3月9日 - 名古屋大学全学技術センター
Contents
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49. 3T3 L1 CRS Fig 1 Denovo formation of L Dsin 3Y 1 cells p e B c 1 i a 1 ae ia 4 e L 9 GE contos A OA 181 control OA 18 1 do x TG e 9 d e LPDS OA A DHA cholesterol o E o OQ LA 18 2 DHA 22 6 LA 182 DHA 226 9 3Y1 fibroblasts were cultured in 2 LPDS for 2 d to deplete LDs and then treated with 100 UM OA LA or DHA for 12 h A Fluorescence microscopy LDs and nuclei were stained with BODIPY493 503 green and DAPI blue respectively after formaldehyde fixation After 2 d of culturing in 2 LPDS LDs were rarely observed control However new LDs formed rapidly after treatment with fatty acids OA induced new LDs most effectively followed by LA and DHA B Conventional electron microscopy of ultrathin sections The electron density of LDs differs prominently in accordance with fatty acid treatment Electron density was the highest in cells treated with DHA followed by LA and OA C Thin layer chromatography of total lipids extracted from 3Y1 cells Two days of culturing in 2 LPDS reduced TG
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85. electron density Fig 3 Thetime dependent change in L D electron density in 3Y 1 cells 3Y1 cells mo RAS DHA 120 min DHA 240 min B OA DHA C OA DHA D DHA OA 1 1 1 i 15 Average 250 DHA only 10s 0 5 Average 25D OA only lOs O 5 10 15 30 60 120 240 min O 5 101530 60120 240 min 0 120 240 min A Cells were cultured with OA for 12 hr rinsed and treated with DHA Representative electron micrographs before and 30 60 120 and 240 min after the DHA addition are shown The LD electron density was low at 0 min but increased gradually after DHA was added to the medium B The average electron density of LDs treated as in A Ten images were taken in random areas and the electron density of all the LDs in each field quantified by the method described for Fig 2A The average electron density increased 1n a time dependent manner Note the small standard deviation at each time point The electron density at any time point is significantly different from that of the adjacent time point e g 0 min vs 5 min p gt 0 01 by Student s t test C The point plot of electron density for all LDs found in ten randomly chosen cells treated as in A The shaded zone indicates the average 7 2 standard deviations of the LD electron density in cells treated with DHA alone for 12 hr Note that LDs of high electron densities did not occur as an independent population D Cells were cultured with
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87. min after the DHA addition 3Y1 cells were cultured with 0 2 mM methyl b cyclodextrin cholesterol complex for 12 hr and then treated with or without DHA for 120 min Representative electron micrographs are shown CE rich LDs were electron lucent in the center but occasionally appeared hazy double arrow and or had compact electron dense materials 1n the rim arrowhead After DHA treatment some LDs became homogenously electron dense whereas others had zones of variously shaped high and low electron density arrows Fig 8 Schemes for three possible mechanisms of LD growth with a uniform incorporation of TGs A Exchange of TGs by mutual fusion of LDs B Exchange of newly synthesized and existing TGs through persistent ER LD conduits C Synthesis of TGs in the mitochondria associated membrane MAM within the local B Exchange of lipid esters through ER LD conduits vicinity of individual LDs A Exchange of lipid esters by mutual LD fusion N C Production of lipid esters in the vicinity of individual LDs O MAM Conclusion IRHS IRHS Infrared High resolution Spectrograph
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90. 7a das O ae co Cc 7al2 7a 7b ee gt 2 re2 3a 5 o E 9a b e 3a 3b 64 E EDI s 6 2 6 9 0 1 lt fa a e oca 3 5 1 3a 1 2 1 3b 9 ni E C g gt 0 2 1 CH 5 S gt lt Pe E i 2 ae 1 4 N Fl ppm CH 4 CH2 3 306 COSY CH2 7 CH 1 7 menthol oH CH 6 CH s 9 CH 10 7 COSY 2 6 NOESY F2 ppm NOE 0 0 Do 8 0 5 1 Ay d e L A am E a 4 EE i 3 a 1 5 b 3b l 72 7 l 4 fr o e 7 7a 2 1 5 573a gs eg 1 OH 5 a 5 A 8 CH 6 3a 1 E 1 menthol Ce Ica 3 0 e gt e 3 5 g 7 la CAITR ce 3 5 340 2 5 2 0 1 5 1 0 Fl ppm 8 NOESY
91. Cl C2 C3 C4 cs C7 C10 3C NMR C IH NMR tt tut Jd F1 ppm 5 i j i f q 1 A E59 i b u 1 2 0 por 5 0 T us 4 0 75 70 65 60 55 50 45 40 35 30 25 20 LS Ix 5 HBTCOR 2 3 DEPT OH OH o KoT OH XX 01 HETCOR H C 3 3 HETCOR H CHR R H Peak at Number SB ppm scheme 34 1 77 1 55 1 63 OH 50 28 1 09 1 15 2 CH 1 95 2 00 pm 0 83 1 03 5 CH 2 14 2 20 2599 CH 1 55 1 63 2555 0 83 1 03 2235 CH 2115 CH 1624 Hs 1 2 3a 83 1 3b 1 65 1 70 4a 0 83 1 03 4b Ta 7b 138148 5 3179 CH CH CH CH CH CH CH CH CH CH 2 5 H HCOSY 2 3 H 6 9 18 10 7b fa 3b 1 6 3a fa 5 2 i _ M n M p m al WM dioc A LAR x i 5 E d SE y
92. DHA for 12 hr rinsed and treated with OA for either 120 or 240 min The point plot of electron density for all LDs before and after OA treatment is shown The shaded zone shows the average 7 2 standard deviations LD electron density in cells treated with OA alone for 12 hr The result indicates that LDs made of the OA ester alone do not form by the OA treatment of 120 or 240 min Fig 4 Serial ultrathin sections of 3Y 1 cells cultured with OA for 12 h and then with DHA for 60 min Serial ultrathin sections of 3Y1 cells The LDs in the field show a homogenous change in density The LD marked by an arrow is more than three times larger than the LD marked by an arrowhead These results show that LDs differing in diameter by 3 4 times or 27 64 times different in volume contain existing OA and newly synthesized DHA esters in comparable ratios 313 11 adipocyte 30 min after the DHA addition o es nas 2 ern aie m made 1 9 om 7 as me gt rm Fig 5 The electron density of L Ds in 3T3 L1 adipocytes treated with DHA A Representative electron micrographs before left and 30 min after right treatment with DHA LDs before DHA treatment were invariably electron lucent whereas those in DHA treated cells were heterogeneous in electron density Small LDs arrowheads generally had a lower electron density than medium sized LDs arrows LDs were categorized into three groups according to their diameter and the
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94. HHHHHHHHHHHHHHL ODO HHHUHHUHHHHHHUHHHHHHHHCADHCAMHHHHUHHUHHHHHHHUHUHHHHLH ODO DO D ID OD OU D U U LIE 12 HHHHHL IlHHHHHHHHHHHLH n 00 Ou ETT IEEE BH TES L1 L 1 ICI 1 1 1 1 NCHHHHH 1 L4 E3 E3 BE E E Q Z UOUUUUU 2 HHHL 21 HHHL
95. L README html SASL zu Ao ab tT gt Be OA RFR
96. LPF HPF BPF 2kHz LPF 2135112 HPF 1810Hz BPF 1 Q10 1943Hz BPF 10 Q20 1858Hz 2kHz BPF Q20 1866Hz LPF 2046Hz HPF 1941Hz BPF 1 QD 1986Hz 2 PE E 2135 2046 BL D BPF Al 0 1943 1986 BPF A10 Q20 1858 1866 Q 20 Q 21 Q 10 X 10 Q R7
97. X700x450 60kg 2 3 80 4 5 6 CHi CH 6 Tektronix TDS 3014C 100MHz 1 25GS s SandiagoCa WAVETEX Model 193 Gd7 20 j HPF LPF mo 1 a BPF A 10 Q 20 BPF A 1 Q 10 100000 Fa 382 ks Hz 7
98. cos 2nd 15553 22 Ist 0 20 169 9 3 39 4 7 Bd 10 gt e 3 2 DA Y f Su m PERS EZ mm oo 5 ss A Ry Ist 3 2nd 0 07mm 20 2mm OR 4 Ry A2017S 20 2mm 2nd 1st 3 7 Ry Y 1st 1 4 20 07mm 2nd cut 20 2mm 4 4 Ry 007mm A2017S 5115304 Ry 3 OSETTREOOEME 0 2mm 20 07mm 2 02mm
99. ir electron density 30 and 60 min after DHA treatment was quantified as for 3Y1 cells After DHA medium sized LDs 2 5 mm in diameter had an electron density higher than that of both smaller less than 2 mm and larger more than 5 mm LDs p 0 05 and p 0 01 by Student s t test respectively Fig 6 The effect of nocodazole on the time dependent change in electron density for 3Y 1 L Ds after DHA treatment 1 0 control 1 0 nocodazole 0 6 0 6 0 4 0 4 48 so 0 60 90 120 min 8 5 O 60 90 120 min O 60 590 120 min A Cells were cultured with OA for 12 hr rinsed and treated with DHA One group of cells was administered 10 mM nocodazole during the last 1 hr of OA culturing and DHA medium containing nocodazole was applied The average electron density of LDs increased at a slightly slower rate in nocodazole treated cells than in control cells but the standard deviation of LD electron density was similarly small in both samples B The point plot of LD electron densities after DHA treatment in the absence left or presence right of 10 mM nocodazole Even in the nocodazole treated sample LDs with high electron densities was not observed as an independent population Fig 7 Incorporation of DHA esters into CE rich L Ds 3Y1 cells 2 A DE Ra gt _ 0 A ud 7 AN xt P im a d ns jim s r 120
100. lingo y 7 gt OD Bat BRET 2 2 6 6 KT VE VIET RU DD E 66 6 6 193 73
101. s and CEs to minimum levels After incubation with 100 UM OA LA or DHA for 12 h a TG band became apparent but CE did not change When cells were treated with 0 2 mM methyl p cyclodextrin cholesterol complex for 12 h CE but not TG became prominent Fig 2 Quantitative measurement of L D electron density in electron micrographs of 3Y 1 cells A B Relative electron density ofLDs Effect of possible variables EM grid bar LD cell extracellular space 8 OA LA DHA Control lead Section citrate thickness staining x1 2 x 1 3 A The method of quantification The electron density of LDs was measured with a relative scale with the extracellular space and a grid bar in the same image field as representing density points of 0 and 1 respectively B The electron density of LDs in cells treated with OA LA or DHA for 12 h was quantified as described in A The average electron density of LDs in LA or DHA treated cells differed significantly from that in OA treated cells Student s t test p 0 01 The numbers in the y axis indicate the relative electron density in this and the following graphs C The effect of possible variables on LD electron density OA treated cell specimens were either electro stained with lead citrate for one third time shorter left or sectioned 1 2 times thicker right than a control sample from the same specimen Neither the length of lead staining nor a difference in section thickness affected LD
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