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Succinate metabolism related to lipid synthesis in the housefly Musca domestica L.

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Archives of insect Biochemistry and Physiology 5:189-199 (1987)
Succinate Metabolism Related to Lipid
Synthesis in the Housefly Musca domestica 1.
Premjit P. Halarnkar, Charles R. Heisler, and Gary J. Blomquist
Department of Biochemistry, University of Nevada, Reno
The metabolism of succinate was examined in the housefly Musca domestica
L. The labeled carbons from [2,3-14C]succinate were readily incorporated into
cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,414C]succinate was not incorporated into either fraction. Examination of the
incorporation of [2,3-14C]succinate, [l-14C]acetate, and [U-14C]proline into
hydrocarbon by radio-gas-liquid chromatography showed that each substrate
gave a similar labeling pattern, which suggested that succinate and proline
were converted t o acetyl-CoA prior t o incorporation into hydrocarbons.
Carbon-I3 nuclear magnetic resonance showed that the labeled carbons from
[2,3-13C]succinate enriched carbons 1,2, and 3 of hydrocarbons with carboncarbon coupling showing that carbons 2 and 3 of succinate were incorporated
as an intact unit. Radio-high-performance liquid chromatographic analysis of
[2,3-14C]succinate metabolism by mitochondria1 preparations showed that in
addition to labeling fumarate, malate, and citrate, considerable radioactivity
was also present in the acetate fraction. The data show that succinate was
not converted t o methylmalonate and did not label hydrocarbon via a
methylmalonyl derivative. Malic enzyme was assayed in sonicated
mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old
insects; higher activity was obtained with NAD" in mitochondria prepared
from thoraces, whereas NADP+ gave higher activity with abdomen
preparations. These data document the metabolism of succinate t o acetylCoA and not to a methylmalonyl unit prior t o incorporation into lipid in the
housefly and establish the role of the malic enzyme in this process.
Key words: hydrocarbon biosynthesis, insect lipids, malic enzyme, acetate, intermediary
The precursors to normal and methyl branched hydrocarbons have been
examined in several insect species [l].In a termite, a combination of radioAcknowledgments: Supported in part by grant DCB-8416558 from the National Science Foundation and a contribution of the Nevada Agricultural Experiment Station. We thank the
Biology Section, S.C. Johnson and Sons, Racine, WI for supplying housefly pupae.
Received December 23,1986; accepted March 5,1987.
Address reprint requests to Dr. Gary J. Blomquist, Department of Biochemistry, University of
Nevada, Reno, NV 89557.
0 1987 Alan R. Liss, inc.
Halarnkar, Heisler, and Blomquist
active and stable isotope studies demonstrated that succinate served as an
efficient precursor for methyl branched alkanes via a methylmalonyl unit
[2,3]. The housefly Musca domestica also readily incorporated labeled succinate into hydrocarbon, but in contrast to the termite, succinate did not
appear to serve as a precursor to the methyl branched unit [4,5]. These
studies prompted questions regarding the metabolic route by which the
labeled carbons of succinate were incorporated into lipid in the housefly.
In an examination of pheromone synthesis in the tsetse fly, Glossina morsitans rnorsitans, Langley and Carlson [6] reported that [2,3-14C]succinate
readily labeled long-chain methylalkanes, presumably as a methylmalonate
unit. The tsetse fly has very high NAD+-specific malic enzyme [L-malate:
NAD oxidoreductase (decarboxylating)] activity, which is involved in the
oxidation of proline to pyruvate and is important in flight energetics [7,8].
Therefore, it was considered possible that succinate could be efficiently
converted to an acetyl unit in certain insect species through a process involving malic enzyme. This possibility was examined in M . domestica, and the
data reported herein demonstrate that carbons 2 and 3 of succinate are
metabolized to an acetate unit, which is then efficiently converted to lipid.
Housefly pupae were supplied by the Biology Section, S.C. Johnson and
Son, Racine, WI. The adults were maintained as described by Dillwith and
Blomquist [9].
Radioactive Substrates
Sodium [l-14C]acetate (57 mCiimmo1) and [U-14C]malic acid (200 mCil
mmol) were purchased from Research Products International, Mount Prospect, IL. [2,3-14C]Succinic acid (15 mCiImmol), [l,4-14C]succinic acid (20.5
mCilmmol), and [U-I4C]L-proline (265 mCilmmol) were purchased from
ICN, Irvine, CA.
In Vivo Radiotracer Studies
Labeled substrates in 0.5-1.0 p1 of water or 10% ethanol were injected into
the thorax of 4- to 5-day-old female houseflies (groups of five). After the
times indicated in the text, usually 2 h, the insects were killed by freezing at
-2O"C, and the cuticular hydrocarbon was extracted in hexane as described
by Dillwith et al. [4]. After the hexane rinses, the internal lipid was extracted
by the method of Bligh and Dyer [lo], in which methanol, chloroform, and
water were added to give a single phase. Then, additional aliquots of chloroform and water were added, and the chloroform layer containing lipid was
removed and analyzed. Hydrocarbon was isolated from the cuticular extracts
and separated into saturated and unsaturated fractions as described [4].
Samples were assayed for radioactivity by liquid scintillation spectrometry in
10 ml of 0.4% (WIV) 2,5-diphenyloxazol in toluene. Radio-gas-liquid chromatography was performed as described [4].
Succinate Metabolism in the Housefly
13C-NMR Experiments
Potassium [2,3-13Cdsuccinate was prepared as described by Blomquist et
al. [3]. Substrate was fed to insects as described by Dillwith et al. [5]. Three
days after including the labeled substrate in the diet, insects were extracted,
hydrocarbons were isolated, and proton decoupled { 'H} 13C-NMR analyses
were performed as described by Dillwith et al. [5].
Mitochondrial Preparations and Malic Enzyme Assay
Mitochondria were prepared from 1-day-old and 4-day-old housefly abdomens and thoraxes (5-8 g each) as described by Halarnkar et al. [ll].
Oxygen uptake rates and respiratory control ratios were determined by using
glutamate-malate as substrates [12].
The mitochondrial malic enzyme activity was determined as described by
Moreadith and Lehninger [13]. Lubrol PX was added from a 2% stock solution in water to give a final concentration of 0.2% in the mitochondrial
suspension. The suspension was sonicated at 140 watts output in an ice bath
for a total of 90 s using six 15-s bursts at 30-s intervals with the microtip
probe of a Braun-Sonic, model 1510 (16 Hz) South San Francisco, CA. This
sonicated mitochondrial suspension was used directly for the enzyme assay
in 2.9 ml of medium containing 50 mM MOPS, 0.5 mM dithiothreitol, 15 mM
L-(-)-malate, 2 mM K+ fumarate, 0.02% NaN3, and 5 mM MnCI2 at pH 7.4
and 30°C. The reaction was started by adding 0.1 ml of 30 mM NAD' or
NADP+. One enzyme unit is defined as the amount of enzyme that catalyzes
the formation of 1 pmol of NAD(P)H/min at 30°C where the millimolar
extinction of reduced NADP at 340 nm equals 6.20 A. In some experiments,
aliquots of the postmitochondrial supernatant were assayed for malic enzyme
Protein was determined by the biuret method [14] using bovine serum
albumin as the standard. All biochemicals were purchased from Sigma
Chemical Co., St. Louis, MO and were used without further purification.
Succinate Metabolism by Mitochondrial Preparations
One microcurie of [2,3-14C]succinic acid was added to 25-pI aliquots of
mitochondrial preparation (total volume 0.225 ml) from 4-day-old females.
Incubation was performed at 37°C in a shaking water bath for the times
indicated in the text. The incubation was stopped by adding 2 ml water, 3
drops of 0.1 N NaOH, and 4 ml of acetonitrile. Precipitate and other debris
were removed by centrifugation at 2,0008 for 5 min. The extract was reduced
to 0.5-1 ml in a 60°C sand bath under a stream of nitrogen. Extract was then
filtered (filter size, 0.2 pm) and analyzed by HPLC".
Organic acids were separated by HPLC on a Bio-Rad HPX-87 (9 pm) ionexchange column using 0.01N H2SO4 as eluent. Organic acids were detected
by an UV-spectrophotometer at 210 nm. The material corresponding to each
component was collected in a scintillation vial. Unlabeled standards were
coinjected with each sample to allow detection by U V absorbance.
*Abbreviations: HPLC = high-performance liquid chromatography; MOPS = morphodinopropane sulfonic acid; radio-CLC = radio-gas-liquid chromatography.
Halarnkar, Heisler, and Blomquist
Ten milliliters of Ready-Solv'" EP (Beckman, Fullerton, CA) were added to
each sample, and radioactivity was assayed on a Beckman liquid scintillation
counter at 80-85% efficiency.
The time-dependent incorporation of [2,3-14C]succinateinto cuticular hydrocarbon and internal lipid is presented in Figure 1A and B. If succinate
were incorporated into hydrocarbon as a methylmalonyl unit, as has been
shown in a termite [2,3] and assumed in the tsetse fly [6], it would be
expected that only the hydrocarbon fraction, which contains methyl branched
alkanes [l5], would be extensively labeled. The relatively high incorporation
of radioactivity from [2,3-14C]succinate into internal lipid, presumably into
fatty acid moieties, suggested that the labeled carbons of [2,3-14C]succinate
were incorporated into lipid after conversion to acetyl-CoA.
To examine this possibility, a comparison of the relative rates and specificity of incorporation of [l-14C]acetate, [2,3-14C]succinate,and other substrates
was performed. The data (Table 1)show that in a 2-h incubation period, the
"7 B
Fig. 1. Time-dependent incorporation of [2,3-14C]succinate into cuticular hydrocarbon (A)
and internal lipids (6).The labeled substrate was administered to 5-day-old female houseflies,
and at the times indicated, the insects were killed, extracted, and radioactivity was determined as described in "Materials and Methods." Error bars represent the SD, n = 3.
Succinate Metabolism in the Housefly
TABLE 1. Incorporation of [l-14qAcetate, [2,3-14C]Succinate, [1,4-14C]Succinate,
[U-14C]Malate, and [U-14CJProlineInto the Cuticular Hydrocarbon and Internal Lipid in
the Housefly*
[I, 4-14Clsuccinate
malate late
malate pro line
into cuticular
0.75 f 0.22a
0.19 f 0.07
< .01
0.25 f 0.04
0.17 f 0.04
Distribution in
YO Saturated
% Unsaturated
57.7 5 1.6
54.4 f 2.0
42.3 f 1.6
45.6 f 2.0
51.3 f 1.4
56.5 f 3.6
48.7 f 12.7
43.5 f 4.1
into internal
5.0 f 0.6
2.9 f 0.3
< .1
2.3 f 0.7
2.0 f 0.1
*Labeled substrates were injected into 4-day-old females, and after 2 h, the insects were killed,
lipids were extracted and separated, and radioactivity was determined as described in
"Materials and Methods."
'Mean f SD. N = 3 groups of 5 insectsigroup.
labeled carbons from [2,3-14C]succinate were incorporated about one-fourth
as efficiently as [l-14C]acetateinto hydrocarbons and about one-half as efficiently into internal lipids. Similar percent incorporation into cuticular hydrocarbons and internal li id as that of [2,3-14C]succinicacid was obtained from
experiments using [U-klmalate and [U-14C]proline (Table 1).Furthermore,
separation of the cuticular hydrocarbons into saturated (which contain normal and methyl-branched alkanes) and unsaturated (contain only n-alkenes)
fractions showed that the distribution of radioactivity from [l-14C]acetate,
[2,3-14C]succinate, [U-14C]malate, and [U-14C]proline gave similar results,
with from 51% to 58% of the radioactivity recovered in the saturated fraction.
If succinate were incorporated as a methylmalonyl unit, it would be expected
to referentially label the saturated fraction, as has been demonstrated with
Il-'C]propionate [4].
Further evidence that the labeled carbons from [2,3-14C]succinateand [U14C]prolinewere incorporated into lipid via acetate was obtained by radioGLC analysis of the hydrocarbon fractions labeled from 12,3-14C]succinate
and [U-14C]prolinecompared to [1-14C]acetate.The results of this experiment
demonstrated that each substrate gave a similar radio-GLC pattern (data not
shown) in which (2;)-9-tricosene and n-tricosane were major labeled hydrocarbons. Methyl branched hydrocarbons and components longer than 23
carbons were somewhat less extensively labeled from [l-14C]acetatein 5-dayold insects of this strain than reported earlier [4] using a different strain of
insects. Nevertheless, the similarity in the radio-GLC profiles from each
substrate are consistent with the conversion of proline and succinate to
acetate prior to incorporation into hydrocarbon.
A comparison of the incorporation of [2,3-14C]succinate and [1,414C]succinate (Table 1) showed that the radioactivity in carbons 2 and 3 of
succinate were readily incorporated into lipid, whereas the radioactivity from
carbons 1and 4 were not. This gave further evidence that succinate was not
incorporated into hydrocarbons as a methylmalonyl unit and suggested that
carbons 2 and 3 of succinate became the carbons of an acetyl unit.
Halarnkar, Heisler, and Blomquist
Direct evidence for this came from a re-examination by {lH} 13C-NMRof
the incorporation of [2,3-13C]succinate into the hydrocarbons of female flies.
An earlier study [5] of methyl branched alkanes gave data indicating that
succinate was not incorporated as a methylmalonyl unit but did not clarify
how it was incorporated. The {lH}13C-NMR spectrum of the total alkane
fraction is presented as Figure 2 and shows that the signals from carbons C1
C,, C2 Cn-l and C3
Cn-2of the alkanes are clearly enriched, and
furthermore, the carbon-carbon coupling shows that carbons 2 and 3 of [2,313C]succinate are incorporated as an intact unit into alkanes. The 13C-13C
coupling constants are from 34 to 36 Hz, which is similar to the 13C-13C
coupling constants reported for methylalkanes [3,5] in earlier studies.
Fig. 2. ('H}I3C-NMR spectrum of the alkanes of the female housefly after enrichment with
[2,3-'3C]succinate. The labeled substrate was included in the insects' diet for 3 days, and then
the insects were killed, cuticular hydrocarbons were extracted, the alkane fraction was
isolated, and {1H)13C-NMR was performed as described in "Materials and Methods." C,
refers to internal carbon atoms; n, No. of carbon atoms in the chain.
Succinate Metabolism in the Housefly
A direct analysis of the water-soluble products of [2,3-14C]succinatewas
obtained after the in vitro incubation of [2,3-14C]succinate.An example of the
type of data obtained is presented in Figure 3, which is from a 15-min
incubation period with mitochondria from 4-day-old female houseflies. The
data clearly show that in addition to the major expected metabolites, fumaric
acid, malic acid, and citric acid, substantial amounts of radioactivity were
associated with acetic acid. A time-dependent examination of succinate metabolism at times up to 2 h showed that the major radiolabeled fractions at
all times were succinate, fumarate, malate, acetate, and citrate. At no time
was appreciable radioactivity associated with the methylmalonic acid fraction. Thus, these data are consistent with the conversion of succinate to an
acetyl unit.
The enzyme most likely associated with the conversion of succinate to
acetyl-CoA, the malic enzyme, has been extensively studied in the tsetse fly
and other insects [7,8,16]. In this study, levels of malic enzyme utilizing
either NAD+ or NADP+ were determined in the housefly. Assay of subcel-
Fig. 3. Radio-HPLC chromatogram of the products after incubation of [2,3-14C]succinatewith
mitochondria isolated from 4-day-old female houseflies. Incubation conditions, extractions,
and HPLC analysis are described in "Materials and Methods."
Halarnkar, Heisler, and Blomquist
lular fractions showed that most of the NAD+- and NADP+-associated malic
enzyme activity from both the abdomen and thorax was associated with the
mitochondria1 fraction (Table 2). The abdomen contained higher specific
activity NADP+ malic enzyme, whereas the thorax contained higher specific
activity NADf malic enzyme, The NAD+ malic enzyme activity was similar
in 1- and 4-day-old flies, whereas the NADP+ malic enzyme activity had
lower specific activity at day 4, particularly in the abdomen.
In contrast to the metabolic fate of succinate in a termite [2,3], in which it
is converted to a methylmalonyl unit and then incorporated into methyl
branched hydrocarbon, the data presented here show that in the housefly,
succinate is converted to an acetyl unit prior to incorporation into lipid (Fig.
4). These observations are consistent with the high levels of vitamin B12
reported in the termite 217 that would presumably be required for the
methylmalonyl-CoA mutase, which converts succinyl-CoA to methylmalonyl-CoA. In contrast, the housefly does not contain detectable levels of
vitamin BIZ [lq,and there is no evidence for the interconversion of succinylCoA and methylmalonyl-CoA.
Langley and Carlson 161 showed that [2,3-14C]succinatewas incorporated
into the long-chain methylalkanes that serve as the contact sex pheromone
of the female tsetse fly, GEossina morsitans morsitans (Westwood). The tsetse
fly does contain vitamin B12 [lq,and it was assumed that succinate preferentially labeled the methylalkanes [6]. However, the data presented here,
which demonstrate that succinate is converted to acetate prior to incorporation into lipid in the housefly, along with the high activity of the malic
enzyme (NAD+-specific)in the tsetse fly [7] suggest the possibility that a
portion of the labeled succinate is converted to acetate prior to incorporation
into hydrocarbon. That the conversion of succinate to acetate is not a major
pathway in the tsetse fly is indicated by the low incorporation of radiolabel
from [2,3-14C]succinate into internal lipid [6], which is in contrast to the
The differences in the metabolism of succinate in the termite (and perhaps
tsetse fly) compared to the housefly suggest another metabolic route prediTABLE 2. Malic Enzvme Activitv in the Houseflv Musca Aomestica L.*
nmolimg proteinimin
1day old
4 days old
1day old
4 days old
*NAD+ and NADP+ malic enzyme activity was assayed as described in "Materials and
Succinate Metabolism in the Housefly
\ J
Occurs in a Termite
Housefly does not convert
succinyl-CoA to methylmalonyl-CoA
Vitamin Blo)
Fig. 4. Selected aspects of intermediary metabolism in the housefly. Succinate is not converted to methylmalonyl-CoA but rather labels lipid via acetyl-CoA.
cated upon the absence of vitamin B12 [17], which precludes a functional
methylmalonyl-CoA mutase, and therefore, have evolved an alternative route
for propionate metabolism. In the housefly, a termite [ll], and the American
cockroach [B], propionate is converted to acetate rather then to succinate as
occurs in mammals [19]. In propionate metabolism, it appears that all insects
examined [20], regardless of vitamin B12 levels, use a pathway that does not
depend upon vitamin B12. In contrast, the termite and housefly metabolize
succinate by different routes, perhaps dictated by vitamin B12 levels.
The finding of higher amounts of the NAD+ malic enzyme activity than
NADP+ malic enzyme activity in the thoraxes of female houseflies is consistent with its postulated role of providing energy for flight. The much higher
NADP+ activity compared to NAD' activity in the abdomen would be in
agreement with its proposed role in this tissue as a source of reduced NADP+
for biosynthetic activity [16]. However, if that were the case, higher activity
would be expected in the soluble fraction (supernatant)than in the mitochondrial fraction. Thus, the relatively high NADP' activity in the mitochondrial
fraction is unusual. Most of the malic enzyme activity in the thoraxes and
abdomens of the housefly was present in the mitochondria, which was
consistent with the subcellular location of this enzyme in most insects 1161
but contrasted to that of the American cockroach in which the NADP+linked enzyme was almost exclusively cytoplasmic.
Halarnkar, Heisler, and Blomquist
Much of the discussion involving malic enzyme in insects has centered on
patterns of substrate utilization in flight metabolism [S] or, less often, on
reducing equivalents for biosynthetic reactions [21]. An additional consideration, which may be important in insects that utilize relatively high protein
diets, could be the conversion of "glucogenic" precursors to acetate. The
relatively high conversion rate of proline and succinate into lipid via acetate
indicates that the pathway in which succinate is metabolized to fumarate,
malate, and thus to pyruvate and acetate (Fig. 4) is of major importance in
the housefly.
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