Northern blot analysis

Transcription

1 Northern blot nlysis RNA SCD RNA SCD FAS C c-9 t-1 C c-9 t-1 PRE PI PDMI PRE PI PDMI PRE PDMI PIM An W c-9, t-11 t-1, c-12 C um C um Angus dipocytes expressed SCD higher thn Wgyu dipocyte fter short term (1 wk) of differentition. Both c9,t11 nd t1,c12 CLA decresed SCD nd FAS expression s the dose incresed. Northern blot nlysis RNA C Arg C Arg C Arg C Arg C Arg C Arg SCD PPAR γ C/EBP β LPL TNF α Pref 1 Control Arg CLA Arg + CLA SCD, PPARγ, nd LPL were greter in predipocytes incubted with rginine thn in control. RNA SCD RNA PPAR γ Control Arg CLA Arg + CLA t1,c12 CLA decresed SCD gene expression nd not overcome by coincubting with rginine. TTU AFS 44/55 1

2 Metbolic nlysis Ftty cid composition Lipogenesis f MUFA/SFA (SCD index) Rtio o SCD index pmol/cell/2h C cette incorportion Control 5uM c9,t11 4uM c9,t11 5uM t1,c12 4uM t1,c12 Control 5uM c9,t11 4uM c9,t11 5uM t1,c12 4uM t1,c12 Postntl Adipose Tissue Models to study deposition Jpnese Blck vs. Angus Beef mrbling score: (Jpnese) 12 CAB crcsses pprox. 4 5 on Jpnese scle 1 12 equls greter thn 2% extrctble lipid in L.D. muscle vs % EE for USDA Prime TTU AFS 44/55 2

3 Animl Growth Rte 7 6 Corn-Angus Corn-Wgyu Hy-Angus Hy-Wgyu Body Weight, Kg st group (corn) 2 nd group (hy) 3 rd group (corn) 4 th group (hy) Trget weight Corn: 1.36 kg/d to 325 kg/8mo Hy:.9 kg/d to 325 kg/12mo 2 1 U.S. Endpoint Jpn Endpoint Dys on feed Crcss chrcteristics ) Percen ntge of ft content (IML 25 Corn Angus Hy Angus Corn Wgyu 2 Hy Wgyu 15 Prime + 1 Prime - Choice + 5 Choice - U.S. endpoint Jpn endpoint Time on Feed (mo) Angus fed corn hd greter IML thn hy nd Wgyu fed corn. Hy-fed Wgyu (Jpn endpoint) hd greter IML thn other diet group. Wgyu hd low finl body weights. P-vlue Endpoint : P<.1 B*E : P<.5 B*D : P<.5 TTU AFS 44/55 3

4 SCD enzyme ctivity vs. Time royl-coa desturse ctivity, mol per 7min per mg protein Ste Nm Hy Angus Corn Angus Incresed between the H A Corn Wgyu Hy Wgyu U.S. endpoint Time on Feed (mo) Jpn endpoint U.S. nd Jpnese endpoint, but not in the hy-fed Angus steers. Incresed most in hy- bsed Wgyu steers. P-vlue Endpoint : P=.6 D*E : P=.8 B*D*E : P=.8 SCD gene expression vs. Time SCD:28S RNA Corn Angus Hy Angus Corn Wgyu Hy Wgyu U.S. endpoint Jpn endpoint Time on Feed (mo) Greter in corn-fed steers Incresed most in Wgyu steers, but decresed with time in Angus steers. P-vlue Diet : P=.6 Endpoint : P=.7 D*E : P=.5 B*E : P=.1 TTU AFS 44/55 4

5 Postntl Adipose Tissue When looking t these nimls to study intrmusculr ft lrger dipocytes more dipocytes these were the predominnt theories they found tht dipocyte size is ctully less in Wgyuthn Angus Postntl Adipose Tissue Numbers of dipocytes re different rte ofpredipoc predipocyte prolifertion ws twice s high in both subcutneous nd intrmusculr dipose tissue from Wgyu vs. Angus cttle Jpnese Blck hve the bility to ccumulte IM lipid seemingly indefinitely Angus hve genetic limittions in predipocyte p differentition/prolifertion TTU AFS 44/55 5

6 Postntl Adipose Tissue Numbers of dipocytes re different rte ofpredipoc predipocyte prolifertion ws twice s high in both subcutneous nd intrmusculr dipose tissue from Wgyu vs. Angus cttle Jpnese Blck hve the bility to ccumulte IM lipid seemingly indefinitely Angus hve genetic limittions in predipocyte p differentition/prolifertion Hormone Production Adipose tissue s n endocrine tissue now more hormones produced in dipose tissue thn the nterior pituitry Leptin 16 kd protein secreted from white dipocytes belongs to clss of helicl cytokines similr to IL 2 nd GH four lph helicl bundle structures 1995 ob protein leptin dministrtion could eliminte obesity in the ob /ob mouse (ob protein = leptin) TTU AFS 44/55 6

7 Hormone Production Long-term signls regulting energy blnce. Insulin nd leptin re the two hormones tht ct s long-term regultors of food intke nd energy blnce. Both insulin nd leptin ct in the centrl nervous system to inhibit food intke nd to increse energy expenditure. Activtion of the sympthetic nervous system (SNS) is likely to contribute to the increse in energy expenditure. (Hvel et l. 2.) Hormone Production AgRP: Agouti-relted protein Arc: rcute nucleus CART: cocine nd mphetmine relted trnscript MC4R: melnocortin 4 receptor NPY: neuropeptide Y POMC: proopiomelnocortin PVN: prventriculr nucleus VMN: ventromedil nucleus. TTU AFS 44/55 7

8 ob muttion Leptin Receptor Receptor high h ffinity it severl splice vrints of single gene long form contins 32 mino cids in intrcellulr domin expressed in vrious regions of the brin ctegorized s clss I cytokine receptor (much like IL 6, LIF) TTU AFS 44/55 8

9 Leptin Signling Signl trnsduction pthwys of clss I cytokine receptors typiclly lck intrinsic tyrosine kinse ctivity ctivtion usully occurs following formtion of homodimers leptin ctully forms homodimers ggregtion ctivtes JAK/STAT pthwy JAK = Jnus Kinse STAT = signl trnsducers nd ctivtors of trnscription Leptin Leptin tretment (exogenous ddition) dose dependent decrese in food intke loss of body weight loss of ft depots incresed metbolic rte TTU AFS 44/55 9

10 Leptin Leptin is synthesized/secreted from white dipocytes exmple of dipose tissue cting s n endocrine tissue cting on other tissues (brin e.g.) promoter of region of Leptin gene contins sites for C/EBP α nd PPAR γ; therefore, dipose specific gene incresed leptin hs negtive feedbck loop on dipocytes to decrese the leptin production discovered in 21 Resistin expression reduced by TZDs C/EBP responsive gene Biologicl ctivities impirment of glucose tolernce ntgonism of glucose uptke inhibition of 3T3-L1 differentition TTU AFS 44/55 1

11 discovered in 1995 Adiponectin secreted by differentited dipocytes models of insulin resistnce hve reduced diponectin levels exogenous diponectin cn stimulte ftty cid oxidtion by skeletl muscle secretion is stimulted by TZDs TNFα TNFα (pro inflmmtory cytokine) synthesized in neutrophils, ctivted T nd B lymphocytes AND dipocytes neutrophils, T nd B lymphocytes endocrine fshion dipocytes prcrine/utocrine effect polypeptide = 157 mino cids(humn) = 17 kd species vrition incresed no signl peptide but it is nchored to membrne nd relesed in soluble form TTU AFS 44/55 11

12 TNFα TNFα Receptors (two distinct) TNFR I (55 kd) single trnsmembrne glycoprotein TNFR II (75 kd) single trnsmembrne glycoprotein both hve similr extrcellulr domin but highly distinct intrcellulr domins tht led to unrelted, different dignl trnsduction pthwys Stellite cell development nd differentition Stellite cell Trnsdifferentition Prolifertion Fuse TTU AFS 44/55 12

13 In vitro mrbling study Stellite cell Prolifertion (MUFA,PUFA) Long chin ftty cid (Ciglitzone, Troglitzone, T-174) Thizolidinedione Horse Serum Long chin ftty cid (PUFA) Trnsdifferentition Differentition Gene experession during trnsdifferentition Muscle derived cell Trnsdifferentition Predipocyte Differentition Adipocyte Erly Intermedite Lte Trnscriptionl ctivtion ADD1/ SREBP1 C/EBPβ Lignds C/EBPα PPAR RXR Fctors binding region Leptin SCD Adipose gene expression Functionl Activtion TNF α FAS TTU AFS 44/55 13

14 BSC trnsdifferentition Arbit. Rtio C/EBP β ( P =.8) Arbit. Rtio PPAR γ ( P <.1) Arbit. Rtio Myogenin ( P <.1) BSC trnsdifferentition nd growth promotnts * Arbit. Rtio * C/EBP β * ( P <.3) E2 : Estrdiol MGA : Melnegesterol Acette M+E: MGA + E2 Tretment TTU AFS 44/55 14

15 BSC trnsdifferentition nd growth promotnts * Arbit. Rtio PPAR γ * ( P <.5) E2 : Estrdiol MGA : Melnegesterol Acette M+E: MGA + E2 Tretment BSC trnsdifferentition nd growth promotnts Arbit. Rtio SCD * ( P =.5) E2 : Estrdiol MGA : Melnegesterol Acette M+E: MGA + E2 Tretment TTU AFS 44/55 15

16 BSC trnsdifferentition nd growth promotnts Arbit. Rtio Myogenin * ( P =.5) E2 : Estrdiol MGA : Melnegesterol Acette M+E: MGA + E2 Tretment BSC trnsdifferentition nd growth promotnts Positive Control : Insulin, Oleic cid, nd Ciglitizone 1nM estrdiol 2nM 17β trenbolone 1nM Melengestrol cette TTU AFS 44/55 16

17 Muscle Biopsy Study Muscle Biopsy Study TTU AFS 44/55 17

18 BSC trnsdifferentition in Biopsy study NA, dnce C/EBPβ mr reltive bund b PPARγ mrn NA, reltive bund dnce b Dy Dy 7 Dy 14 Dy 28 Dy Dy 7 Dy 14 Dy 28 Muscle biopsy dte Muscle biopsy dte e SCD mrna, reltive bundnce 5 b 4 b e Myogenin mrna, reltive bundnce Dy Dy 7 Dy 14 Dy 28 Dy Dy 7 Dy 14 Dy 28 Muscle biopsy dte Muscle biopsy dte BSC trnsdifferentition in Biopsy study C/EBPβ mrn NA, reltive bund dnce b A, dnce PPARγ mrn reltive bund b b Cont E2 TBA T+E Tretment Cont E2 TBA T+E Tretment e SCD mrna, reltive bundnce b e Myogenin mrna, reltive bundnce 2 1 Cont E2 TBA T+E Cont E2 TBA T+E Tretment Tretment TTU AFS 44/55 18