matla a ho itaola ka mokhoa o hlakileng, joalo ka ha ho bonts'itsoe ho Setšoantšo sa 4. (Hlokomela hore moputso o mong le o mong oa sucrose o na le caloric ea 0.1 kcal, ka hona ts'ebetso ea ho itaola e fana ka likhalori tse sa tsotelleng ho fihlella ka botlalo.) Lethathamo 6 e bonts'a data ea paramente ea boikitlaetso ho pholletsa le paradigm ea matsatsi a 9, e nang le AGRP kapa aCSF e kentseng ICV ka Matsatsi a 2, 5 le a 8. Litsing tse tšoaroang ke AGRP, palo ea mechine ea khatiso e sebetsang e ile ea eketseha ka kakaretso ho pholletsa le Matsatsi a PR- 2 (p = 10), le ka matsatsi a sa enteloang (p = 0.03) ka mokhoa o lebisang keketseho matsatsing a ente (averaged). Ho ekelletsa moo, Nako ea Stop (e bonts'ang nako eohle e sebelisitsoeng mosebetsing oa ho itaola) e ile ea eketseha haholo matsatsing a ho se ente (p = 0.048) ka mekhoa e lebisang keketseho ka kakaretso, le matsatsing a ente. Palo ea meputso ea sucrose e ile ea eketseha ka kakaretso ho pholletsa le Matsatsi a PR 0.02-2 (p = 10). Ha ho na phello ea kalafo ea AGRP khatellong e sa sebetseng ea lever, ha e bapisoa le li-control tse tšoaroang ke aCSF, kapa lipakeng tsa matsatsi a ente- le a sa ente. Liphetho li ts'ehetsa ho hlalosoa ha ts'ebetso e tsitsitseng ea AGRP ho eketsa boitšoaro ba boits'oaro: likhoto li hatelletse ho mofani ea fanang ka moputso, a fumana meputso e eketsehileng, 'me a qeta nako e eketsehileng mosebetsing.

  

Setšoantšo sa 4

Lekhetlo la boraro la cyricular (ICV) AGRP (0.01 nmol) e matlafatsa boikemelo ba ho itaola ho paradigm ea PR, empa ha e na tšusumetso ho litekanyetso tsa lijo tsa letsatsi le letsatsi ho pholletsa le nako ea ho ithuta (PR Matsatsi a 2-10, ka liente ka Matsatsi a 2, 5, le 8) . Lintlha tsa AGRP (n = 9) li hlahisitsoe ...

  

Lethathamo 6

Kameho ea ICV AGRP vs. aCSF mabapi le Ts'ebetso ea Ratios ea Ts'ebetso ea Ts'ebetso ea Ts'ireletso

Phello ea boemo ba bophelo ba hau ho khethoe le sepheo sa ho ikemela

Thutong ea hoqetela, re lekotse hore na sepheo sa ho iketla se fapana pakeng tsa likhoto tsa pubertal le tsa batho ba baholo. Qalong, likhoto tsa khale tsa 5-le 10-wk li ile tsa fuoa tlhahlobo ea khetho ea likhetho ka khetho ea tharollo ho tloha ho isa ho 0% sucrose, pele ho tlhahlobo ea boits'oaro le koetliso. Joalokaha ho bontšitsoe Setšoantšo sa 5a, 'me e lumellana le liphumano tse tlalehiloeng ka har'a lingoliloeng, likhoto tsa pele ho selemo li bonahala li rata tharollo e monate ho feta likhoto tse kholo tsa batho ba bangata: likhoto tse ngata tsa pele ho nako li ne li le tekanyo e phahameng ea tharollo ea 20%, athe litoeba tsa batho ba baholo li bonts'a moferefere. ea 15% sucrose. Kamora nako, lihlopha tsena ka bobeli li ile tsa aroloa pakeng tsa ratop le lijo tse phahameng tsa mafura nakong ea boikoetliso le liteko tsa ho ipusa. Ho bile le keketseho e nyane empa e le ea lipalo-palo palong ea likhatiso tsa lever tse sebetsang ke litoeba tsa batho ba baholo (45 ± 3 vs 37 ± 2, p = 0.05) ka karolelano mekhahlelong ea FR, ntle le phapang ea palo ea meputso ea sucrose kapa lipampiri tsa khatiso ho lever e sa sebetseng. Joalokaha ho bontšitsoe Setšoantšo sa 5b, ho bile le phello e kholo ea kakaretso ea lilemo, ho pholletsa le linako tsa PR, ka khatello e matla ea lever e sebetsang bakeng sa pubertal (n = 15) dhidi ea motho e moholo ea tsofetseng (n = 14) likhoto (2-way ANOVA, PRDay × age; phello ea lilemo, p = 0.017, ha ho na phello e ikemetseng ea PRDay, ha ho na tšebelisano ea bohlokoa). Tthere e ne e le tšekamelo ea phello e kholo ea botsofaling maemong a ho fepa ka mafura a mangata empa sena ha sea ka sa fihlela bohlokoa ba lipalo (p = .13). Lethathamo 7 e thathamisa likarolo tsa boits'oaro tsa PR: ho kenyelletsa mechini e hatellang ea lever, likhoto tsa peri-pubertal li amohetse moputso o motle haholoanyane, mme li bonts'itse mokhoa o lebisang ho Stop Nako. Ho ekelletsa moo, likhoto tsa peri-pubertal li bile le keketseho e nyane empa e kholo ea likhatello ho "lever" tse seng li sa sebetse, le ha e le bakeng sa likhakanyo tsa bobeli tsa peri-pubertal le tsa batho ba baholo. ea likhatiso tsa khatiso tse matla tse sebetsang. Liphetho tsena li bontša hore likhoto tsa peri-pubertal li rata ebile li tla batla lijo tse latsoang tse monate haholo, 'me phello e ka ntlafatsoa ka semelo sa lijo tse nang le mafura a mangata.

  

Setšoantšo sa 5

Likhoto tsa bana li eketsehile tšusumetso ea moputso oa sucrose ha o bapisoa le likhoto tsa batho ba baholo. 5a. Liteko tsa likhetho tsa Sucrose bakeng sa bana (li-peri-pubertal, n = 15) le batho ba baholo ba baholo (n = 14). Likhoto li ne li e-na le metsotso e 30 ea ho noa ho tloha ho mefuta e fapaneng ea likhopolo (0-20% sucrose). ...

  

Lethathamo 7

Phello ea botsofe ho Tsoelo-pele ea Litekanyetso tsa Rogos^a bakeng sa Sucrose

Patlisiso ena e tšehelitsoe ke thuso ea NIH DK40963. Dianne Figlewicz Lattemann ke Setsebi sa Lipatlisiso sa Tlhahlobo ea Boithuto bo Holimo, Lenaneo la Patlisiso ea Li-biomedical Laboral, Lefapha la Litaba tsa Veterans Puget Sound Health System, Seattle, Washington. Stephen Benoit o tšehelitsoe ke NIH DK066223 le Ethicon Endosurgery Inc. Bangoli ba leboha Dr. Tami Wolden-Hanson ka tšehetso ka litekanyo tsa sebopeho sa 'mele; Ngaka William Banks le Lucy Dillman bakeng sa tšehetso ka litekanyo tsa triglyceride; le Amalie Alver, le Samantha Thomas-Nadler bakeng sa thuso lithutong tsa boits'oaro.

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