Departments of Nutrition and Medicine, University of Toronto, Toronto, Ontario, Canada, M5S 1A8
Submitted 6 August 2003 ; accepted in final form 29 October 2003
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ABSTRACT |
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lipopolysaccharide; refeeding; tumor necrosis factor-; tumor necrosis factor receptors
Previously, we showed that nutritional status altered the metabolic response to TNF- infusion as well as distribution of labeled TNF in rats. TNF-
caused anorexia and weight loss in orally fed rats without any significant metabolic abnormalities (9). When total parenteral nutrition was administered to TNF-
-treated rats to prevent weight loss, severe metabolic abnormalities, namely hyperglycemia and azotemia, were observed (18). Correspondingly, in another study the retention of TNF-
in circulation was significantly increased in weight-gaining animals and reduced in weight-losing animals (11). These differences were due to the effect of nutrition on the concentration of both membrane receptors in the liver and circulating receptors (26). Weight-gaining rats showed an increase in membrane and soluble receptors, suggesting stabilization of TNF-
in circulation, thereby augmenting its activity and prolonging its action, resulting in greater degree of metabolic abnormalities as seen in our previous studies (18, 26).
There is evidence that increased energy intake increases the incidence of complication in sepsis (33). On the contrary, high protein intake may even be protective (23). Despite energy intake being low, in adults increased amino acids and protein intake increase protein synthesis (19) and preserve body nitrogen (3). Increased sensitivity to septic shock has been reported in protein-malnourished patients (6). Li et al. (15) reported that protein malnutrition is a direct cause of increased LPS-induced NF-B activation and transcription levels of its downstream genes IL-1
and TNF-
. Lyoumi et al. (17) reported the presence of the
2-macroglobulin in the serum of protein-deficient rats returning to normal levels after reintroduction of the normal protein diet.
In line with the observations above, we have found that feeding energy increases morbidity (18, 26). However, the effect of protein deficiency on TNF- and TNFRs in animals fed with adequate energy has not been studied. Because high protein intake may protect against sepsis (23), the present study was designed to test the hypothesis that low protein intake may stabilize TNF-
in circulation mediated through increased TNFR expression in liver, spleen, and gastrocnemius muscle of endotoxic rats, which, upon refeeding protein, may improve or reduce the expression of TNFRs, resulting in decreased TNF-
activity.
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MATERIAL AND METHODS |
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Diet fed during study. Seven days after acclimatization to the new environment after reaching an average weight of 250 g, the rats were randomly allocated to different groups (as described below) and were placed on a liquid defined-formula diet (Table 1) for 10 days. This liquid diet allowed the control rats (C) to grow at the same rate as animals fed a nonpurified diet ad libitum in a previous study (9). The rats in C and low-protein (LP) groups were fed diets of different protein levels, with the total energy and micronutrient (trace elements, vitamins, and electrolytes) concentrations kept the same in both groups. The amino acid mix in the LP diet was reduced by 75% compared with the C diet. The loss of protein calories in the LP group was compensated by increasing the carbohydrate and fat content by 10 and 19%, respectively. Refed (RF) groups were also run concurrently.
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The details of the groups are as follows. C (n = 6) received orally ad libitum a defined-formula diet with an energy density of 0.35 MJ, protein energy density of 0.047 MJ/60 ml, and daily subcutaneous injections of normal saline (NS) in a total volume of 0.25 ml. LP (n = 6) rats, treated daily with LPS injections (5 mg/kg body wt sc in a total volume of 0.25 ml), received orally ad libitum a defined-formula diet with an energy density of 0.35 MJ, but the protein-energy density was reduced to 0.012 MJ/60 ml. RF (n = 6) rats, treated with LPS, were initially depleted on the LP diet for 10 days and then repleted on the C diet for an additional 10 days. Because LPS causes anorexia, it was necessary to compare LPS-treated groups with comparably fed non-LPS-treated groups. Thus P-F (n = 12) rats receiving NS injections were paired with individual LP and RF rats and given the exact volume of liquid diet consumed by these two groups. Food intake was staggered from that of the experimental group by 1 day to match the food intake for each study day.
All of the animals were observed carefully, weighed daily, and allowed to take water freely throughout the experiment. A daily record of dietary intake was maintained for 10 days. The protocol was approved by the University of Toronto Animal Care Committee.
Organ and muscle preparations for Western analysis and RT-PCR. After 10 days of the study diets, rats were anesthetized with pento-barbital sodium (50 mg/kg ip), exsanguinated by abdominal aortic puncture, and then killed by cervical dislocation. Their organs and muscle were removed immediately after death, frozen quickly in liquid nitrogen, and stored until used. Tissue lysate and RNA were prepared from liver, spleen, and gastrocnemius for protein and mRNA expressions, respectively, from 30 rats (6 in each group, as described in Diet fed during study).
Protein measurements. Total protein in lysate was measured by DC Protein Assay Kit (Bio-Rad) on the basis of the reaction similar to that of the Lowry assay (16).
Western blot analysis for TNF-, TNFR-I, and TNFR-II. Liver, spleen, and gastrocnemius muscle were homogenized in lysis buffer (1x PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSF, 45 µg/ml aprotinin, 184 µg/ml sodium orthovanadate) and incubated on ice for 30 min. Homogenate was centrifuged at 10,000 g for 10 min at 4°C. Protein concentration was determined in the supernates as described in Protein measurements. Total protein (160 µg) was separated in a denaturing 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Nonspecific binding was blocked with 1x PBS (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, and 150 mM NaCl, pH 7.4) containing 5% nonfat dry milk for 2 h at room temperature. Membranes were then incubated in a 1:500 dilution of primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS with 0.1% Tween-20 (PBST) overnight at 4°C. After six washes in PBST, membranes were incubated in a 1:5,000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology). To prevent aggregate formation in the HRP-conjugate, it was diluted and filtered through a 0.2-µm filter before use in the blotting procedure. Immunoreactive protein was detected by SuperSignal West Femto maximum sensitivity chemiluminescence substrate (BioLynx, Brockville, ON, Canada) (14).
The primary and secondary antibodies used for TNF were goat polyclonal IgG and bovine anti-goat IgG-HRP; for TNFR-I, mouse monoclonal IgG2b and anti-mouse IgG2b-HRP; and for TNFR-II, mouse monoclonal IgG2a and goat anti-mouse IgG-HRP, respectively.
The specificity of protein expression was confirmed by using a positive control with every gel run. Cruz Marker molecular weight standards were used to identify the band sizes.
RNA isolation and RT-PCR. Total RNA was extracted from liver, spleen, and gastrocnemius by the acid guanidinium-phenol-chloroform method, using TRIzol reagent (GIBCO-BRL, Gaithersburg, MD) according to the manufacturer's instructions. This protocol is based on the method described by Chomczynski and Sacchi (2). Quantification and purity check of total RNA were performed spectrophotometrically. The isolated RNA samples were treated with DNase I, Amp Grade (Invitrogen, Burlington, ON, Canada) to remove genomic DNA contaminant. The expression of TNF- mRNA, TNFR-I mRNA, TNFR-II mRNA, and GAPDH mRNA was determined by RT-PCR of total RNA. Total cellular RNA was reverse-transcribed using SuperScript RNaseH Reverse Transcriptase (Invitrogen). Blank reactions with no RNA were performed in all experiments. Aliquots of 1 µl of cDNA were added to 49 µl of PCR mix containing 2.5 U of Platinum Taq DNA Polymerase (Invitrogen), PCR buffer (10x, minus Mg), 0.2 mM dNTP mixture, 1.5 mM Mgcl2, and primer mix (0.2 µM each). PCR amplifications of a fragment of GAPDH were also performed as internal control for each sample. For each set of primers the PCR conditions are specified in Table 2. The number of PCR cycles was adjusted carefully to avoid saturation of the amplification system. The amplification products were analyzed by electrophoresis on 2% agarose gels, visualized by 2UV Transilluminator (UVP; BioDoc-It System, Diamed) with ethidium bromide staining.
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Densitometric assessment of PCR products was performed using commercially available Quantity One Quantitation software (Bio-Rad). Semiquantitative PCR results were generated by grading a ratio between the densitometry results of the target gene (TNF-, TNFR-I, TNFR-II) and the housekeeping enzyme (GAPDH).
Statistical analysis. Data were expressed as means ± SE. Differences in weight, cumulative dietary intake, and mRNA levels among groups were analyzed by one-way analysis of variance (ANOVA) and were considered statistically significant at P < 0.05. This was followed by Duncan's new multiple range test (28).
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RESULTS |
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The cumulative total energy intake over 10 days was significantly lower (P < 0.01) in LP (474.92 ± 10.57 kcal) and its corresponding P-F (439.95 ± 7.25 kcal) group compared with C (822.36 ± 10.68 kcal), respectively. Upon refeeding, the cumulative total kilocalorie value was significantly (P < 0.01) increased in RF (725.49 ± 10.7) and its corresponding P-F (735.93 ± 12.7) group compared with LP (474.92 ± 10.57).
The cumulative protein kilocalorie value over 10 days was significantly (P < 0.01) lower in LP (15.98 ± 0.36) and its corresponding P-F (14.8 ± 0.24) group compared with C (110.75 ± 1.44). Upon refeeding, the cumulative protein kilocalorie value was significantly (P < 0.01) increased in RF (97.7 ± 1.44) and its corresponding P-F (99.12 ± 1.71) group compared with LP (15.98 ± 0.36).
The cumulative total kilocalorie value and protein kilocalorie value in the LP and RF groups were not significantly different from their respective P-F controls because the diet provided to each P-F rat matched the food intake of the respective experimental rat.
The intake of an LP diet with LPS reduced protein and energy intake, whereas RF increased intake close to C. LPS-treated groups did not differ significantly from the corresponding P-F groups.
Body weight. Body weight was measured to determine the relative effects of diets and the contribution of LPS to weight change. The changes in body weight were consistent with the dietary manipulations. The weight gain in C was 27.43 ± 4.8 g as opposed to a fall of -46.2 ± 6.11 g in LP and -47.33 ± 1.89 g in P-F rats. This difference between C and LP was statistically significant (P < 0.01). The weight gain in RF and its corresponding P-F group was 31.17 ± 4.99 and 47.00 ± 5.4 g, respectively, which were markedly increased compared with LP (-46.2 ± 6.11 g) and its corresponding P-F (-47.33 ± 1.89 g) group. The difference between RF and LP was statistically significant (P < 0.01).
The LP and RF diets resulted in the expected change in weight. However, weight change in the LPS groups was not significantly different from their corresponding P-F groups, showing that weight change was largely mediated by nutrient intake.
TNF- activity in C and LP groups. The aim of these measurements was to determine the effect of diet and LPS on the expression of TNF-
in liver, spleen, and muscle. Western blotting showed a marked increase in TNF-
activity in liver lysate in the LP group (Fig. 1A, lanes 4-6) injected with LPS compared with C (lanes 1-3) and P-F (lanes 7-9) groups injected with NS. Similarly, spleen lysate showed a marked increase in TNF-
in the LP rats injected with LPS (Fig. 1B, LP lanes 4-6) compared with controls (C, lanes 1-3; P-F, lanes 7-9) injected with NS.
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In contrast, gastrocnemius muscle did not show any difference in the TNF- activity between the LP and the C groups (Fig. 1C: C, lanes 1-3; LP, lanes 4-6; P-F, lanes 7-9).
In liver and spleen, but not muscle, the expression of TNF- increased only in LPS-treated animals on the LP diet.
TNF- activity in RF groups. The intent of these measurements was to determine whether the increase in the expression of TNF-
was due to LPS alone or to the combined effect of LPS and LP. Upon repletion of protein in the diet of the RF group injected with LPS, there was a marked reduction in TNF-
activity in liver (Fig. 1A, lanes 10-12) and spleen (Fig. 1B, lanes 10-12) compared with the LP group injected with LPS (Fig. 1, A and B, lanes 4-6). No difference in TNF-
activity was seen between RF and its P-F group in both liver (Fig. 1A, lanes 13-15) and spleen (Fig. 1B, lanes 13-15). Gastrocnemius muscle did not show the same change consistently as in liver and spleen. There was increased TNF-
activity in RF (Fig. 1C, lanes 10-12) injected with LPS compared with its P-F group injected with NS (Fig. 1C, lanes 13-15).
Refeeding reduced the LPS-induced increase in TNF- in both the liver and spleen in animals receiving the LP diet. Therefore, LP and LPS interact to increase the expression of TNF-
. In contrast, muscle showed the opposite effect.
TNFR-I and TNFR-II protein expressions in C and LP groups. The aim of these measurements was to determine the effect of diet and LPS on TNFR expression in liver, spleen, and muscle. Western blotting showed an increase in TNFR-I expression in liver (Fig. 2A), spleen (Fig. 2B), and gastrocnemius (Fig. 2C) lysate in LP rats (lanes 4-6) injected with LPS compared with control rats injected with NS (Fig. 2, A, B, and C; lanes 1-3; P-F, lanes 7-9).
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TNFR-II expression also showed an increase in liver (Fig. 3A), spleen (Fig. 3B), and gastrocnemius (Fig. 3C) lysate in LP rats injected with LPS (lanes 4-6) compared with control rats injected with NS (Fig. 3, A, B, and C: C, lanes 1-3; P-F, lanes 7-9).
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In general, TNFR expression was markedly increased by a combination of LPS injection and LP diet in liver, spleen, and muscle.
TNFR-I and -II protein expression in RF groups. The aim was to determine whether the increase of TNFR seen in the previous measurements was due to LPS alone or to LPS and the LP diet.
Upon repletion of protein in the diet of the RF group injected with LPS, there was reduced expression in TNFR-I in liver (Fig. 2A, lanes 10-12), spleen (Fig. 2B, lanes 10-12), and gastrocnemius muscle (Fig. 2C, lanes 10-12) compared with the LP group injected with LPS (Fig. 2, A, B, and C, lanes 4-6). Although lower than LP, there was a slightly greater TNFR-I expression in RF (injected with LPS) compared with its P-F counterpart in liver (Fig. 2A, lanes 13-15) spleen (Fig. 2B, lanes 13 and 14), and gastrocnemius muscle (Fig. 2C, lanes 13-15).
TNFR-II expression followed a similar pattern. Upon repletion of protein in the diet of the RF group injected with LPS, there was a reduction in TNFR-II expression in liver (Fig. 3A, lanes 10-12), spleen (Fig. 3B, lanes 10-12), and gastrocnemius muscle (Fig. 3C, lanes 10-12) compared with the LP group injected with LPS (Fig. 3, A, B, and C, lanes 4-6). Although lower than LP, there was a slightly greater TNFR-II expression in RF (injected with LPS) compared with its P-F counterpart in liver (Fig. 3A, lanes 13-15), spleen (Fig. 3B, lanes 13 and 14), and gastrocnemius muscle (Fig. 3C, lanes 13-15).
The results show that, in general, a combination of LPS and LP is required to markedly increase TNFR expression.
TNF- and TNFR-I and -II mRNA expression in C, LP, and RF groups. The aim of these measurements was to show the contribution of transcriptional changes to the expression of TNF-
and TNFR induced by LPS and dietary manipulations. TNF-
mRNA was significantly increased (P < 0.05) in gastrocnemius (Figs. 4A and 5) of LP rats compared with control (C, P-F) and RF groups. TNF-
mRNA was not significantly different in any of the groups in liver and spleen (Fig. 4A). TNFR-I (Fig. 4B) and TNFR-II (Fig. 4C) mRNA expression showed a significant increase (P < 0.05) in spleen and gastrocnemius of LP rats compared with C and RF groups. Liver showed no significant changes in TNFR-I and TNFR-II mRNA expressions (Figs. 4, B and C). The changes in expression were due to a combination of altered transcription and translation.
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DISCUSSION |
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The present study showed that protein depletion of animals treated with LPS resulted in increased TNFR-I and -II expression in the tissues. Feeding protein suppressed the increase despite receipt of LPS. Although the energy intake was lower in the LP group and higher in the RF group, the observed effects cannot be due to differences in energy intake, as we had previously shown (26) that a lower energy intake suppressed TNFR expression, exactly opposite to the current effects of reducing the protein-energy ratio in LP rats. TNF- expression was similarly increased in liver and spleen with protein depletion and reduced by refeeding a higher-protein diet. Gastrocnemius muscle behaved differently from liver and spleen. There was an increase in TNFR-I and TNFR-II protein expressions, but not in TNF-
, in the rats fed the LP diet compared with controls (C, P-F). Upon refeeding, TNFR-I and TNFR-II protein expressions were reduced, but TNF-
expression was increased. In addition, mRNA expressions of TNF-
, TNFR-I, and TNFR-II were significantly increased in LP rats compared with C, which returned to normal levels in the RF and P-F groups. Therefore, in the gastrocnemius, the effects were transcriptional/translational for the TNFR-I and TNFR-II. In contrast, although TNF-
mRNA transcription increased, muscle TNF-
protein expression was reduced by a lower protein intake. Increasing protein intake increased TNF-
expression but suppressed transcription. To the best of our knowledge, these effects of modulating protein intake on TNF-
and TNFR-I and -II have not been described previously and may have implications in the nutrient-sepsis interaction.
In the present study, we also observed an increase in TNF-, TNFR-I, and TNFR-II protein expressions in spleen and liver of rats placed on LP (injected with LPS) compared with C (injected with NS). Upon refeeding, TNF-
, TNFR-I, and TNFR-II protein expressions were reduced in liver and spleen. On the other hand, liver mRNA expressions of TNF-
, TNFR-I, and TNFR-II and spleen mRNA expression of TNF-
did not show significant changes in any of the groups. But spleen mRNA expressions of TNFR-I and TNFR-II showed a significant increase in LP rats compared with C, which was restored to normal levels in the RF and P-F groups. Therefore, in the liver, the increases of TNF-
, TNFR-I, and TNFR-II in the LP group were due to changes in translation. In the spleen, the effects were due to a combination of translation (TNF-
) and transcription. These data suggest that nutritional manipulations alter the differential expression of protein and mRNA of TNF-
and its receptors in organs and muscle and that the polarization of cytokine and the receptors' response to endotoxin are regulated at the level of individual organs and muscle.
Difference in TNF- and its receptors' expression pattern between organs and muscle could also be as a result of the difference in cell population between organ and muscle. Kupffer cells of the liver represent the largest population of macrophages in the mammalian body (4). It has been shown that Kupffer cells are the major source of TNF-
in the liver and that LPS is a strong elicitor of TNF-
expression (4, 24). Spleen, being a large lymphoreticular organ, also consists of a large population of macrophages (20), but the cell population in spleen is more homogenous than in liver. This may explain the difference observed between liver and spleen in TNF-
and its receptor expressions at both the transcription and translation levels.
The role of muscle cells in immunological reactions is controversial. Some studies show that myoblasts can induce tolerance in vivo, possibly because of the lack of costimulatory molecules (12, 32), whereas others demonstrate proliferative responses in T-cell lines (7). In the present study, a greater degree of consistency between the transcription and translation levels of TNF- and its receptors was seen in the gastrocnemius muscle.
Our results indicate involvement of both TNFRs in protein-deficient, normal-energy, endotoxic rats. The data presented in a recent report (22) reveal the importance of both p55 and p75 TNFRs in the induction of PA-1 mRNA in ob/ob mice, a model for elevated endogenous TNF- or chronic inflammation. A ligand-passing mechanism or a cooperation between signals transmitted independently by p55 and p75 may explain the requirement for both these receptors in protein malnutrition normal-energy endotoxic rats. Ligand-induced formation of p55 and p75 heterocomplexes has been reported on intact cells (25). It is not known yet whether a similar phenomenon occurs in protein malnutrition normal-energy septic rats. Use of p55 and p75 receptors for TNF-
-mediated response may depend on various factors, including tissue-specific receptor/ligand availability and the state of inflammation, namely chronic vs. acute.
Apart from the study in kwashiorkor children, there are few data on the effect of protein deficiency alone on TNF receptors. Our study suggests that preventing protein deficiency will reduce TNF- response. Our previous study (26) showed that energy deficiency, in contrast, reduced the expression of TNF receptors. Taken together, our studies suggest that a higher-protein low-energy intake will reduce TNF-
activity. This effect may be desirable in patients with sepsis, diabetes, and insulin resistance.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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