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¸(µ!ÍÌÖÈÑÓÐÈÑ!ÓÐ© êä¾ + .»n+Fig. 1.

De novo formation of LDs in 3Y1 cells

3Y1 fibroblasts were cultured in 2% LPDS for 2 d to deplete LDs, and then treated with 100 µM 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 mosteffectively, followed by LA and DHA.
B. Conventional electron microscopy of ultrathin sections. The electron density of LDs differs prominently inaccordance 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 TGs and CEs to minimum levels. After incubation with 100 µM 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-!-cyclodextrin-cholesterol complex for 12 h, CE, but not TG, became prominent.

Fig. 2.

Quantitative measurement of LD electron density in electron micrographs of 3Y1 cells.

A. The method of quantification. The electron density of LDs was measured with a relative scale, with theextracellular 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-treatedcells (Student’s t test; *p < 0.01). The numbers in the y-axis indicate the relative electron density in this andthe following graphs.
C. The effect of possible variables on LD electron density. OA-treated cell specimens were either electro-stainedwith lead citrate for one-third time shorter (left) or sectioned 1.2 times thicker (right) than a control samplefrom the same specimen. Neither the length of lead staining, nor a difference in section thickness, affected LDelectron density.

Fig. 3.

The time-dependent change in LD electron density in 3Y1 cells.

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 0min, 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 electrondensity increased in 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. 5min (p < 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 shadedzone indicates the average (Ă 2 standard deviations) of the LD electron density in cells treated with DHA alonefor 12 hr. Note that LDs of high electron densities did not occur as an independent population.
D. Cells were cultured with DHA for 12 hr, rinsed, and treated with OA for either 120 or 240 min. The point plotof electron density for all LDs before and after OA treatment is shown. The shaded zone shows the average (Ă2 standard deviations) LD electron density in cells treated with OA alone for 12 hr. The result indicates thatLDs made of the OA ester alone do not form by the OA treatment of 120 or 240 min.
Fig. 4.

Serial ultrathin sections of 3Y1 cells cultured with OA for 12 h and then with DHA for 60 min.

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.
Fig. 5. The electron density of LDs in 3T3-L1adipocytes 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. SmallLDs (arrowheads) generally had a lower
electron density than medium-sized LDs(arrows).
B. LDs were categorized into three groups
according to their diameter, and theirelectron density 30 and 60 min after
DHA treatment was quantified as for 3Y1cells. 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 5mm) 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 3Y1 LDs after DHAtreatment.

A. Cells were cultured with OA for 12 hr, rinsed, and treated with DHA. One group of cells was administered 10mM 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 incontrol 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 anindependent population.
Fig. 7.

Incorporation of DHA esters into CE-rich LDs.

3Y1 cells were cultured with 0.2 mM methyl-b-cyclodextrin-cholesterol complex for 12 hr, and then treated with orwithout DHA for 120 min. Representative electron micrographs are shown. CE-rich LDs were electron-lucent in thecenter, but occasionally appeared hazy (double arrow) and/or had compact electron-dense materials in the rim(arrowhead). After DHA treatment, some LDs became homogenously electron-dense, whereas others had zones ofvariously shaped high and low electron density (arrows).

Fig. 8. Schemes for three possible
mechanisms of LD growth with auniform incorporation of TGs.
A. Exchange of TGs by mutual fusionof LDs.
B. Exchange of newly synthesizedand existing TGs throughpersistent ER-LD conduits.
C. Synthesis of TGs in the
mitochondria-associatedmembrane (MAM) within the local
vicinity of individual LDs.