Abstract
The impact of dietary soybean oil, lard and fish oil on physiological
responses in middle age is little studied. In this study, we
investigated the changes of oxidative stress, inflammatory cytokines,
telomere length, and age-related gene expression in the liver of
middle-aged rats in response to the above three fat diets. Male Sprague
Dawley rats (12 months old) were fed AIN-93M diets for 3 months, in
which soybean oil was equivalently replaced by lard or fish oil. As
compared to the lard diet, intake of fish oil diet significantly
decreased body weight gain, white blood cell count, and levels of
hepatic triacylglycerol, total cholesterol, fat accumulation,
low-density lipoprotein, oxidative stress and inflammatory cytokines
(P < 0.05), but increased telomere length (P < 0.05). On the other
hand, lard diet and soybean oil diet showed great similarity in the
above variables. PCR array analysis further indicated that fish oil
diet significantly down-regulated gene expression related to
inflammatory response, apoptosis, DNA binding, proteostasis and
telomere attrition. Differentially expressed genes were enriched in the
complement and coagulation cascades pathways. Such physiological and
molecular responses could be due to different fatty acid composition in
fish oil, lard and soybean oil.
Introduction
Fat is an important component of human diet and its nutritional value
is largely dependent on the fatty acid composition^[48]1. Different
sources of fat have different applications. Lard was mainly applied to
bakery and food homemaking in western countries many years ago and it
has been replaced by plant oils, including soybean, sunflower and olive
oils in terms of health concerns^[49]2, [50]3. However, lard is still
widely consumed in some developing countries. Some people even believe
that lard is not bad as we image^[51]4. Fish oil is usually applied as
a supplement agent because it contains high levels of n-3 fatty acids
and can prevent some diseases^[52]5.
Fatty acid composition in fat diets has been shown associated with
oxidation and inflammation. Saturated fatty acids (SFAs) are more
resistant to oxidation because they do not contain unsaturated bonds.
However, high-saturated-fat diets may increase the risk to inflammation
level in murine models^[53]6. On the other hand, n-3 polyunsaturated
fatty acids (PUFAs) have anti-oxidative activity because NF-κB pathway
can be inhibited by eicosahexaenoic acid (EPA), docosahexaenoic acid
(DHA) and their metabolites^[54]7. The deficiency of n-3 PUFAs may
promote lipogenic gene expression and hepatic steatosis through the
liver X receptor^[55]8. On the contrary, n-6 PUFA-rich diets may lead
to higher levels of lipid peroxidation and DNA oxidative breaks in rat
tissues and blood during aging, while MUFA-based diets showed less
oxidation^[56]9. The n-3 PUFAs are thought to systemically decrease
inflammatory responses by down-regulating inflammatory gene expression
via NF-κB inactivation^[57]10. However, n-6 fatty acids, in particular
to linoleic acid (18:2 n-6), may exert a pro-inflammatory
effect^[58]11. Monounsaturated fatty acids (MUFAs), including oleic
acid (OA), have been shown to play a dual role in inflammation^[59]6,
[60]12–[61]14.
Oxidative stress has been recognized as a critical contributor to many
physiological changes, in particular to the aging process^[62]15,
[63]16, which is characterized as a chronic and subclinical
inflammatory state^[64]17. The aging process is normally accompanied
with genomic instability, telomere attrition, epigenetic alterations,
loss of proteostasis, mitochondrial dysfunction and cellular
senescence^[65]18. Middle age is an earlier stage of the aging process,
during which gradual physical changes and some chronic illness may
occur^[66]19. Such changes would affect the outcomes at old
ages^[67]20. Many studies have emphasized the impact of diets on aging.
However, few data are available on the impact of dietary fats on
physiological responses at middle ages.
To this end, we investigated how intake of soybean oil, lard and fish
oil affected oxidative and inflammatory status, age-related gene
expression, telomere length and other related parameters of middle-aged
rats. The underlying mechanism was also proposed.
Results
Fat accumulation
Histological observations with Oil Red O staining indicated that the
quantity and size of hepatic fat droplets in the lard group were
greater than those in the soybean oil and fish oil groups
(Fig. [68]1a–c). Correspondingly, hepatic triacylglycerol (TAG) and
total cholesterol (TC) in the lard group were significantly higher than
those in the fish oil group (Fig. [69]1d, P < 0.05). No significant
difference existed between the lard group and the soybean oil group
(Fig. [70]1d, P > 0.05). The lard group also showed greater body weight
gain and liver index than the fish oil and soybean oil groups
(P < 0.05, Table [71]1), although the actual body weight and feed
intake were not significantly different among the three diet groups
(P > 0.05, Table [72]1). In blood, TAG, TC and low-density lipoprotein
(LDL) were higher in the lard group than those in the fish oil and
soybean oil groups (P < 0.05, Table [73]2). Insulin resistance index
was not significantly different among the three diet groups (P > 0.05).
Figure 1.
Figure 1
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Hepatic fat accumulation of rats fed with soybean oil, lard and fish
oil diets. (a) Hepatic section stained with Oil Red O from the soybean
oil group. (b) Hepatic section stained with Oil Red O from the lard
group. (c) Hepatic section stained with Oil Red O from the fish oil
group. Adipocytes were stained red and nuclei were stained blue.
(Magnification ×200). (d) Hepatic triacylglycerol and total
cholesterol. Values are shown as means ± SE (n = 11). Different letters
indicate significant difference (P < 0.05).
Table 1.
Food intake, body weight gain and indices of rats after feeding for 3
months (means ± standard errors, n = 11).
Items Soybean oil Lard Fish oil
Average daily feed intake (g) 21.74 ± 0.96^a 23.31 ± 0.81^a
24.12 ± 0.64^a
Initial body weight (g) 784.40 ± 13.35^a 800.60 ± 11.49^a
816.20 ± 16.29^a
Final body weight (g) 881.10 ± 17.49^a 933.20 ± 21.10^a
900.00 ± 16.79^a
Body weight gain (g) 96.70 ± 6.98^b 132.60 ± 11.73^a 83.80 ± 7.16^b
Liver index (mg/g) 24.42 ± 0.39^b 26.17 ± 0.58^a 23.58 ± 0.57^b
[75]Open in a new tab
Table 2.
Blood parameters of rats fed with soybean oil, lard or fish oil diets
(means ± standard errors, n = 11).
Items Soybean oil Lard Fish oil
Glu (mmol/L) 5.27 ± 0.20^a 5.47 ± 0.27^a 4.89 ± 0.18^a
TAG (mmol/L) 1.59 ± 0.11^b 1.99 ± 0.14^a 1.09 ± 0.13^c
TC (mmol/L) 3.06 ± 0.12^b 3.50 ± 0.17^a 2.76 ± 0.11^b
HDL (mmol/L) 1.20 ± 0.06^a 1.02 ± 0.04^b 1.00 ± 0.05^b
LDL (mmol/L) 1.68 ± 0.06^b 1.94 ± 0.09^a 1.48 ± 0.06^b
White blood cell count (WBC, 10^9/L) 8.88 ± 0.51^b 10.68 ± 0.70^a
8.20 ± 0.28^b
Insulin resistance index (IRI) 13.96 ± 2.47^a 15.16 ± 1.55^a
12.29 ± 1.09^a
[76]Open in a new tab
Oxidative status
Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase
(GSH-PX), and total antioxidant capacity (T-AOC) were analyzed to
indirectly evaluate reactive oxidative species (ROS) production in the
liver (Fig. [77]2). The activities of SOD and T-AOC in the fish oil
group were higher than those of the lard group (P < 0.05, Fig. [78]2b
and d). Although a strict Student-Newman-Keuls test indicated no
significant difference in CAT and GSH-Px activities among three diet
groups (P >0.05, Fig. [79]2a and c), the moderately strict Duncan’s
multiple comparisons showed a significant difference in CAT and GSH-Px
activities between fish oil and lard groups (Figure [80]S1). The
soybean oil group did not exhibit any significant difference in these
variables from the fish oil group (P > 0.05, Fig. [81]2). This
indicates that intake of lard may impair the antioxidant activity to a
greater extent than fish oil.
Figure 2.
Figure 2
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Antioxidant enzyme activities level of rats fed with soybean oil, lard
and fish oil diets. (a) Catalase activity; (b) Superoxide dismutase
activity; (c) Glutathione peroxidase activity; (d) Total antioxidant
capacity. Values are shown as means ± SE (n = 11). Different letters
indicate significant difference (P < 0.05).
Inflammatory status
White blood cell count (WBC) in the soybean oil and fish oil groups
were significantly lower than the lard group (P < 0.05, Table [83]2).
The RT-PCR results indicated that mRNA levels of NF-κB, IL-1β, IL-6 and
TNF-α were lower in the fish oil group than those in the lard group
(P < 0.05, Fig. [84]3). No significant difference was observed in NF-κB
and IL-6 mRNA levels between the soybean oil group and the fish oil
group (P > 0.05, Fig. [85]3a and c). The IL-1β and TNF-α mRNA levels
did not differ between the soybean oil group and the lard group
(P > 0.05, Fig. [86]3b and d).
Figure 3.
Figure 3
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Hepatic mRNA levels of inflammatory cytokines of rats fed with soybean
oil, lard and fish oil diets. (a) NF-κB; (b) IL-1β; (c) IL-6; (d)
TNF-α. The mRNA levels were determined by RT-PCR. Different letters
indicate significant difference (P < 0.05).
Aging-related gene expression
As shown above, intake of lard induced the middle-aged rats to
substantially different physiological responses from the other two fat
diets. To further explore the underlying molecular mechanisms,
aging-related PCR array analyses were performed and mRNA data were
compared in which the lard group was set as control. The results
revealed that 41 and 32 genes were down-regulated in the fish oil group
and the soybean oil group, respectively, as compared to the lard group
(P < 0.05, Fig. [88]4a and b). Of these genes, caspase 1 and c1qc
showed the greatest changes in the soybean oil group (actual change
folds were −3.83 and −3.76, respectively, Fig. [89]4a). In the fish oil
group, the absolute change folds of 20 genes were greater than 2.0
(Fig. [90]4b). The Venn plot further indicated that 12 and 21
differentially expressed genes in the soybean oil group and the fish
oil group were specific for the diets, respectively (Fig. [91]4c). In
addition, forkhead box O 1 (FOXO1) was down-regulated by 1.35 folds in
the soybean oil group (P >0.05) and 2.13 folds in the fish oil
group(P < 0.05).
Figure 4.
Figure 4
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Aging-related PCR array of rat livers in response to soybean oil and
fish oil as compared to the lard group. (a) 32 differentially expressed
genes in the soybean oil group as compared to the lard group
(P < 0.05); (b) 41 differentially expressed genes in the fish oil group
as compared to the lard group (P < 0.05); (c) Venn plot of
differentially expressed genes; (d) Gene set network of hepatic aging
PCR array analysis of the soybean oil group as compared to the lard
group. (e) Gene set network of hepatic aging PCR array analysis of the
fish oil group as compared to the lard group. The networks were
generated by STRING10 ([93]http://string-db.org/) and Cytoscape 3.3.0.
The node color change from blue to orange represents an increase of P
value from −2.83 to −1.20 in the soybean oil group, and −2.92 to −1.20
in the fish oil group.
Gene set analysis of PCR array data demonstrated that 16, 3, 2, 4 and3
of 32 differentially expressed genes in the soybean oil group genes
were related to inflammatory responses, apoptosis, DNA binding,
proteostasis and telomere attrition, respectively (Fig. [94]4d). The
gene set enrichment analysis further indicated that those genes were
involved in the pathways of complement and coagulation cascades,
oxidative damage, complement activation and toll-like receptor
signaling (Supplementary Table [95]S1). Of 41 differentially expressed
genes in the fish oil group, 19, 2, 2, 3, 2, 2 and 4 genes were
associated with inflammatory responses, apoptosis, DNA binding,
telomere attrition, laminopathies, neurodegeneration, oxidative stress
and mitochondrial dysfunction respectively (Fig. [96]4e). The
enrichment analysis matched these genes with six pathways, i.e.,
complement and coagulation cascades, oxidative damage, complement
activation, toll like receptor signaling, mitochondrial gene
expression, and FAS pathway and stress induction of HSP regulation
(Supplementary Table [97]S2).
Telomere length
Telomere length was considered as a good indicator for the aging
process^[98]18. The hepatic absolute telomere length (aTL) was measured
by RT-PCR. The results indicated that the aTL values were significantly
greater (P < 0.05, Fig. [99]5) for the fish oil group than those of the
lard group, but no difference was observed for the soybean oil group
from any of the other two groups (P > 0.05). Thus, intake of fish oil
may retard telomere attrition for the middle-aged rats as compared to
the intake of lard.
Figure 5.
Figure 5
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Hepatic absolute telomere length of rats fed soybean oil, lard and fish
oil diets. Values are shown as means ± SE (n = 11). Different letters
indicate significant difference (P < 0.05).
Discussion
Increasing evidence indicates that the type of dietary fatty acids has
a certain effect on animal health by altering lipid
metabolism^[101]21–[102]23. For example, intake of stearic acid leads
to dramatically reduced visceral fat^[103]24, even if stearic acid can
be more easily stored in liver at intermediate postprandial time points
(24~48 h) than linolenic or oleic acid^[104]25. Palmitoleic acid and
conjugated linoleic acid may prevent fat deposition in the
liver^[105]26, [106]27. Furthermore, intake of DHA and arachidonic acid
(ARA) can significantly reduce hepatic weight, serum TAG and TC,
epididymal fat as compared to palmitic acid, which would be associated
with enhanced fatty acid β-oxidation, down-regulated mRNA fatty acid
synthase and sterol regulatory element binding protein-1c
expression^[107]28. And thus, intake of long-chain saturated fatty
acids (the number of carbon skeletons greater than 12) may increase
lipid deposition, while monounsaturated or polyunsaturated fatty acids
would inhibit lipid deposition. Such an effect could be dependent upon
the age of studied subjects and the type of fatty acids.
Age effect is relatively complex, because the level of inflammation
varies greatly with age during which Toll-like receptors (TLRs) play a
critical role in the induction of inflammatory and immune
responses^[108]29. In young rats, SFAs may have a greater contribution
to pro-inflammatory processes than other fatty acids, because SFAs can
be served as ligands for TLR-2 and TLR-4, resulting in induction of
proinflammatory gene transcription via activation of NF-κB signaling
cascades^[109]30. The n-6 fatty acids (e.g., linoleic acid), have been
shown to exert proinflammatory effect^[110]31 and it could be converted
into ARA. In young TLR4 knockout mice, EPA and DHA were found to
inhibit the TLR-4 signaling pathway. However, n-3 PUFA supplementation
(1.68 g EPA and 0.72 g DHA/d) in old subjects for 3 month showed
greater potential to decrease inflammatory cytokines (IL-1β, IL-6,
TNF-α) than for young ones^[111]32. The n-3 PUFA may alleviate
age-related diseases by decreasing the level of inflammation^[112]33.
The above results focused on the diet or age effects in different
models, including high–fat-diet model. The diet formulations were quite
different from the normal diet.
Therefore, the present study was intend to reveal how fatty acids in
diet affect fat accumulation, oxidative status, inflammatory status,
aging-relate gene expression and telomere length in the middle-aged
rats.
Soybean oil, lard and fish oil showed significantly different fatty
acid composition. Soybean oil is characterized by high levels of n-6
PUFAs (53.67%) and fish oil contains high levels of n-3 PUFAs (15.79%
in EPA and 12.28% in DHA), while lard is largely composed of SFAs and
MUFAs (43.59% and 39.99% respectively, Supplementary Table [113]S3).
The different fatty acid composition in diets may have associations
with hepatic fat accumulation as well as body weight gain. Histological
observations indicated a relatively moderate hepatic fat accumulation
in the soybean oil group compared to lard and fish oil group. OA was
the most abundant MUFAs in diet and blood^[114]34. OA can increase fat
accumulation in hepatocytes by combining more specifically with
acyl-CoA to form TAG than SFAs^[115]34. In the present study, the OA
contents were 23.44%, 37.81% and 17.78% in soybean oil, lard and fish
oil, respectively (Table [116]S3). This may partially account for the
highest hepatic fat accumulation and body weight gain in the lard
group. The fish oil was more competent to reduce fat accumulation and
body weight gain than soybean oil and lard just because of higher
levels of n-3 fatty acids. Similar findings were observed that
supplementation of 5% n-3 PUFA (EPA + DHA) in diet resulted in a
complementary decrease in total body weight gain in young rats^[117]35.
The n-3 PUFAs can not only increase insulin sensitivity, but also
enhance lipid oxidation under the regulation of PPAR-α^[118]7.
Long-chain n-3 PUFAs, e.g. EPA and DHA in fish oil may decrease the
levels of plasma triglycerides^[119]36, [120]37. In this regard, it
seems better for fish oil to prevent fat accumulation as compared to
lard and soybean oil. This is because the deficiency of n-3 PUFAs may
increase gene expression involved in lipogenesis under the regulation
of liver X receptors^[121]8. The highest liver index and body weight
gain in the lard group could be attributed to those of hepatic fat
accumulation. In addition, higher SFAs in lard could cause fat
accumulation. The SFAs, e.g. palmitic acid, may inhibit autophagy by
inducing caspase-dependent Beclin 1 cleavage, and speed up the process
of apoptosis^[122]38. Inhibition of autophagy in cultured hepatocytes
and mouse liver might increase lipid storage^[123]39. Reduced autophagy
in the liver may contribute to hepatic fat accumulation and further
increase the incidence of metabolic syndromes in aged subjects^[124]40.
The underlying mechanism may be that autophagy can not only decrease
TAGs and lipid formation, but also increase TAG breakdown because TAGs
and lipid droplet structural proteins co-localize in autophagic
compartments^[125]39. And thus autophagy may protect against fatty
acid-induced lipotoxicity, including aging and metabolic
disorders^[126]38, [127]41. Autophagy also preserved a subset of old
haematopoietic stem cells from replication stress by preventing entry
into an activated state, which may improve old haematopoietic stem cell
function therefore improve health and longevity^[128]42. The
associations between autophagy and intake of soybean oil, lard and fish
oil need further studies. Transmission electron microscopy and
quantification of Atg8/LC3 and Atg6/Beclin1 might be useful ways to
monitor autophagy^[129]43.
It has been recognized that western-style diets, which are
characteristic of high levels of n-6 PUFAs, SFAs and trans fatty acids
but low levels of n-3 PUFAs, may be associated with an increasing
incidence of metabolic syndromes that were induced by inflammatory
responses^[130]44. However, substitution of SFAs with MUFAs would
reduce inflammatory responses^[131]6, and a MUFA–enriched Mediterranean
diet can reduce the incidence of metabolic syndromes^[132]13.
Inflammation could be induced by ROS, which are produced during the
process of oxidative phosphorylation. However, ROS are essential for
several physiological functions, e.g., maintaining immune function and
acting as regulatory mediators in signaling processes^[133]16, [134]45.
Antioxidant systems play a role in reducing the ROS to a balanced
level, and the major antioxidant systems are CAT, SOD and
GSH-PX^[135]46. The fish oil group showed the lowest level of oxidative
stress and the highest antioxidant activities in the liver. The soybean
oil group showed a great similarity to the fish oil group in the levels
of oxidative stress and antioxidant activities. This is because n-3
fatty acids can increase CAT and SOD activities^[136]47.
Supplementation with adequate α-linolenic acid (ALA) or EPA + DHA
increased hepatic SOD activity in young rats^[137]48. It was also
reported that EPA or DHA supplementation enhanced the total
anti-oxidant status and resistance to lipid peroxidation in young
rats^[138]49. ROS may induce the oxidization of DNA, proteins and
lipids during the progression of chronic inflammatory and degenerative
diseases^[139]10. NF-κB is a transcription factor that plays an
important role in various inflammatory signaling pathways. It regulates
several cytokines, chemokines, adhesion molecules and inducible
effector enzymes^[140]50. The n-3 fatty acids may inhibit NF-κB
activation^[141]7. Glutathione would enhance the n-3 PUFA-induced
inhibition to NF-κB activation^[142]51. SFAs in lard may activate
toll-like receptor 4 (TLR4) and NF-κB^[143]52. NF-κB -induced proteins
include COX-2, TNF-α, IL-1β, and IL-6, which are in turn potent NF-κB
activators, forming an auto-activation loop^[144]53. Lower NF-κB mRNA
level in the soybean oil group was probably due to lower SFAs (16.87%)
compared to the lard group (43.59%). A human study confirmed that
natural killer cell activity and in vitro secretion of IL-1β and TNF–α
in young healthy men were significantly reduced by supplementation with
6 g DHA/d for 90 d^[145]54.
In the present study, dietary fats triggered significant phenotype
changes of inflammation in middle-aged rats. The rats fed lard seemed
more susceptible to inflammation by counting white blood cells and
platelets than those of soybean and fish oils. In fish oil, a
relatively high level of palmit oleic acid (16:1) could be involved in
the regulation of insulin sensitivity, the inhibition of inflammation
and the prevention of fat accumulation in the liver^[146]55. In
addition, ARA can be converted to prostaglandins and leukotrienes that
are important pro-inflammatory mediators and can induce ROS
production^[147]56. Linoleic acid in soybean oil can be converted to
ARA^[148]57. Moreover, supplementation with olive oil (high in OA) may
cause severe inflammation by up-regulating intrahepatic gene expression
of proinflammatory molecules^[149]12. Higer oleic and linoleic acids in
soybean oil might induce greater level of inflammation than fish oil.
The higher level of LDL in the lard group could be associated with
inflammation. LDL is rich in cholesterol and cholesteryl ester, which
are easily oxidized and aggregated. Oxidized LDL would induce
TLR4-mediated signaling pathway (CD36–TLR4–TLR6 inflammasome), and
activate NF-κB and Pro-IL-1β that can be cleaved by caspase 1 to IL-1β
and further induces inflammation^[150]58.
PCR array results further confirmed that dietary fats induced different
levels of inflammation. As compared to the lard group, intake of
soybean oil caused the down-regulation of 16 genes involved in
inflammatory response, and 19 inflammation-related genes. The pathway
enrichment analysis indicated that intake of soybean and fish oils
might reduce inflammatory response by down-regulating gene expression
involving complement activation and toll-like receptor signaling. In
addition, genes involving oxidative stress and mitochondrial
dysfunction were also down-regulated in the fish oil group, and this
was in line with the changes of hepatic antioxidant enzyme activities.
The intakes of soybean and fish oils resulted in the down-regulation of
FOXO 1, which might be due to less hepatic fat accumulation, and thus
decrease the insulin level. However, higher insulin may activate NF-κB
by the phosphorylation of FOXO via the PI3K/Akt signaling pathway, and
result in the down-regulation of MnSOD and CAT. This would increase ROS
level, which may in turn activate NF-κB, and as a result, increase
inflammation^[151]53.
Oxidative-stress-induced inflammation is commonly accompanied with
telomere dysfunction^[152]17. Telomeres are long hexamer (TTAGGG)
repeats that protect the genome against chromosomal instability and
cellular senescence^[153]59. Exogenous and endogenous ROS can cause
telomere attrition and senescence^[154]60. Senescence would compromise
tissue repair and regeneration, and further induce tissue and organism
to aging. Telomere length (TL) is considered a potential biomarker of
aging^[155]61. In the present study, the fish oil group had higher aTL
than the lard group, indicating that fish oil may prevent DNA damage.
This could be attributed to the roles of n-3 PUFAs^[156]59.
Docosapentaenoic acid (DPA) and EPA were also shown to exert a
protective effect in old rats by significantly decreasing age-related
microglial activation and 8-OHdG (marker of DNA oxidative damage)
compared to the young^[157]62. During aging, inflammation and ROS may
activate caspase and further induce cellular apoptosis and
senescence^[158]63. Caspase 1 (CASP 1) and clusterin (CLU) can activate
apoptosis. Lower CASP 1 and CLU mRNA levels were observed in the fish
oil and soybean oil groups, which is indicative of less senescence.
Age-related ROS generation can stimulate NF-κB activation, and ROS
production may be in turn activated by cellular senescence^[159]53. The
present study also showed that the genes related to DNA binding,
apoptosis, proteostasis, laminopathies and neurodegeneration were
down-regulated by intake of fish oil. In this regard, the substitution
of lard for fish oil may retard the aging process at middle ages.
It is noting that effect of dietary fats on inflammation at normal dose
was relatively small. However, the diet effect may be enhanced if diet
fat dose increases. Even so, the present study still provided
significant results of some physiological responses. To a certain
extent, the results reflected gradual oxidative stress and inflammation
induced by diet fats at middle ages. Further work should be done to
compare diet fat effects among young, middle and old ages, and to
evaluate the diet effect at higher doses, In addition,
autophagy-related markers should be quantified to evaluate whether
different source of fats induce autophagy at middle ages.
In summary, an underlying mechanism for the fat-induced
oxidation-inflammation in middle-aged rats was proposed (Fig. [160]6).
Fat accumulation may increase ROS production, which can induce the
oxidation of proteins, lipids, and DNA, and consequently the cellular
apoptosis, senescence and aging. SFAs, oxidized fatty acids and n-6
PUFAs in diets could be transported into the cytoplasm by TLRs, and
then activate NF-κB and induce inflammation. However, n-3 PUFAs could
suppress TLR-induced NF-κB activation. ROS may directly activate NF-κB,
and induce CASP-1 to cleaving pro-IL-1β to produce IL-1β. TNF-α, IL-6
and IL-1β could in turn activate NF-κB and further aggravate
pro-inflammation. TNF-α, apoptosis and senescence would induce the ROS
production. In addition, insulin could activate FOXO 1, but inhibit
MnSOD and CAT, and as a consequence enhancing ROS production.
Figure 6.
Figure 6
[161]Open in a new tab
A proposed underlying mechanism for fat-induced
oxidation/inflammation/aging. ROS, reactive oxygen species; FA: fatty
acids; n-3: n-3 fatty acids; n-6: n-6 fatty acids; SFA: saturated fatty
acid; TLR: toll- like receptor; NF-κB: nuclear transcription factor κB;
FOXO 1: fork head box O 1; Pro-IL-1β: pro-interleukin 1β; MnSOD: Mn
superoxide dismutase; CAT: catalase; IL-6: interleukin 6; TNF-α: tumor
necrosis factor α; IL-1β: interleukin 1β; purple line indicates the
CASP-1 mediated pathway; green line indicates the insulin-induced
pathway; red line indicates TNF-α, cellular apoptosis and senescence
pathways which increase ROS production; blue dot line indicates
auto-activation loop induced by IL-6 and IL-1β.
Materials and Methods
Ethics statements and animals
Animal experimental protocols were reviewed and approved by the Ethical
Committee of Experimental Animal Center of Nanjing Agricultural
University. All experiments were performed in accordance with the
relevant guidelines and regulations of the Ethical Committee of
Experimental Animal Center of Nanjing Agricultural University, and all
efforts were made to minimize animal pains. Thirty-three male Sprague
Dawley rats (800.4 ± 8.08 g, 12 months of age) were purchased from the
Academy of Military Medical Science Laboratory Animal Center (Beijing,
P.R. China, SCXK < Jun > 2012-0004). All the rats were housed
individually in plastic cages in a specific pathogen-free animal center
(SYXK < Jiangsu > 2011-0036). The room was kept at a temperature of
20 ± 0.5 °C and humidity of 60 ± 10% on a 12 h light–dark schedule. All
the rats were given free access to diets and water.
Diets
The diets were formulated according to the AIN-93M protocol and
prepared by Trophic Animal Feed High-Tech Co., Ltd (Nantong, China).
The diets differed in the nature of the fats, which were soybean oil,
lard and fish oil. Soybean oil was purchased from Shanghai Jiali Oil
Industry Co. Ltd (Shanghai, China), and lard was obtained from Tianjin
Lihongde Fat Products Inc. (Tianjin, China). Fish oil was obtained from
Rongcheng Ayers Ocean Bio-technology Co, Ltd (Weihai, China). The fatty
acid profiles of three fats are shown in Table [162]S3. The diets were
formulated as (per 1000 g diet): 465.69 g cornstarch, 140.0 g casein,
155.0 g dextrinized cornstarch, 100.0 g sucrose, 40.0 g soybean oil,
lard or fish oil, 50.0 g fiber, 1.8g L-cystine, 2.5 g choline
bitartrate, 10.0 g vitamin mixture, 35.0 g mineral mixture and 0.014 g
tertbutylhydroquinone. The diets were vacuum packaged, stored at –20 °C
and allowed to reach room temperature before being served.
Animal feeding and sampling
The rats were randomly assigned to one of the three diet groups, i.e.
soybean oil, lard and fish oil (n = 11, each). Feed intake was recorded
daily and body weights were recorded weekly. After 90-day feeding, all
rats were decapitated. Blood was collected into EDTA-Na[2] treated
tubes and plasma was centrifuged at 1750 × g for 10 min. The liver was
weighed and the largest hepatic lobe was cut into 5 10 × 10 mm cubes at
the same part. One cube was fixed in 4% paraformaldehyde for Oil Red O
staining, and the other cubes were snap-frozen and stored at −80 °C
until analysis.
Hepatic fat accumulation analysis
After 12 h fixation in 4% paraformaldehyde at 4 °C, the liver samples
were embedded in paraffin and 10 μm - thick sections were cut. The
sections were stained with Oil Red O according to the procedures of
Goto-Inoue et al.^[163]64. The sections were examined under a light
microscope with a magnification × 200. Triacylglycerol and total
cholesterol were analyzed with commercial kits according to the
manufacturer’s protocols (Nanjing Jiancheng Bioengineering Institute,
Nanjing, China).
Blood profiling analysis
Plasma parameters were measured under an automatic biochemical analyzer
(DXC − 800, Beckman Coulter Inc., Fullerton, CA, USA) and an automatic
hematology analyzer (SYSMEXLAS, Sysmex Corporation, Kobe, Japan).
Insulin was determined with a radioimmunoassay kit according to the
manufacturer’s instruction (Beijing North Institute of Biological
Technology Company, Beijing, China). IRI was calculated as
follows^[164]65:
[MATH: IRI=(fastinginsulininmU/L×fastingglucoseinmM)/22.5. :MATH]
1
Hepatic oxidative stress analysis
Liver samples (0.5 g) were homogenized in 4.5 mL ice-cold physiological
saline for 1 min and then centrifuged (2700 × g, 4 °C, 10 min). The
supernatants were aliquoted and the enzymatic activities of CAT, SOD,
GSH-PX, and T-AOC were analyzed with commercial kits (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China) according to the
manufacturer’s protocols.
RNA isolation and RT- PCR analysis of inflammatory cytokines
Total RNA was extracted from liver samples using RNeasyPlus Mini Kit
(Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
The purity and quantity of total RNA were measured by a NanoDrop 2000
spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 260
and 280 nm. The integrity of RNA samples was evaluated by agarose gel
electrophoresis. First-strand cDNA was synthesized from 250 ng of total
RNA using RT^2 First Stand kit (Qiagen, Hilden, Germany) in a Veriti®
Certified Refurbished Thermal Cycler (Thermo Scientific, USA) according
to the manufacturer’s instructions. The RT-PCR reactions were run using
SYBR Premix Ex Taq Kit (Takara Biotechnology Co. Ltd., China) in
QuantStudio™ 6 Flex Real-Time PCR System (Thermo Scientific, USA).
Primers were designed according to the public database at the National
Center for Biotechnology Information (NCBI)
([165]http://www.ncbi.nlm.nih.gov/RefSeq/) and were synthesized by
Sangon Biotech Co., Ltd (Sangon, Shanghai, China). Primers used for
RT-PCR were presented in Supplementary Table [166]S4. The amplification
was performed in a total volume of 20 μL, containing 10 μL of SYBR
Premix Ex Taq, 0.4 μL of each primer (10 μM), 0.4 μL of ROX Reference
Dye II (Takara Biotechnology Co. Ltd., China), 2 μL of cDNA and 6.8 μL
of sterilized doubled-distilled water. The RT-PCR program was as
follows: 95 °C for 10 min, 35 cycles of 95 °C for 30 s, 60 °C for 30 s
and 72 °C for 30 s, holding at 72 °C for 10 min, and the fluorescence
signals were collected at 60 °C. Each sample was performed in
triplicate. Relative mRNA levels were calculated using the 2^−ΔΔCt
method. β-actin was applied as reference gene to determine NF-κB, IL-1β
and TNF-α levels, and GADPH was applied to determine IL-6 level.
Aging - related RT-PCR array analysis
After RNA isolation of liver and first-strand cDNA synthesis (see
subsection “RNA isolation and RT-PCR analysis of inflammatory
cytokines”), a RT² Profiler™ PCR array rat aging kit (330231
PARN-178ZA, Qiagen, Hilden, Germany) and a RT^2 SYBR Green ROX qPCR kit
(330522, Qiagen, Hilden, Germany) were applied to evaluate
aging-related gene expression on an ABI 7500 Real-Time PCR System
(Applied Biosystems, Foster City, CA, USA) according to the
manufacturer’ protocols. All RNA samples were normalized to
250 ngbefore first-strand cDNA synthesis. The RT^2 Profiler PCR array
profiled the expression of 84 target genes besides housekeeping and
control genes (Supplementary File [167]S1). The RT-PCR cycling
conditions were as follows: 95 °C for 10 min, 40 cycles of 95 °C for
15 s, 60 °C for 1 min, 95 °C for 1 min, and 65 °C for 2 min to collect
fluorescence signals. Differentially expressed genes were obtained by
using an online program (Geneglobe; Qiagen, Redwood City, CA;
[168]http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php) in
which the lard group was set as control, and RPLP1 was set as
housekeeping gene for normalization. The genes with greater than
1.2-fold changes were defined as differentially expressed genes. The
protein-protein interactions of significant gene products were
evaluated by the STRING 10 (http://string-db.org/) and visualized by
the Cytoscape 3.3.0.
Telomere length
Absolute telomere length (aTL) was measured with a RT-PCR method
according to O’Callaghan and Fenech^[169]66 with minor modifications.
Briefly, DNA was isolated with a QIAamp DNA mini kit (Qiagen, Hilden,
Germany) according to the manufacturer’s protocols. The RT-PCR
reactions were run in QuantStudio™ 6 Flex Real-Time RCR System (Thermo
Scientific, Carlsbad, USA) using a SYBR Premix Ex Taq Kit (Takara
Biotechnology Co. Ltd., China). The cycling profile consists of
denaturation at 95 °C for 10 min, denaturation at 95 °C for 15 s,
annealing at 60 °C for 60 s, extension at 72 °C for 30 s, holding at
72 °C for 10 min with data collection, and 40 cycles with fluorescence
data collection. Standard oligomers and primers were shown in
Supplementary Table [170]S5. The aTL was calculated as follows:
[MATH: aTL=amount of telomere sequence per
reaction(kb)/genome copy number
:MATH]
2
Statistical analysis
The PCR array data was conducted by t-test between two variances with
an online program
([171]http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php,
Geneglobe, Qiagen, Redwood City, CA). The other variables were analyzed
using the analysis of variance under the SAS program (version 9.2, SAS
Institute Inc., Cary, NC, USA). The differences between diet groups
were compared by the procedure of Student-Newman-Keuls Test. The values
were considered significantly different if the P value was less than
0.05.
Electronic supplementary material
[172]Supplemetary Information 1^ (404.3KB, pdf)
[173]Supplementary File S1^ (70KB, xls)
Acknowledgements