(Received for publication, July 7, 1995; and in revised form, September 7, 1995)
From the
Cultured fibroblasts from patients affected with the genetic metabolic disorder named neutral lipid storage disease (NLSD) exhibit a dramatic accumulation of cytoplasmic triacylglycerols (Radom, J., Salvayre, R., Nègre, A., Maret, A., and Douste-Blazy, L.(1987) Eur. J. Biochem. 164, 703-708). We compared here the metabolism of radiolabeled short-, medium- and long-chain fatty acids in these cells. Short/medium-chain fatty acids (C4-C10) were incorporated into polar lipids (60-80%) and triacylglycerols (20-40%) at a lower rate (5-10 times lower) than long-chain fatty acids. Pulse-chase experiments allowed to evaluate the degradation rate of cytoplasmic triacylglycerols in normal and NLSD fibroblasts and to discriminate between two catabolic pathways of cytoplasmic triacylglycerols. Short/medium-chain (C4-C10) triacylglycerols were degraded at a normal rate in NLSD fibroblasts, whereas long-chain (C12 and longer) triacylglycerols remained undegraded. These data are confirmed by mass analysis. The use of diethylparanitrophenyl phosphate (E600) and parachloromercuribenzoate (PCMB) inhibitors allows to discriminate between the two triacylglycerol degradation pathways. E600 inhibited selectively the in situ degradation of short/medium-chain triacylglycerols without inhibition of the degradation of long-chain triacylglycerols, whereas PCMB inhibited selectively the in situ hydrolysis of long-chain triacylglycerols without affecting the degradation of long-chain triacylglycerols. This was correlated with the in vitro properties of cellular triacylglycerol-hydrolyzing enzymes characterized by their susbtrate specificity and their susceptibility to inhibitors; the neutral lipase specific to long-chain triacylglycerols is inhibited by PCMB, but not by E600, in contrast to short/medium-chain lipase, which is inhibited by E600 but not by PCMB. The data of in vitro and in situ experiments suggest the existence in fibroblasts of two separate acyl chain length-dependent pathways involved in the degradation of cytoplasmic triacylglycerols, one mediated by a neutral long-chain lipase and another one mediated by a short/medium-chain lipase.
In human fibroblasts, triacylglycerols are degraded in two
separate and independent subcellular (lysosomal and cytoplasmic
non-lysosomal) compartments(1) . In the lysosomal compartment,
triacylglycerols and cholesteryl esters of low density lipoprotein and
very low density lipoprotein taken up by cells are degraded by the acid
lysosomal lipase(2, 3) , which is genetically
deficient in Wolman disease(4) . In the cytoplasmic compartment
of fibroblasts, triacylglycerols are degraded by a lipase system (yet
poorly characterized) different from the other known cellular
(hormone-sensitive lipase) or secretory lipases (1, 5) and different from the cholesteryl ester
degradation pathway(6) . As shown by using radiolabeled oleic
acid or pyrene-containing fluorescent fatty acids, this degradative
pathway of cytoplasmic triacylglycerols is deficient in the neutral
lipid storage disease
(NLSD)()(1, 5, 7, 8, 9, 10) ,
a rare inherited metabolic disease generally characterized by the
association of muscular weakness, ichthyosis, and multisystemic
triacylglycerol storage(11, 12) . We have recently
reported that the pool of cytoplasmic triacylglycerols accumulated in
fibroblasts from neutral lipid storage disease is constituted by
triacylglycerols endogenously biosynthesized (7, 8, 9, 10) and by
triacylglycerols taken up from high density lipoproteins(13) .
An increased biosynthesis of phosphatidylethanolamine has also been
reported(14) .
Comparative studies of the uptake and metabolic utilization of short-chain and long-chain fluorescent pyrene fatty acids in cultured lymphoblasts (15) and in fibroblasts (16) have shown that short-chain (pyrene-butanoic) acid was incorporated into phospholipids but not in triacylglycerols, whereas long-chain (pyrene-decanoic and pyrene-dodecanoic) fatty acids were incorporated into phospholipids and triacylglycerols. In lymphoblasts from neutral lipid storage disease pulsed with short-chain pyrene fatty acid, we detected no significant accumulation of fluorescent triacylglycerols(15) . These results led us to speculate that replacing natural long-chain fatty acids by short-chain fatty acids could be of interest to slow down the triacylglycerol accumulation in neutral lipid storage disease cells. However, the conclusions obtained with fluorescent pyrene fatty acids cannot be directly transposed to natural fatty acids because natural and pyrene fatty acids exhibited several known metabolic differences (5, 8, 17) . The apparent lack of incorporation of short-chain fluorescent fatty acid (pyrene-butanoic) in triacylglycerols and of accumulation of pyrene-butanoic containing triacylglycerols in NLSD cells could be due either to a lack of biosynthesis of pyrene-butanoic-containing triacylglycerols (despite the biosynthesis of pyrene-butanoic-containing glycerophospholipids) or to a rapid degradation of pyrene-butanoic-containing triacylglycerols by a cytoplasmic pathway independent of the neutral lipase system (deficient in intact NLSD cells). This latter hypothesis is supported by the existence in fibroblast homogenates of five different enzymes able to hydrolyze in vitro long- or short-chain fluorescent triacylglycerols. Both groups of enzymes referred to as long-chain lipases or short/medium-chain lipases, respectively, can be discriminated by their enzymatic properties (heat stability, effect of inhibitors)(1, 18) , with short/medium-chain lipases exhibiting properties of (nonspecific) carboxylesterases(19) .
This prompted us to investigate the metabolism of short- and long-chain fatty acids in cultured fibroblasts from controls and from neutral lipid storage disease to examine the metabolic fate of short-chain fatty acids and their potential influence on the triacylglycerol accumulation in neutral lipid storage disease.
The data reported here showed that, in comparison to long-chain fatty acids, short/medium-chain fatty acids 1) are incorporated into cellular lipids at a much lower rate, 2) induce a lower accumulation of triacylglycerols in NLSD cells, and 3) contained in triacylglycerols are hydrolyzed at a normal rate through a catabolic pathway involving a short/medium-chain lipase activity not deficient in situ in NLSD cells (in contrast to the defect of the long-chain lipase activity).
Cell viability was assessed by 3-(4,5-dimethyl thiazol-2-yl)-2,5diphenyl tetrazolium bromide test (20) or by trypan blue test and by morphological examination as previously used (21) , which shows that the number of necrotic and apoptotic cells did not exceed 8-10% during the metabolic experiments.
Cells were pulsed with fatty acids, used at
the concentrations indicated in the text, generally 30 nmol/ml and
10 dpm, solubilized in Me
SO (0.5% final
concentration), and preincubated with the culture medium (for 30 min at
37 °C) before addition to the cell culture. At the end of the pulse
period (12 or 24 h, as indicated in the text), the fatty acids were
removed by washing the cells twice with phosphate-buffered saline
containing 10 nmol/ml bovine serum albumin (essentially fatty
acid-free) and twice with phosphate-buffered saline. One batch of cells
was harvested at the end of the pulse (time 0 of the chase); the other
batches were grown in a fresh medium containing 2% Ultroser G (but no
additional fatty acids) and harvested at the indicated time of this
chase period. Under the experimental conditions used here, fatty acids
and Me
SO had no adverse effect on cell viability.
Mass analysis of cellular lipids was
performed by gas liquid chromatography. After adding tripentadecanoin
(50 µg used as standard), cellular lipids were extracted according
to the Folch procedure. After drying of the chloroformic phase, lipids
were partitioned in the Dole's biphasic solvent system under the
above indicated conditions. After evaporation of the heptane phase,
neutral lipids were solubilized in 50 µl of hexane, and
triacylglycerols were determined by gas liquid chromatography under the
previously used conditions (24) (Carlo-Erba GC-8000; 6 M CP-Sil-5CB, 0.32-mm diameter capillary column; oven
temperature programmed from 220 to 350 °C, 5 °C per min, flame
ionization detector 370 °C, carrier gas N, 35
pK
). Phospholipids (of the chloroformic phase of
the Folch extract) were evaluated by their phosphorus content
determined according to the method of Chen et
al.(25) . Alternatively, gross triaglycerol mass was
estimated by a method derived from Jefferson et al.(26) (briefly, after TLC separation of neutral lipids on
Silica Gel G plates as above indicated (petroleum ether/diethyl
ether/acetic acid, 80/20/1 (v/v/v) as developing system) and staining
by dipping in 1 N sulfuric acid in methanol, followed by air
drying for 15 min and heating at 180 °C for 15 min). Lipid
component were quantified with a Biocom Image Station (Biocom-France)
using a standard of tripentadecanoin treated under the same conditions
on the same TLC plate (standard was linear between 5 and 50 µg).
Figure 6:
Effect of E600 and PCMB on the in situ degradation of triacylglycerols endogenously biosynthesized from
radiolabeled decanoic and oleic acids by fibroblasts from normal
subjects. During the pulse period, cells were incubated at 37 °C
for 12 h in RPMI 1640 medium supplemented with 2% Ultroser G and
radiolabeled fatty acids, decanoic (A, C) or oleic (B, D) acids (10 dpm/ml and 30 nmol/ml
non-labeled fatty acids). At the end of the 12-h pulse period (time 0
of the chase), total radioactivity levels, in cell batches used without
(control) or with E600 and without (control) or with PCMB, were 0.67
± 0.04, 0.69 ± 0.06, 0.70 ± 0.04, and 0.71
± 0.05 with radiolabeled decanoic acid and 6.1 ± 0.4, 6.8
± 0.8, 6.3 ± 0.6, and 6.2 ± 0.7 (as 10
dpm/mg cell protein) with radiolabeled oleic acid, respectively.
After washing, cells were incubated in RPMI 1640 containing 2% Ultroser
G for a 48-h chase period. Inhibitors, E600 (1 µmol/liter) (A, B) or PCMB (10 µmol/liter) (C, D), were added to the culture medium only during the chase
period. Mean ± S.E. of three
experiments.
Figure 1: Influence of lipids of the culture medium on the amounts of triacylglycerols in fibroblasts or lymphoblasts from a patient with NLSD(N1) and from normal subjects (no1 and no2). Cells were grown under the standard culture conditions either in the presence (FCS+, hatched bars) or absence (FCS-, black bars) of fetal calf serum for 15 days. Then, lipids were extracted, and the triacylglycerol mass was analyzed as indicated under ``Materials and Methods.'' Mean ± S.E. of three experiments. In insets are shown the thin layer chromatography of lipids extracted from fibroblasts (N1, NLSD; no1, normal) treated as indicated above, in the presence (+) or absence(-) of fetal calf serum.
Figure 2:
Incorporation of radiolabeled short-,
medium-, and long-chain fatty acids into cellular lipids of cultured
fibroblasts from controls and patients with NLSD and Wolman disease at
the end of the pulse period. Cells were incubated for 12 h in RPMI 1640
medium supplemented with 2% Ultroser G and fatty acids (radiolabeled
fatty acids, 10 dpm/ml, were mixed with non-labeled fatty
acid, 30 nmol/ml, i.e. 33,000 dpm/nmol). At the end of the
pulse, fibroblasts were washed and harvested. Lipids were extracted by
the procedure of Folch, and the radioactivity of the chloroformic phase
was determined by liquid scintillation counting. The level of
radiolabeled fatty acids incorporated into cellular lipids was
calculated assuming that the radiolabeled and non-labeled fatty acids
have the same metabolic fate (and on the basis of 33,000 dpm/nmol).
Mean ± S.E. of four experiments.
Figure 3:
Degradation of triacylglycerols and polar
lipids (insets) endogenously biosynthesized from radiolabeled
short-, medium-, and long-chain fatty acids incorporated in the culture
medium of fibroblasts from normal subjects (filled squares and circles) and from patients with NLSD (empty squares and circles) and Wolman disease (triangles).
Cells were incubated at 37 °C for 12 h (pulse period) in RPMI 1640
supplemented with 2% Ultroser G, and radiolabeled fatty acids
(10 dpm/ml and 30 nmol/ml non-labeled fatty acid) are as
described under ``Materials and Methods.'' At the end of the
pulse period, cells were washed and incubated for an additional 24 or
48 h (chase period) in RPMI 1640 supplemented with 2% Ultroser G. At
the indicated times, cells were washed and harvested, and the lipids
were extracted and analyzed as described under ``Materials and
Methods.'' Mean ± S.E. of three experiments. D and H, comparison of the levels of undegraded radiolabeled
triacylglycerols in the various cell types at the end of the pulse (whole bars) and at the end of the 48-h chase period (black sections) with various fatty
acids.
Figure 4:
Microautoradiography of fibroblasts from a
normal subject (A, B, E, F) and
from NLSD (C, D, G, H) pulsed for
12 h with radiolabeled fatty acids (30 nmol/ml and 2.5 µCi/ml),
[C]decanoic acid (A-D), or
[
C]oleic acid (E-H) and chased
for an additional 48-h chase period in a fatty acid-poor medium, as
described under ``Materials and Methods.''
Microautoradiographies were performed (under the procedure described
under ``Materials and Methods''; exposure times were 4 and
1.5 days for [
C]decanoic- and
[
C]oleic-labeled cells, respectively) after
fixing the cells, either at the end of the pulse period (upper
panel, A, C, E, G) or at the
end of the chase period (lower panel, B, D, F, H). Magnification,
800
.
It is noteworthy that, under the used experimental
conditions of the pulse (12 or 24 h), elongation of C10 incorporated
into cellular lipids was negligible. Reversely, we did not detect any
appreciable amount of short derivatives of
[H]oleic acid incorporated into cellular lipids.
Similar data were observed with lipids extracted at the end of the
chase (data not shown). These data allow to conclude that the chain
lengths of the main part of fatty acids incorporated into cellular
lipids remain unaltered during the time of the experiments reported
here.
These conclusions were also supported by morphological
(microautoradiographic) studies of the cellular radioactivity during
pulse-chase experiments (Fig. 4). At the end of the pulse
period, the level of radioactivity incorporated into the cells was
higher with [C]oleic acid than with
[
C]decanoic acid, and the radioactivity was
localized in the membranes and in the cytoplasmic compartment (Fig. 4, A, C, E, and G).
These morphological features are in agreement with the metabolic
studies (Fig. 3), which showed the incorporation of radiolabeled
fatty acids into phospholipids and triacylglycerols. At the end of the
chase, when cells were labeled with [
C]oleic
acid, the cytoplasmic radioactivity was lost in normal cells (Fig. 4F) but was still apparent in NLSD cells (Fig. 4H). This was quite consistent with the
degradation of oleyl-containing triacylglycerols in normal but not in
NLSD cells (Fig. 3G). In contrast, in cells labeled
with [
C]decanoic acid, the cytoplasmic
radioactivity disappeared almost completely at the end of the chase in
normal cells and in NLSD cells as well, the radioactivity being
persistent only in cell membranes (Fig. 4, B and D). This was consistent with the normal degradation of
[
C]decanoic acid-containing triacylglycerols in
NLSD cells (Fig. 3D).
As the accumulation of long-chain triacylglycerols in NLSD cells is due to a defect of the degradation pathway, it was suggested that, in the cytoplasmic compartment, short/medium-chain triacylglycerols could be degraded by an enzymatic system specific to short/medium-chain triacylglycerols and different from that hydrolyzing long-chain triacylglycerols (neutral lipase).
Figure 5: Mass analysis of cellular triacylglycerols in pulse-chase experiments with a fixed concentration of decanoic (A) or oleic acid (B) in lymphoblasts from NLSD patient or normal subject. Cells were grown in a serum-free RPMI medium for 15 days, under the standard conditions of Fig. 1. Then, cells were pulsed with 30 nmol/ml decanoic acid for 24 h or oleic acid for 12 h. After washing, one batch was harvested for analysis (p), and another batch was grown in lipid-free medium (i.e. serum-free and fatty acid-free medium) for a 24-h chase period and washed and harvested for analysis (ch). Lipids were extracted and analyzed either by gas liquid chromatography or by thin layer chromatography as described under ``Materials and Methods.'' Hatched and black bars represent triacylglycerols with carbon number <45 and >48, respectively (note that no significant level of triacylglycerols with the carbon number <45 was detected in cells fed with oleic acid (panel B)). Mean ± S.E. of three experiments. In insets are shown the triacylglycerol TLC spots of the relative cells treated as indicated above.
Lymphoblasts grown in the presence of 30 nmol/ml C10 or C18:1 (for 24 and 12 h, respectively) showed that the levels of triacylglycerols, at the end of the pulse, were much lower in cells grown in the presence of C10 than in cells grown in the presence of C18:1 (Fig. 5). In normal cells, the levels of triacylglycerols were considerably reduced at the end of the 48-h chase (Fig. 5, A and B). In NLSD cells grown in the presence of oleic acid, the triacylglycerol level was almost unchanged (Fig. 5B), whereas it was reduced by about 50% in cells fed with C10 (Fig. 5A). Analysis of the triacylglycerol species in NLSD cells fed with C10 showed that triacylglycerols with a carbon number lower than 45 (triacylglycerols containing C10 or short/medium-chain fatty acid) were degraded during the chase, whereas triacylglycerols with a carbon number higher than 48 (long-chain triacylglycerols) were not degraded (Fig. 5A). In cells fed with C18:1, the major part of triacylglycerols have a carbon number higher than 48 and were not degraded in NLSD cells (Fig. 5B). These data confirm the results observed with radiolabeled fatty acids, supporting the idea that only long-chain containing triacylglycerols accumulate in NLSD cells, whereas medium-chain containing triacylglycerols do not accumulate in these cells.
Figure 7: In vitro determination of activities of enzymes degrading radiolabeled decanoic acid- or oleic acid-containing triacylglycerols (A-D and E, respectively) in fibroblast homogenates (prepared in distilled water as described under ``Materials and Methods''). A, linearity of the enzyme activity versus time (standard assay conditions using 100 µg of protein of the homogenate per assay); B, linearity of the enzyme activity versus enzyme concentration (expressed as µg of homogenate protein/assay; incubation time, 2 h); C, effect of increasing concentrations of the substrate (100 µg of protein/assay; incubation time, 2 h); D and E, hydrolytic activity when varying pH from 3.5 to 9.0 using decanoic- or oleic-containing triacylglycerols as substrates (D and E, respectively). Mean ± S.E. of three experiments.
When intact normal cells were incubated in the presence of 1 µmol/liter E600, the in situ hydrolysis of short/medium-chain triacylglycerols was completely blocked (Fig. 6A), whereas that of long-chain triacylglycerols was not affected (Fig. 6B). In contrast, when intact normal cells were incubated in the presence of 10 µmol/liter PCMB, the in situ hydrolysis of long-chain triacylglycerols was severely impaired (Fig. 6C), whereas that of medium-chain triacylglycerols was not altered (Fig. 6D). The separate inhibition of the degradation pathways of short/medium- and long-chain triacylglycerols by these two inhibitors supports the hypothesis of two separate degradation pathways.
The activities of the two candidate enzymes were evaluated
in homogenates of cells treated with the irreversible inhibitors under
conditions of Fig. 6, using in vitro assays containing
either [C]decanoic-containing triacylglycerols
(prepared from cells loaded either with radiolabeled
[
C]decanoic acid or
[
H]triolein). The enzyme hydrolyzing
C10-containing triacylglycerols was inhibited in cells treated by E600,
whereas the enzyme hydrolyzing triolein was not inhibited in the same
cells (Fig. 8, C and D). Reversely, in cells
treated with PCMB, the enzyme hydrolyzing triolein was inhibited,
whereas the enzyme hydrolyzing C10-containing triacylglycerols retained
its activity (Fig. 8, C and D).
Figure 8: Comparison of inhibition of enzymes degrading radiolabeled decanoic acid- or oleic acid-containing triacylglycerols (A, C and B, D, respectively) by the irreversible inhibitors E600 (A, B) or PCMB (C, D) in vitro or ex situ. The enzyme activities were determined in vitro under the standard assay conditions, using radiolabeled decanoic acid- or oleic acid-containing triacylglycerols as substrate and fibroblast homogenates as enzyme sources, in the absence(-) or presence (+) of inhibitors used either in vitro (inhibitors added in the test tube) or ex situ (determination in vitro of the in situ inhibition: in this case, the intact living cells were incubated for 12 h with 1 µmol/liter E600 or 10 µmol/liter PCMB; then cells were harvested and homogenized, and the enzyme activity was determined under the standard conditions in the absence of any additional inhibitor). Mean ± S.E. of three experiments.
These results support the hypothesis that cytoplasmic C10-containing triacylglycerols are degraded in situ by an E600-sensitive short-chain lipase (not deficient in NLSD cells), which is clearly discriminated from the neutral lipase activity (specific to long-chain triacylglycerols) involved in the liberation of long-chain fatty acids of cytoplasmic triacylglycerols and deficient in NLSD cells.
The data previously reported (8, 9, 10, 31, 32) and those reported here clearly show that the cytoplasmic triacylglycerol accumulation in cultured fibroblasts is largely dependent on the extracellular lipids, since it is supplied by (at least) two separate pathways, the first one being the intracellular pathway of triacylglycerols endogenously biosynthesized from the cytoplasmic pool of fatty acids (8, 9, 10) and the second one (probably minor) resulting from the cellular uptake of triacylglycerols contained in high density lipoproteins(13) . Both sources of triacylglycerols have been shown to participate to the storage of cytoplasmic triacylglycerols in NLSD(8, 9, 10, 13) .
In the experiments reported here, the difference between the rates of incorporation of fatty acids with various acyl chain length into cellular lipids (under the used conditions, long-chain fatty acids were incorporated around 10 times faster than short- or medium-chain fatty acids) cannot be attributed to any cytotoxic artefactual effect (as assessed by the viability of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide or trypan blue tests). The selectivity of incorporation of long-chain natural fatty acids into cellular lipids is consistent with the results observed with fluorescent fatty acid analogs (15, 33, 34) and with natural fatty acids(35) . As the ratio of triacylglycerols/polar lipids is nearly constant whatever the acyl chain length, it is suggested that the limiting step of short-chain fatty acid incorporation is probably an early metabolic step, such as transmembrane transport or biosynthetic pathway (for instance, acyl-CoA synthases are known to be acyl chain length-dependent) (36) .
Natural short-chain fatty acids were really incorporated into triacylglycerols, in contrast to short-chain fluorescent fatty acids (pyrene-butanoic acid), which were incorporated into phospholipids but not in triacylglycerols(15) . Therefore, the mechanism of the lack of accumulation of short/medium-chain triacylglycerols in NLSD cells is different with fluorescent fatty acids (lack of biosynthesis) and with natural fatty acids (lack of degradation block).
On the basis of genetic and inhibitor-induced metabolic blocks, it was suggested that cytoplasmic triacylglycerols are degraded through two separate catabolic pathways, one being specific to long-chain triacylglycerols (long-chain specific lipase activity) and the other one specific to short-chain triacylglycerols (short-chain specific lipase or carboxylesterase activity).
The genetic defect of the in situ triacylglycerol degradation in NLSD cells is specific to triacylglycerols containing dodecanoic and longer chain fatty acids, thus suggesting an in situ defect of the long-chain lipase activity. As the activity of the long-chain neutral lipase is apparently not deficient in vitro(10, 15) , it may be speculated that the mutation affects either a site involved in the lipase activity in situ (for instance, site for routing or enzyme activation) or a co-factor necessary to the lipase activity in situ by analogy with the co-lipase for the pancreatic lipase (37) or with the activator proteins (saposins) for several lysosomal enzymes(38, 39) . The normal degradation of short/medium-chain triacylglycerols (up to 10 carbons) in NLSD cells suggests that these lipids are degraded through a catabolic pathway different from and unable to compensate the in situ deficient activity of the long-chain lipase.
The hypothesis of the existence of two separate hydrolytic pathways is also supported by experiments with irreversible inhibitors specific to each degradation pathway and able to inhibit the enzyme activities in situ and in vitro as well. E600 inhibited concomitantly the degradation of short/medium-chain triacylglycerols in situ and the short-chain lipase activity in vitro but not the long-chain lipase in vitro activity nor the in situ degradation of long-chain triacylglycerols. Reversely, the sulfhydryl reagent PCMB inhibited the in situ degradation of long-chain triacylglycerols and the long-chain lipase activity in vitro, but not the in situ degradation of short/medium-chain triacylglycerols and the short/medium-chain lipase activity. Moreover, the inhibition of the enzymes (activities determined in vitro by using their respective substrates) persisting after lysis of cells treated with irreversible inhibitors, E600 and PCMB (this study), and the in vitro studies of enzymatic properties previously reported (1, 40, 41, 42) are consistent with the idea that the short-chain lipase is different from the long-chain lipase. The enzymatic properties (specificity to long-chain triacylglycerols, heat stability, susceptibility to PCMB but not to E600) of the neutral long-chain lipase (1, 5, 18, 42) are similar to those of the microsomal neutral lipase(43, 44, 45) , which has not been cloned to date, to our knowledge, and is different from the hormone-sensitive lipase (no activation of triacylglycerol degradation by dibutyryl-cAMP) (data not shown) and other known lipases by its enzymatic properties in vitro(1, 5) . The neutral short/medium-chain lipases exhibit some enzymatic properties (specificity to various short-chain lipophilic esters, heat lability, susceptibility to organophosphorous compound E600, but relative resistance to PCMB) similar to those of carboxylesterases(1, 40, 41, 42) , some of them having been cloned in liver or other tissues (45, 46, 47, 48) . As the enzymes are not identified at the molecular level, the alternative hypothesis that there is one lipase with differential substrate specificity cannot be excluded.
Finally, from the data observed in intact cells, i.e. degradation at a normal rate of short-chain triacylglycerols in NLSD (in contrast to the accumulation of long-chain triacylglycerols), it is suggested to use diets enriched with short/medium-chain lipids (and poor in long-chain lipids) to tentatively slow down the long-chain triacylglycerol accumulation in NLSD patients.