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The effect of calorie restriction on growth and development in silkworm Bombyx mori.

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A r t i c l e
THE EFFECT OF CALORIE
RESTRICTION ON GROWTH AND
DEVELOPMENT IN SILKWORM,
Bombyx mori
Yijia Li, Keping Chen, Qin Yao, and Jun Li
Institute of Life Sciences, Jiangsu University, 301 Xuefu Road,
Zhenjiang 212013, P.R. China
Yong Wang
School of Food and Biological Engineering, Jiangsu University, 301
Xuefu Road, Zhenjiang 212013, P.R. China
Haijun Liu, Chiyu Zhang, and Guoping Huang
Institute of Life Sciences, Jiangsu University, 301 Xuefu Road,
Zhenjiang 212013, P.R. China
Caloric restriction (CR) is known to extend the life span in different species
from yeast to mammals. In this report, a simple organism silkworm
(Bombyx mori) was used to study the effect of moderate CR on the growth
and development processes of insects. Here we show that an extension of
life span upon moderate CR was observed in the silkworm. The total
protein level in the 5th instar larvae hemolymph appeared to decline
significantly under CR. SDS-PAGE analysis showed that the influence of
CR was sex-dependent. The CR effects on female animals were much more
significant than on the males. The MALDI-TOF MS study identified 16
proteins that expressed differentially among six groups of the male or
female larvae fed at different time frequencies. Four of them, storage
protein 1 (SP1), arylphorin (SP2), imaginal disk growth factor (IDGF),
and 30-kDa lipoprotein, showed significant differences. It was demonstrated that these four proteins were up-regulated when the larvae were
C 2009 Wiley
over-fed and down-regulated when the larvae were less-fed. Periodicals, Inc.
Grant sponsor: National Basic Research Program of China; Grant number: 2005CB121000; Grant sponsor:
National ‘‘863’’ Project of China; Grant number: 2008AA10Z145.
Correspondence to: Keping Chen, Institute of Life Sciences, Jiangsu University, 301 Xuefu Road, Zhenjiang
212013, P.R. China. E-mail: kpchen@ujs.edu.cn
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 71, No. 3, 159–172 (2009)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2009 Wiley Periodicals, Inc. DOI: 10.1002/arch.20313
160
Archives of Insect Biochemistry and Physiology, July 2009
Keywords: Bombyx mori; calorie restriction (CR); growth and development; MALDI-TOF MS
INTRODUCTION
Aging and longevity are of major concern in the field of biology. Every animal’s
average and maximal life span is relatively steady and is species-specific. There have
been dozens of hypotheses trying to account for the complicated aging process.
Examples are immunology theory, neuroendocrine theory, radical theory, protein
synthesis error accumulation theory, gene regulation theory, DNA damage repair
theory, mitochondrion damage theory (Silva and Larsson, 2002), and telomere theory
(Kondo et al., 1998).
In the 1930s, McCay, a nutritionist at Cornell University (Ithaca, New York), found
that moderate calorie restriction (CR) extended the life span of laboratory rodents
(Guarente, 2005). Since then, a wealth of dietary, physiological, and medical
information has emerged from many carefully designed studies. These data suggest
that CR can reduce or prevent the occurrence of many different diseases, and, as a
consequence, can extend the life span of different animal species including rodent,
fish, fly, worm, and yeast (Berrigan et al., 2002; Heilbronn and Ravussin, 2003;
Hursting et al., 2003; Lane et al., 1999; Masoro, 2000; Stern et al., 2001). In yeast
Saccharomyces cerevisiae, Jiang et al. (2000) found that reduction of glucose concentration in culture medium led to an extension in life span and a delay in appearance of
aging phenotype. It was discovered that the extension of life span was inversely related
to the glucose level. Life extension was also elicited by decreasing the amino acids
concentration in the medium (Jiang et al., 2000). In fruit flies, a reliable method of
dietary restriction has been developed involving dilution of the yeast and glucose that
make up flies’ food. Over a range of food dilutions, the life span was extended, but
eventually the dilution point became too severe and the life span shortened again
(Guarente, 2005). Kealy et al. (2002) utilized 48 Labrador Retrievers to perform the
paired feeding study and found that 25% restriction in food intake increased the
median life span and delayed the onset of signs of chronic diseases in these dogs. Most
importantly, in human beings, many physiological changes have been associated with
CR, including total plasma cholesterol, triglyceride, and high- and low-density
lipoprotein levels (Verdery and Walford, 1998).
Dozens of genes related to longevity have been found, such as Sir-2, Daf-2,
Pit-1, Amp-1, Clk-1, p66Shc, Hst2, and Pha-4 (Chen and Guarente, 2007; Kaeberlein
et al., 2006; Wang et al., 2006), among which Sir-2 and Daf-2 genes have been
fully studied. Sir-2, also known as sirtuin, comprises an ancient family of NAD1dependent deacetylases that are conserved from bacteria to human and play
an important role in a wide variety of biological processes, including development,
heterochromatin formation, transcriptional silencing, DNA recombination and
repair, genome stability, apoptosis, axonal protection, fat mobilization, metabolic
regulation, and longevity (Avalos et al., 2005; Hoff et al., 2006). In Rogina and
Helfand’s (2004) findings, in Drosophila, CR did not extend the life
span in Sir-2 null mutants. Further, strains that over-expressed Sir-2 had an
extended life span on normal diet, which could not be further extended by CR (Rogina
and Helfand, 2004). Daf-2 is an insulin receptor–like protein that regulates metabolism,
development, and aging in Caenorhabditis elegans. In a quantitative proteomic study,
Archives of Insect Biochemistry and Physiology
Calorie Restriction in Bombyx mori
161
Dong et al. (2007) identified 86 proteins that were more or less abundant in long-lived
Daf-2 mutant worms than in wild-type worms. Genetic studies on a subset of these
proteins indicated that they acted in one or more processes regulated by Daf-2,
including entry into the dauer developmental stage and aging (Dong et al., 2007).
At present, there are still many disputes about the mechanisms that would account
for how CR works. Over the past 70 years, there have been at least ten plausible
theories to explain how CR works but almost all of them have fallen out of favor
(Sinclair, 2005). It seems that the effect of CR is too complex to be described by a single
hypothesis. Some early hypotheses are about developmental delays and reduced
metabolic rates. Current hypotheses concern glucocorticoid cascade, reduced reactive
oxygen species, cell survival hypothesis, protein turnover, insulin/IGF-1 signal
pathway, target of rapamycin (TOR) pathway, and the hormesis hypothesis (Sinclair,
2005). Herein, insulin/IGF-1 signal pathway theory is one of the most important
hypotheses to explain how CR works. In insect, Bombyxin, also called prothoracicotropic hormone (PTTH), is the first discovered insect insulin-like peptide. Bombyxin is
secreted from silkworm brain and has a similar structure and the same function as
insulins and IGFs in mammals.
Nowadays, researchers utilize microarray to measure the differences in gene
expressions after CR is applied to organisms (Cao et al., 2001). The effect of CR was
detected at the mRNA level and many genes were found to be related to the increase of
the life span. However, there have been no related reports about CR studied at the
protein level, especially at the proteomics level. Silkworm, Bombyx mori, is a good model
organism for Lepidopterean insects. It is an ideal material for CR studies owing to its
uniformity of growth and development, ease of handling and maintaining, relative
short life cycle, and consistency of food and environmental control. In this study, we
explored the effects of CR by feeding silkworm larvae at various time frequencies.
MATERIALS AND METHODS
Silkworm Strain and Feeding Methods
Silkworm strain Qiufeng was used in this study. Six hundred newly hatched larvae were
selected at random and separated into four groups: A, B, C, and D. Feeding was carried
out using fresh mulberry leaves at a frequency of 1, 6, 12, 24 hours per day (hours/day)
for Groups A, B, C, and D respectively. All larvae were kept in a chamber with a
controlled temperature of 251C, a relative humidity of 80%, and a 12-h light/dark cycle.
On Day three of the fifth instar, 10 female and 10 male larvae were sampled from
Groups B, C, and D, respectively. Individuals in Group A were all dead in the 1st instar
and were not able to be sampled. Their hemolymph was collected and centrifuged for
15 min at 12,000 rpm at 41C. The supernatant was collected and kept at 701C for
later use. Furthermore, 25 larvae were kept for further observation on the remaining
life processes.
Protein Concentration in Hemolymph
The total protein concentration of the hemolymph from Groups B, C, and D was
measured according to the instruction of the Bio-Rad protein assay kit (Bio-Rad,
Archives of Insect Biochemistry and Physiology
162
Archives of Insect Biochemistry and Physiology, July 2009
Hercules, CA). Bovine serum albumin (BSA) (0, 0.3, 0.6, 0.9, 1.2, 1.5 mg/ml) was used
to prepare the standard curve.
SDS-PAGE of Hemolymph
The SDS-PAGE was carried out to look for differentially expressed proteins. One
hundred and eighty micrograms of each protein sample was applied to the gel.
Electrophoresis was performed on 12% polyacrylamide gel at 41C under 90 V for
30 min and then under 140 V for 8 h.
In-Gel Digestion and Protein Identification
The main differential protein bands found in hemolymph were subjected to
identification of proteins using peptide mass fingerprinting (PMF). The band was
excised with circular plugs 2–3 mm in diameter, and transferred to 1.5-ml Eppendorf
tubes. Coomassie blue–stained gel pieces were first destained with 50 ml of 50% 50 mM
NH4HCO3 and 50% acetonitrile for 10 min, followed by three washes with 50 ml of
MilliQ water. The gel pieces were then dehydrated with 100% acetonitrile for 5 min,
and dried in a SpeedVac (Thermo Savant) for 30 min. The dried gel particles were
rehydrated at 41C for 30 min with 2.5 ml/well trypsin (sequencing grade; Promega,
Madison, WI) in 50 mM NH4HCO3 (20 mg/ml), and then incubated at 371C overnight.
Peptide mixture (1 ml) was mixed with 1 ml 10 mg/ml a-Cyano-4-hydroxycinnamic
acid (Sigma) and pointed on the MTP Anchor Chip (Bruker) and analyzed by
matrix-assisted laser desorption/ionization-time of flight mass spectrometry
(MALDI-TOF MS) (ultraflex tof/tof, Bruker, Germany). The mass spectra were
interpreted using the Mascot peptide mass fingerprint engine available on the web site
(http://www.matrixscience.com) for protein identification.
Data Analysis
The food intake and the newly exuviated larvae were weighed in each group. And the
larval periods among different groups was recorded, respectively. The results were
analyzed using Fisher LSD test by SPSS software. Differences between individual
means were deemed to be significant at Po0.05. The regression analysis among the
food intake, the body weight, and the larval period was performed by SPSS software.
RESULTS
Development of the Larvae
The food intake, body weight, and development processes in each group are
presented in Table 1. Figure 1 shows the value of natural logarithm (Ln) in Table 1.
Larvae in each group consumed more than 95% leaves in grown silkworm stage. The
amount of ingested leaves of the larvae in Group D was 3.18 times than that in Group
B and 1.47 times than that in Group C. In each group, the body weight of larvae
increased when the food intake increased, but the larval period shortened instead. The
larval period in Group B was 1.13 and 1.45 times that in Group C and D, respectively.
Compared with Group D, when the food intake decreased by 32.10%, the larval stage
was extended by 28.87%; and when the food intake decreased by 68.59%, the larval
Archives of Insect Biochemistry and Physiology
Archives of Insect Biochemistry and Physiology
b
6.35170.08647ab
c
13.7370.08523ac
0.052370.001087
0.086470.001187ac
0.355270.004562c
1.94170.1189ac
11.3770.04ac
C
bc
B
C
D
20.2270.8076bc
1.88970.01305ab
2.20370.006245ac
ab
18872.52
14573.00ab
15872.52ab
17371.73b
25272.52ab
B
ac
14871.73
12072.00ac
14471.00ac
16271.53c
23072.65ac
C
D
11071.53bc
74.071.00bc
11472.08bc
13972.52bc
19071.53bc
Larval period (h)
3.22570.005568bc 910713.0ab 80879.61ac 62778.00bc
0.111970.005510
0.0004
0.0004
0.0004
0.122170.003251bc 0.00389272.54E-05ab 0.00537470.000131ac 0.00623470.000139bc
0.54870.1603bc
0.0206870.00065b
0.0218172.65E-05c 0.0280070.000757bc
3.73470.2139bc
0.0956374.36E-05b 0.0967073.51E-05c
0.125270.00225bc
15.770.9296bc
0.518070.01253ab
0.561770.004528ac 0.612770.02181bc
D
Body weight (g)
week and could not develop into the second instar, so no data are shown here.
Values in a column with different superscripts (a–c) are significantly different (Po0.05, Fisher LSD test). Data represent the mean7SD. All larvae in Group A died within one
Total
1st instar 0.0491170.001798
2nd instar 0.0795970.0009113ab
3rd instar 0.196470.009102b
4th instar
0.96670.04443ab
5th instar
5.0670.08379ab
B
Food intake (g)
Table 1. Food Intake, Body Weight and Development Process of Each Instar in Groups B, C, and D
Calorie Restriction in Bombyx mori
163
164
Archives of Insect Biochemistry and Physiology, July 2009
Figure 1. Food intake, body weight and development process of each instar in Groups B, C and D. The
figures are made by the value of natural logarithm (Ln) of Table 1. All larvae in Group A died within one
week and could not develop into the second instar, so no data was showed here. (a) Food intake of larvae in
each instar. (b) Body weight of larvae in each instar. (c) Developmental process of the laevae. Each data point
is the mean7SD. All the data are significantly different within a column (Po0.05, Fisher LSD test).
stage was extended by 45.13%. On the other hand, almost all larvae in Group A died
within one week and did not develop into the second instar. Ninety percent of the
individuals in Group D could spin cocoons, metamorphose, mate, and lay eggs
normally, while in Group C and B, the proportions decreased to less than 50%,
respectively. Furthermore, compared to Group D, the number of eggs of Group B and
C were also reduced by 30–40%.
SPSS software was utilized to analyze the relationship of the food intake, the body
weight, and the larval period. The body weight was positively correlated with food
intake (r40.9), while the larval period negatively correlated with the food intake
(r40.9). Therefore, we chose the food intake as the independent variable X, the larval
period duration as the dependent variable Y, and obtained a linear regression
equation: Y 5 1,053.776–20.256 X, which suggests that the larval stage was prolonged
when the food intake decreased.
Protein Concentration of Hemolymph
According to the standard curve and the absorbance of hemolymph protein on
750 nm, the protein concentration in three groups was calculated, respectively (Fig. 2).
Archives of Insect Biochemistry and Physiology
Calorie Restriction in Bombyx mori
165
Figure 2. Protein concentrations in hemolymph. Larvae in Group B, C and D were fed 6, 12, 24 hours/day,
respectively. Each data column is the mean7SD. (a-f) indicate that the data is significantly different within a
column (Po0.05, Fisher LSD test). All larvae in Group A died within one week and we could not sample the
hemolymph, so no data was showed here.
Figure 3. SDS-PAGE of hemolymph from female and male larvae of Groups B, C and D. Lane a, b, c, d, e
and f represent females in Group B, males in Group B, females in Group C, males in Group C, females in
Group D, males in Group D, respectively. Arrowheads 1 to 16 indicate the differential protein bands
identified by MALDI-TOF MS.
The results showed that in the same group total hemolymph protein concentration in
the females was slightly higher than that in the males. The average protein
concentration in both females and males was the highest in Group D and the lowest
in Group B. In the females, the total protein concentration of Group D was 3.00 times
higher than that of Group B and 2.20 times higher than that of Group C. Moreover,
Archives of Insect Biochemistry and Physiology
166
Archives of Insect Biochemistry and Physiology, July 2009
the protein concentration of Group C was 0.23 times higher than Group B. In the
males, the total protein concentration of Group D was 2.70 times higher than Group B
and 1.70 times higher than Group C, and Group C was 0.36 times higher than Group
B. Compared with Group D, when the food intake decreased by 32.10%, the protein
concentration decreased by 66.61%; and when the food intake decrease by 68.59%, the
protein concentration decreased by 74.10%.
SDS-PAGE of Hemolymph
The result of SDS-PAGE is shown in Figure 3. Proteins corresponding to 16 bands
(Bands 1 to 16) were found to be altered in abundance or presence in six groups.
Protein band 11 was present only in Group D females (Lane e); Band 12 was absent
from Group B females (Lane a) and down-expressed in Group B males (Lane b);
Bands 13, 15, and 16 were down-expressed in Group B and C, and Band 3 was
present only in Group B males (Lane b).
Mass-Spectrum Identification of Differentially Expressed Proteins
The 16 bands cited above were isolated for MS analysis by MALDI-TOF MS (Fig. 3).
Table 2 summarizes the peptide mass fingerprinting and database searching results.
We successfully identified 16 bands corresponding to eight distinct gene products.
Namely, Bands 1, 4, 6, 7, 8, 9, 10, and 12 represent arylphorin, a silkworm storage
protein (SP), also named storage protein 2 (SP2); Band 11 represents storage protein 1
(SP1), which is a female-specific protein. Bands 5 and 13 belong to imaginal disk
growth factor (IDGF) and Band 15 is a 30-KDa small lipoprotein in silkworm.
The representative mass spectra of interest, i.e., Bands 5, 11, 12, and 15 of IDGF,
SP1, arylphorin, and 30-KDa lipoprotein, are shown in Figure 4, and the results of a
database search for these proteins are shown in Figure 5.
DISCUSSION
Individuals in Group A died within one week and could not develop into the second
instar. This indicated that an extreme control on food intake played a severe role on
normal physiological functions and development process and finally led to the death of
the experimental animals. Meanwhile, mulberry leaf is the only energy source for
silkworms containing both nutritive substance and water. Silkworms cannot intake
water as freely as rodent animals when we control their energy source. As a result, we
could not rule out the possibility that the death of the individuals may have been
caused by extreme water depletion, which might lead to a severe physiological
functional disorder. But one result is that moderate food restriction extends the life
span of silkworm larvae significantly, consistent with that observed in many other
organisms including rat, mouse, fish, fruit fly, nematode, and yeast. It was shown that
CR can prevent many different diseases and will extend the life span of different
rodent species and strains, which die of different causes (Hursting et al., 2003). CR
reduces metabolic rate and oxidative stress, improves insulin sensitivity, and alters
neuroendocrine and sympathetic nervous system function in animals (Heilbronn and
Ravussin, 2003).
Our results showed that although individuals in Group B had longer life, their
fecundities were reduced relative to Group C and Group D. Furthermore, the eggArchives of Insect Biochemistry and Physiology
Archives of Insect Biochemistry and Physiology
Arylphorin
Hypothetical protein GSPATT00015163001
Acyl-CoA dehydrogenase
Arylphorin
Imaginal disk growth factor
Arylphorin
Arylphorin
Arylphorin
Arylphorin (partial)
Arylphorin
SP1
Arylphorin
Imaginal disk growth factor
Antitrypsin
30-KDa lipoprotein PBMHPC-23 precursor
Hypothetical protein
Protein name
Bombyx mori
Paramecium tetraurelia strain d4-2
Streptomyces avermitilis MA-4680
Bombyx mori
Bombyx mori
Bombyx mori
Bombyx mori
Bombyx mori
Bombyx mandarina
Bombyx mori
Bombyx mori
Bombyx mori
Bombyx mori
Bombyx mori
Bombyx mori
Klebsiella pneumoniae
Source
Gi
Gi
Gi
Gi
Gi
Gi
Gi
Gi
Gi
Gi
Gi
Gi
Gi
Gi
Gi
Gi
124430725
145523666
29828457
124430725
152061158
124430725
124430725
124430725
624791
124430725
1335609
124430725
152061158
112983770
126419
53987112
Gene no.
5.70
6.12
5.51
5.70
7.64
5.70
5.70
5.70
7.79
5.70
6.55
5.7
7.64
5.41
8.46
9.11
pI
83,569
49,863
44,270
83,569
48,362
83,569
83,569
83,569
14,776
83,569
86,440
83,569
48,362
43,528
30,609
40,688
Mr
28
23
23
14
33
18
19
17
58
17
24
23
44
26
33
33
Sequence
coverage (%)
130
81
82
85
102
82
99
90
99
90
98
118
92
110
82
93
Score
hemolymph identified by MS. We successfully identified 16 bands corresponding to 8 distinct gene products that come from four different species. The proteins corresponding
to bands 5, 11, 12, and 15 are significant.
Shown here are the band number, protein name, source, gene no., calculated pI value, nominal mass (Mr), sequence coverage, and score of the differential protein bands of
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Band no.
Table 2. Differential Protein Bands of Hemolymph Identified by MS
Calorie Restriction in Bombyx mori
167
168
Archives of Insect Biochemistry and Physiology, July 2009
Figure 4. Peptide mass fingerprints for IDGF, SP1, arylphorin and 30-KDa lipoprotein. MALDI-TOF MS
spectrometry was done with in-gel-digested peptides of bands 5, 11, 12 and 15.
laying ability of the females was impaired and the quality of eggs was downgraded
accordingly. We conjectured that, just like many other organisms, silkworms could not
do everything well at the same time. The energy used for growth, maintenance, and
reproduction is inevitably limited and living beings must distribute resources
effectively to find the best allocation scheme among their conflictive requirements.
Obviously, food availability, nutritional status of the organism, and their effects upon
reproductive rate play a key role in life span determination and are central to
evolutionary life history theory (Piper et al., 2005).
The fact that the protein concentration of hemolymph in Group B was a little
lower than Group C but both groups were much lower than Group D showed that the
total hemolymph protein concentration changed significantly by CR applied to
silkworm. This was related to the decrease of one or several proteins that might have
functions in CR.
MALDI-TOF MS identified four significant proteins, SP1, arylphorin, IDGF, and
30-KDa lipoprotein, which were found to be altered in abundance or presence in six
groups. Both SP1 and arylphorin belong to the silkworm storage protein. SP1
exhibited female-specific expression in the hemolymph of the fifth instar larvae
containing an exceptionally high proportion of methionine, whereas SP2 is an
arylphorin-type storage protein rich in phenylalanine and tyrosine (Fujii et al., 1989).
The storage proteins of insect are synthesized by fat bodies and secreted into the
Archives of Insect Biochemistry and Physiology
Calorie Restriction in Bombyx mori
169
Figure 5. Results of NCBInr Database Search for the identification of band 5, 11, 12 and 15. Band 5, 11,
12 and 15 represent IDGF, SP1, arylphorin and 30-KDa lipoprotein, respectively. There listed the start and
end position of the matched peptide, the observed molecular mass, expective Mr, calculative Mr, the delta
value between expective Mr and calculative Mr and the matched sequences.
hemolymph. The final instar larvae accumulate large quantities of specific proteins in
their hemolymph, but they are selectively taken up by the fat body cells at the end of
the feeding period. These proteins are presumed to be used as the amino acid depot
required for the development of adult tissues at later stages and the formation of eggs
in the females (Hwang et al., 2001; Song et al., 1997; Tungjitwitayakul et al., 2008).
It was obvious that SP was highly expressed in Group D, but down-expressed in
Group B and C. We also found that SP was degraded in Group B and C. We conjectured
that hungry silkworms took SP as an energy source and degraded them for low molecular
segments, so proteins represented by bands 1 and 6–10 would exist. However, until
recently, the effect of CR on nutrition storage and the relationship between insect storage
proteins and longevity are still unknown. Resource allocation theory suggests that
longevity is enhanced by increasing somatic storage, at the expense of current
reproduction (Kirkwood, 2002). When individuals extend their life span caused by CR,
the storage of the protein increases. But Cunha et al. (2005) found that sugar-fed workers
of the honey bee Apis mellifera responded to the lack of dietary proteins by significantly
diminishing the amount of HEX 70b subunits in hemolymph. Apparently, they use HEX
70b to compensate for the lack of dietary proteins (Cunha et al., 2005). Our research
found that reduced food intake without malnutrition reduced the protein concentration
of hemolymph by 66–75%. These reduced proteins were storage proteins SP1 and SP2,
which indicates that the nutrition situation would affect the protein concentration,
especially the concentration of storage proteins in hemolymph. Reduced food intake leads
to the decrease of storage protein concentration, which may compensate its effect.
Another hypothesis to explain how CR works is the apoptosis mechanism. CR may
affect the life span of organisms through regulating apoptosis. A number of
researchers found that CR works via Sir, which regulates the activity of many other
important proteins in the cell (e.g., p53, FOXO, Ku 70, etc.). Sir can enhance the
ability of repair mechanisms of the cell and reduce apoptotic cell death, especially in
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Archives of Insect Biochemistry and Physiology, July 2009
the non-regenerated tissues like heart and brain. Consequently, CR may slow the rate
of apoptosis and extend the longevity of organisms. However, findings in a variety of
experimental models suggested that CR enhances rates of apoptosis concomitant with
decreases in DNA synthesis, markedly reducing the number and volume of
preneoplastic lesions (Hursting et al., 2003). Recently, Rhee et al. (2007) found that
in Hela cells, SP2 protein suppressed nuclear fragmentation and apoptotic body
formation and suppressed apoptosis further. So does the 30-KDa protein (Band 15,
Fig. 3), a specific type of plasma lipoprotein, which is synthesized in the larval fat body
and then released into the hemolymph (Kim et al., 2003). In our study, 30-KDa
protein was up-expressed in Group D but down-expressed in Group B and C.
Additionally, 30-KDa protein in the hemolymph of silkworms also exhibited apoptosisinhibiting activity. Obviously, our research supports the latter point. We speculated
that CR decreases arylphorin and 30-KDa protein in hemolymph, which may weaken
the anti-apoptosis effects induced in arylphorin and 30-KDa protein and may
accelerate the rate of apoptosis. Thus, the mechanism employed by organisms to clear
redundant cells could work well, which may decrease the risk of being a target for
some lethal diseases.
Another protein we found is IDGF, which is a soluble polypeptide growth factor first
identified in Drosophila melanogaster. IDGF cooperates with insulin in stimulating the
growth of imaginal disc cells, suggesting that IDGF might function as a cofactor of
Drosophila insulin or insulin-like peptide (Varela et al., 2002). Apparently, IDGF (Bands 5
and 13, Fig. 3) was down-expressed in Group B compared with Group D, especially in the
females. We conjecture that IDGF may work similarly to Bombyxin, the first discovered
insect insulin–like peptide, through the insulin/IGF-1 signal pathway. Insulin or IGF binds
to the Insulin receptor (Inr) or IGF-R, which may trigger the downstream signaling
pathways, and eventually inhibit the transcriptional activity of aging-related transcriptional
factors such as DAF16 and FOXO. These transcriptional factors can activate longevity
genes and subsequently inhibit aging genes. We suppose that nutrition regulates the
secretion of IDGF. The starvation of the silkworm larvae causes a decline in the level of
IDGF in the blood. Therefore, the insulin/IGF-1 signal pathway is disturbed, which causes
longevity genes to be activated and aging genes to be inhibited. Consequently, the life span
is extended when CR is applied to the larvae.
Interestingly, we found that the effect of CR on the life span was sex-dependent.
Apparently, the changes in the females in response to CR were much more than that of
the males. The reasons for this sex difference are not clear. To our knowledge, the
nutritional economy of the two sexes is different. The females undertake a high level of
anabolism in the production of eggs and thus respond to CR by reducing egg
production, whereas the males do not have that high level of anabolism and thus need
less food, so CR will not affect them as significantly as the females.
There are still many debates about the mechanisms that would explain how CR
can extend the life span of organisms. Our findings provide more information on the
effect of CR in silkworm, which can be an important reference for related studies using
other organisms.
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