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Am J Physiol Gastrointest Liver Physiol 294: G1120-G1129, 2008. First published March 27, 2008; doi:10.1152/ajpgi.00407.2007
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HORMONES AND SIGNALING

Tumor necrosis factor-{alpha} directly stimulates the overproduction of hepatic apolipoprotein B100-containing VLDL via impairment of hepatic insulin signaling

Bolin Qin,1,2 Richard A. Anderson,2 and Khosrow Adeli1

1Department of Laboratory Medicine and Pathobiology, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada; 2Beltsville Human Nutrition Research Center, United States Department of Agriculture, Beltsville, Maryland

Submitted 9 September 2007 ; accepted in final form 19 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin-resistant states are commonly associated with both increased circulating levels of tumor necrosis factor (TNF)-{alpha} and hepatic overproduction of very low density lipoproteins (VLDL). Here, we provide evidence that increased TNF-{alpha} can directly stimulate the hepatic assembly and secretion of apolipoprotein B (apoB) 100-containing VLDL1, using the Syrian golden hamster, an animal model that closely resembles humans in hepatic VLDL-apoB100 metabolism. In vivo TNF-{alpha} infusion for 4 h in chow-fed hamsters induced whole-body insulin resistance on the basis of euglycemic hyperinsulinemic clamp studies. Immunoprecipitation and immunoblotting analysis of livers from TNF-{alpha}-treated hamsters indicated decreased tyrosine phosphorylation of insulin receptor (IR)-β, IR substrate-1 (Tyr), Akt (Ser473), p38, ERK1/2, and JNK but increased serine phosphorylation of IRS-1 (Ser307) and Shc. TNF-{alpha} infusion also significantly increased hepatic production of total circulating apoB100 and VLDL-apoB100 in both fasting and postprandial (fat load) states. Ex vivo experiments, using cultured primary hepatocytes from hamsters, also showed TNF-{alpha}-induced VLDL-apoB100 oversecretion, an effect that was blocked by TNF receptor 2 antibody. Unexpectedly, TNF-{alpha} decreased the sterol regulatory element-binding protein-1c mass and mRNA levels but significantly increased microsomal triglyceride transfer protein mass and mRNA levels in primary hepatocytes. In summary, these data provide direct evidence that TNF-{alpha} induces whole-body insulin resistance and impairs hepatic insulin signaling accompanied by overproduction of apoB100-containing VLDL particles, an effect likely mediated via TNF receptor 2.

TNF-{alpha}; liver; insulin resistance; lipid; lipoprotein; apoB

THERE IS GROWING EVIDENCE suggesting a strong link between systemic inflammation and type 2 diabetes mellitus (type 2 DM) (13, 21). Tumor necrosis factor (TNF)-{alpha}, a major proinflammatory cytokine, has been implicated in metabolic disorders, such as obesity and insulin resistance (33, 34), indicating that perturbations of TNF-{alpha} metabolisms may affect the onset of type 2 DM and play a role in the development of cardiovascular disorders. Plasma TNF-{alpha} concentration has been shown to positively correlate with very low density lipoprotein (VLDL) triglyceride in healthy middle-aged men (56) and patients who are postinfarction (38) and is associated with early atherosclerosis. Elevated levels of lipoproteins containing apolipoprotein B (apoB) are widely thought to contribute to the development of atherosclerosis in human and experimental animals, even in the absence of other risk factors (25). ApoB100 is the major protein component of plasma lipoproteins and is required for the synthesis and secretion of triglyceride-rich circulating lipoproteins such as VLDL (41). Numerous studies have shown that the insulin resistance observed in type 2 DM is associated with an overproduction of apoB100-containing lipoproteins and reduced VLDL fractional catabolism (10, 35). Furthermore, the excess production of hepatic VLDL1 particles is the main abnormality of lipoprotein metabolism in type 2 DM (2, 3, 23).

Although a great deal of information on hepatic lipogenesis and VLDL production has been obtained in TNF-{alpha}-treated normoglycemic and diabetic rats (15–17), data available are conflicting and are limited to VLDL-triglyceride (TG) production; also rodent models employed have limitations when attempting to delineate the effect of TNF-{alpha} on VLDL synthesis and secretion in humans. TNF-{alpha} has been shown to induce an increase in in vivo VLDL-TG production in rats (15, 17), which was thought to be the result of decreased lipoprotein lipase activity and increased hepatic lipogenesis (15). However, these studies did not investigate the role of TNF-{alpha} on hepatic VLDL-apoB production, nor did they explore the underlying mechanisms. In addition, rat hepatocytes synthesize VLDL particles containing either apoB48 or apoB100, unlike human hepatocytes, which synthesize VLDL containing solely apoB100 (39). There are also conflicting data published on the effect of TNF-{alpha} on hepatic lipid metabolism in rat models. In contrast to the above studies, in vivo continuous TNF-{alpha} infusion of supraphysiological doses has been reported to reduce plasma TG levels in rats (58). Consequently, further investigation in this area is clearly needed to delineate the link between TNF-{alpha} and hepatic VLDL metabolism and explore the underlying mechanisms.

In the present study, we hypothesized that TNF-{alpha} can potentially induce hepatic VLDL overproduction via induction of hepatic insulin resistance. Systemic TNF-{alpha} infusion during hyperinsulinemic-euglycemic clamp has been shown to induce insulin resistance (46); however, no direct study exists on the link between TNF-{alpha}, hepatic insulin signaling, and hepatic lipoprotein production. In the present report, we examined the effects of TNF-{alpha}, hepatic-insulin signaling, and hepatic apolipoprotein production by using the Syrian Golden hamster, a model that closely resembles humans in hepatic VLDL metabolism. We present both in vivo and ex vivo evidence that TNF-{alpha} not only induces hepatic insulin resistance, but also stimulates fasting and postprandial overproduction of hepatic apoB100-containing VLDL1 particles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. Male Syrian golden hamsters (Mesocriceus auratus) weighing between 130–150 g were obtained from Charles River (Montreal, QC, Canada). All animals were housed individually and given free access to food and water. Following a 1-wk acclimatization period, the animals either underwent the in vivo protocol or were euthanized for isolation of hepatocytes for the ex vivo protocols. All animal protocols were approved by the animal ethics committee at the Hospital for Sick Children, University of Toronto.

Euglycemic-hyperinsulinemic clamp study. The right jugular vein and the left carotid artery were exposed under isoflurane anesthesia, and hamsters were inserted with catheters encased in silastic tubing that were exteriorized to the back of the neck (48, 49). Following an overnight recovery period and a 16-h fast, a 4-h infusion of 0.9% normal saline (vehicle) or TNF-{alpha} (0.5 µg/kg per h) was administered by the venous catheter. With the infusion of saline or TNF-{alpha} continuing, a baseline blood sample was drawn at the 2-h time point. The hamster was then submitted to a euglycemic clamp procedure for the final 2 h to assess the whole-body insulin sensitivity. The venous catheter was then used for the infusion of glucose (20%) and insulin (3 mU/kg per min). TNF-{alpha} was dissolved in saline and administered at a dosage shown to be sufficient to effectively inhibit insulin action (66, 67). During the euglycemic clamp, blood glucose concentrations were measured every 10 min, and the euglycemia was maintained at around 4.2 ± 0.1 mM of baseline. The glucose infusion rate (GIR) was calculated every 10 min (48). Finally, the liver tissues were excised and stored at –80°C until analyses.

In vivo Triton WR1339 infusion and preparation of VLDL-apoB100 fractions. Hamsters were fasted overnight for 16 h, and the catheters were inserted as described (48). Following a 4-h recovery period, hamsters were infused with saline or TNF-{alpha} (0.5 µg/kg per h) for 4 h. After the first 2-h and 20-min infusion, an intravenous bolus of Triton WR1339 (0.5 g/kg) was administered. Triton WR1339 effectively blocks the activity of lipoprotein lipase in vivo and therefore blocks the VLDL particle clearance, such that the secretion rate of VLDL-apoB is proportional to the rate of increase in plasma VLDL-apoB over time (57). After Triton WR1339 administration, 300 µl of blood were collected (as baseline). An additional blood sample was taken at the 90-min time point following Triton WR1339 treatment. Studies performed in the postprandial state were as described above except that hamsters were manually administered a 200-µl olive oil (1 g polyunsaturated, 2 g saturated, 11 g unsaturated; 120 kcal) load via oral gavage. The bolus of Triton WR1339 was administered without prior fasting, and blood samples were drawn as described above.

To isolate the VLDL fraction, the serum samples were fractionated by rate flotation ultracentrifugation. A serum sample (100 µl) was mixed with 4 ml of 1.10 g/ml NaBr solution and loaded onto the bottom of a Beckman SW41 centrifuge tube. The sample was overlaid with 3 ml each of 1.065, 1.02 g/ml, and 2 ml of 1.006 g/ml NaBr solutions. After ultracentrifugation at 151,000 g at 4°C for 70 min, the top layer (1 ml) containing the triglyceride-rich lipoprotein (TRL) was removed, and 1 ml of 1.006 g/ml NaBr solution was added and centrifuged at 151,000 g at 4°C for 18 h. The top 2-ml layer was further fractionated to isolate VLDL (VLDL1 and VLDL2) particles, essentially as described (40). After immunoprecipitation with antiserum against hamster apoB antibody, samples were resolved in SDS-PAGE and subjected to immunoblotting using hamster apoB antiserum. ApoB bands were visualized and quantified using an imaging densitometer. To determine the total serum apoB100, the serum samples were diluted (200-fold) and then treated as described above.

Hamster primary hepatocytes. Hamsters were anesthetized by isoflurane. After achieving complete general anesthesia, the liver was perfused and hepatocytes were isolated as described (59, 60).

Preparation of nuclear extracts from primary hepatocytes. Crude nuclear extracts were prepared from primary hepatocytes, essentially as described (62). The hepatocytes are washed twice in 2 ml cold PBS and lysed in 500 µl of cold buffer A (10 mM HEPES pH 7.9, MgCl2 1.5 mM, KCl 10 mM, DTT 0.5 mM, aprotinin 100 µg/ml, leupeptin 5 µg/ml, pepstatin 1 µg/ml, and PMSF 0.5 mM). After a 15-min incubation on ice, 0.5% NP-40 in final concentration was added to the homogenates and the tubes were vortexed for 10 s. The nuclei were pelleted at 6,500 rpm (tabletop microfuge) for 20 s. The nuclear pellet was suspended in 150 µl of cold buffer B (20 mM HEPES pH 7.9, MgCl2 1.5 mM, NaCl 420 mM, EDTA 0.2 mM, glycerol 25% vol/vol, aprotinin 100 µg/ml, leupeptin 5 µg/ml, pepstatin 1 µg/ml, and PMSF 0.5 mM) and incubated on ice for 30 min. Nuclear extracts were recovered after centrifugation for 10 min at 12,000 rpm at 4°C. Protein concentration was determined by Bradford assay, and the aliquots were stored at –80°C until analysis.

Immunoprecipitation and immunoblotting. Hepatocytes were lysed, and the immunoprecipitation/immunoblotting protocols were performed as described (49, 59, 60).

Metabolic labeling of primary hepatocytes and measurement of VLDL-apoB secretion. Hamster primary hepatocytes were used for pulse-chase experiments as described (59) with some modifications. In brief, hepatocytes were first treated with TNF-{alpha} (10 ng/ml) in methionine/cysteine-free minimal essential medium at 37°C for 1 h and then used for pulse-chase experiments. After labeling with 100 µCi/ml [35S]methionine/cysteine for 45 min, the pulse-labeled cells were treated with TNF-{alpha} (10 ng/ml) in the chase medium. Following the pulse, the cells were washed with PBS and the radiolabel was chased by the addition of attachment media supplemented with 10 mM methionine. At various chase times, media were collected and cells were harvested and lysed in solubilization buffer. Radiolabeled apoB100 was immunoprecipitated from media and cell lysates with an anti-hamster apoB antibody, analyzed by SDS-PAGE and fluorography, and the radiolabeled apoB was quantified by scintillation counting as described (59).

To measure VLDL-apoB secretion, hepatocytes were preincubated in methionine/cysteine-free MEM and TNF-{alpha} (10 ng/ml) for 1 h and pulsed chased for 2 h with 100 µCi/ml [35S] protein-labeling mix. Culture media were collected for VLDL isolation, and cells were harvested and lysed. The density of the culture media was adjusted to 1.006 g/ml, and VLDL was isolated by ultracentrifugation (18 h, 151,000 g). The VLDL fraction was collected and solubilized. Radiolabeled VLDL-apoB100 was immunoprecipitated, analyzed by SDS-PAGE/fluorography, and quantified as detailed above.

Effects of anti-TNF-receptor-1/2 antibodies on TNF-{alpha}-induced apoB100 secretion ex vivo. Hepatocytes were pulse labeled as described (50) with minor alterations. Briefly, hepatocytes were pretreated with the antibodies against the p55 TNF receptor (TNFR)-1 and p75 TNFR-2 for 30 min and then stimulated with TNF-{alpha} (10 ng/ml) for 60 min in methionine-free DMEM at 37°C. Cells were [35S] methionine/cysteine labeled for 2 h. The cells were harvested and lysed, and apoB100 was immunoprecipitated as described (50, 59). Media were then fractionated using discontinuous KBr gradient ultracentrifugation as detailed above.

SREBP-1c, and MTP mRNA abundance. RT-PCR analyses were carried out as described (49). Hepatocytes were treated with TNF-{alpha} (10 ng/ml) at 37°C for 24 h. The mRNA levels were assessed by real-time quantitative RT-PCR using an ABI Prism 7700 sequence detector. The primers used for PCR were as follows: sterol regulatory element-binding protein (SREBP)1c primers: 5'-GCGGACGCAGTCTGGG-3' and 5'-ATGAGCTGGAGCATGTCTTCAAA-3'; microsomal triglyceride transfer protein (MTP) primers, 5'-GTCAGGAAGCTGTGTCAGAATG-3' and 5'- CTCCTTTTTCTCTGGCTTTTCA-3'; and 18S primers, 5'-TAAGTCCCTGCCC TTTG TACACA-3' and 5'-GATCCGAGGGCCTCACTAAAC-3'.

Other laboratory methods. Measurement of glucose, serum insulin, cholesterol, and TG levels were performed as described (49).

Statistical analysis. Statistical significance was calculated with a two-tailed paired Student's t-test analysis or one-way ANOVA. P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of TNF-{alpha} infusion on whole-body insulin sensitivity. Normal, chow-fed hamsters were used to perform euglycemic-hyperinsulinemic studies following TNF-{alpha} infusion. Table 1 summarizes metabolic profile of hamsters during clamp studies. Four-hour TNF-{alpha} infusion induced a significant decrease in GIR compared with the saline-treated controls. The cholesterol and TG levels were significantly increased by 90-min Triton WR1339 treatment (Table 2), compared with baseline levels in control hamsters, in both fasting and postprandial conditions (P < 0.001, respectively). There was a trend for TNF-{alpha} infusion-treated hamsters to have higher cholesterol and TG levels, but, perhaps because of high inter-animal variability, the differences were not statistically significant (P = 0.13 and P = 0.09, respectively). TNF-{alpha} treatment significantly increased serum TG levels (P < 0.05) compared with controls in the postprandial state at the end of the 90-min Triton treatment, but the change in cholesterol levels was not significant (P = 0.11). Additionally, TNF-{alpha} infusion did not significantly affect serum insulin concentrations in fasting or postprandial state (Table 2).


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  		Table 1. Body weights, the blood glucose and serum insulin levels, and GIR during the euglycemic clamp procedure

 

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  		Table 2. The serum TG, cholesterol, and insulin levels, before and after Triton WR-1339 treatment in the fasting and fat loading studies

 
Effect of TNF-{alpha} infusion on hepatic signal transduction. The effects of TNF-{alpha} on hepatic insulin signaling after euglycemic clamp are shown in Fig. 1. The total protein mass of hepatic insulin receptor (IR)-β, IR substrate (IRS)-1, Shc, Akt, p38, ERK1/2, and JNK did not differ significantly between saline- and TNF-{alpha}-treated hamsters. The tyrosine phosphorylation levels of hepatic IR-β and IRS-1 in the TNF-{alpha}-treated hamsters were significantly lower, however, when compared with the controls (Fig. 1, A and B, 60 ± 17% and 64 ± 16% of saline, P < 0.05, respectively). Likewise, significantly reduced phosphorylation of Akt (Ser473), p38, ERK1/2 (44/42 kDa), and JNK (55/46 kDa) was observed in the TNF-{alpha}-treated groups (Fig. 1, D, F, G, and H, 57 ± 7%, 65 ± 3%, 57 ± 4%, and 75 ± 14% of saline, P < 0.05, respectively). In addition, there were significant increases in the phosphorylation of IRS-1 (Ser307) and Shc (52/46 kDa), both of which have been linked to insulin resistance (Fig. 1, C and E, 170 ± 18%, 130 ± 6% of saline, respectively, P < 0.05).


Figure 1
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  		Fig. 1. Effect of tumor necrosis factor (TNF)-{alpha} infusion on in vivo hepatic insulin signaling cascade during euglycemic-hyperinsulinemic clamp. A–H: effects of TNF-{alpha} infusion on the mass and phosphorylation of insulin receptor (IR)-β, IR substrate (IRS)-1 (Tyr), IRS-1 (Ser307), Shc, Akt (Ser473), and MAPKs in the liver tissue of hamsters from the euglycemic clamp study. Cell lysates were analyzed by immunoblotting with the corresponding antibodies against either phosphorylation or total mass. Data are means ± SE (n = 4 or 5); open bars, saline; shaded bars, TNF-{alpha}, *P < 0.05 and **P < 0.01 vs. control. IB, immunoblotting; pTy, phosphotyrosine; p, phosphorylation.

 
TNF-{alpha} infusion increases the accumulation of apoB100-containing lipoproteins in both fasted and postprandial states. To investigate the effect of TNF-{alpha} on the accumulation of circulating apoB100-containing lipoproteins, fasted hamsters were infused 4 h with either saline or TNF-{alpha}, and serum was collected at baseline and 90 min after Triton WR1339 treatment. Total and VLDL-apoB100 were significantly increased at the 90-min time point in both groups compared with baseline (P < 0.001). There was significantly higher accumulation of both total serum apoB100 (Fig. 2A) and VLDL-apoB100 (Fig. 2B) in the TNF-{alpha}-infused group, compared with controls at 90-min time points (P < 0.05). We also compared the effect of TNF-{alpha} on VLDL1 versus VLDL2 distribution; the result suggests that VLDL1-apoB100 was 80% higher at the 90-min time point compared with the control (P < 0.05, Fig. 2C). Mean VLDL2-apoB100 did not significantly change in either group.


Figure 2
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  		Fig. 2. Effect of TNF-{alpha} infusion on fasting and postprandial apolipoprotein B (apoB)100-containing lipoproteins. The total serum- and very low density lipoproteins (VLDL)-apoB100 as well as VLDL1 and VLDL2-apoB100 were immunoblotted using anti-hamster apoB primary antibody. Immunoblots were analyzed using densitometry and expressed as a proportion of time 0. A–C: representative experiments on the total serum- (A) and VLDL-apoB (B) and the distribution of VLDL1- vs. VLDL2-apoB100 (C) in the fasting state. Results of densitometric analysis of replicate experiments are shown (means ± SE, n = 6, respectively). D–F: representative experiments on the total serum- (D) and VLDL-apoB (E) and the distribution of VLDL1 and VLDL2-apoB100 (F) in the postprandial state (after fat loading). The values given are means ± SE and are representative of several replicate experiments (n = 6, respectively). Open bars, saline; shaded bars, TNF-{alpha}; {dagger}P < 0.001 vs. baseline; controls *P < 0.05 vs. controls.

 
The in vivo postprandial hepatic lipoprotein production was also assessed in TNF-{alpha}-treated hamsters following an oral fat load. Following Triton infusion, the levels of total serum apoB100 (Fig. 2D) and VLDL-apoB100 (Fig. 2E) were significantly higher in the TNF-{alpha}-infused group at 90 min (P < 0.05), compared with the controls, suggesting that TNF-{alpha} infusion results in increased hepatic secretion of apoB-containing VLDL lipoproteins in the postprandial state (following an oral fat load). VLDL1-apoB100 was 93% higher in TNF-{alpha}-treated hamsters at the 90-min time point compared with the control (P < 0.05, Fig. 2F), but no change was observed in VLDL2-apoB100.

TNF-{alpha} stimulates hepatic apoB secretion ex vivo. To investigate whether TNF-{alpha} directly affects the production of apoB100-containing lipoprotein, primary hepatocytes were freshly isolated from chow-fed hamsters and then incubated ex vivo with TNF-{alpha}. Cells were then subjected to pulse-chase labeling experiments to assess the stability and secretion of apoB. The hepatocytes were treated with TNF-{alpha} for 1 h and radiolabeled, and the radiolabel was chased for 1-h and 2-h periods. Radiolabeled apoB100 was immunoprecipitated from cells and media. Figure 3 shows quantitation of radiolabeled apoB100 in cells and media (Fig. 3A) and total (cells + media) radiolabeled apoB100 recovered (Fig. 3B) (apoB remaining at each chase time expressed as a percent of the labeled apoB at time 0). The radiolabeled apoB (counts per minute) at the zero time point was not significantly different between control and TNF-{alpha}-treated hepatocytes (6,533 ± 145 vs. 6,676 ± 176, P = 0.29). Percent apoB remaining in TNF-{alpha}-treated hepatocytes was significantly higher at both 1-h and 2-h chase times, compared with untreated control cells (P < 0.05, respectively). The data suggest that intracellular degradation of apoB100 in cells treated with TNF-{alpha} was less pronounced than that in basal conditions, perhaps due to increased apoB100 stability.


Figure 3
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  		Fig. 3. Ex vivo effect of TNF-{alpha} on the synthesis and secretion of apoB100 in primary hepatocytes. Primary hepatocytes were pretreated with saline or TNF-{alpha} (10 ng/ml) for 1 h at 37°C, then were pulsed with 100 µCi/ml [35S] methionine and chased for 0, 1, and 2 h in the chase medium supplemented with TNF-{alpha} (10 ng/ml). The media and cell were collected at each chase time point, and these were subjected to immunoprecipitation using anti-hamster apoB antibody and then analyzed by SDS-PAGE and fluorography; to investigate the effect of TNF-{alpha} on VLDL-apoB production, primary hepatocytes were pretreated with TNF-{alpha} (10 ng/ml) for 1 h, and pulsed for 2 h with 100 µCi/ml [35S] methionine. Media was collected and subjected to the ultracentrifugation protocol described in MATERIALS AND METHODS. The VLDL was collected and immunoprecipitated using anti-hamster apoB antibody. A and B: distribution of labeled apoB100 in cells and media as well as total recovered apoB100 (cell + media). C: secreted levels of VLDL-apoB100 in control and TNF-{alpha}-treated hepatocytes, respectively. Data are means ± SE; open bars, saline; shaded bars, TNF-{alpha}; *P < 0.05 vs. controls.

 
TNF-{alpha} stimulates hepatic VLDL-apoB secretion ex vivo in primary hepatocytes. To determine the effects of TNF-{alpha} on VLDL-apoB secretion, we performed ex vivo steady-state labeling experiments in which hepatocytes were treated with TNF-{alpha} for 1 h and then radiolabeled for 2 h. Culture media containing secreted lipoprotein particles were then collected and subjected to ultracentrifugation to isolate VLDL. Figure 3C shows the immunoprecipitable VLDL-apoB secreted by vehicle and TNF-{alpha}-treated hepatocytes. There was a significant elevation in the amount of VLDL-apoB secreted into the media in TNF-{alpha}-treated hepatocytes (1.72-fold of control, P < 0.05). The result suggests the secretion of a considerably higher number of VLDL particles by TNF-{alpha}-treated hamster hepatocytes. Taken together, these data suggest an overall increase in apoB100 stability that is translated into an increase in apoB-containing VLDL secretion.

Effects of TNF-{alpha} on mass and mRNA expression of MTP and SREBP-1c in hepatocytes. MTP is an essential factor for lipid transfer to nascent hepatic of apoB-containing VLDL. Mass and mRNA levels were therefore measured to determine whether changes in MTP were responsible for the changes observed in VLDL secretion. Our results show that the MTP mass increased by 163 ± 17.8% (Fig. 4A) and mRNA increased by 130 ± 10% TNF-{alpha} (Fig. 4B) in hepatocytes treated with TNF-{alpha} (P < 0.05, respectively).


Figure 4
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  		Fig. 4. Effect of TNF-{alpha} on the mass and mRNA levels of microsome triglyceride transfer protein (MTP) in primary hamster hepatocytes. A: hepatocytes were treated with TNF-{alpha} (10 ng/ml) at 37°C for 24 h; then the cells are washed twice in 2 ml cold PBS, and the cell lysates were prepared as described previously (49, 59), subjected to SDS-PAGE, immunoblotted with an anti-mouse MTP antibody (BD Biosciences), washed, and then incubated with a secondary antibody conjugated to peroxidase. Proteins were detected by commercially available ECL kit (Amersham Biosciences). B: MTP mRNA levels of 24-h TNF-{alpha} (10 ng/ml)-treated hepatocytes at 37°C were determined with RT-PCR using cDNA made from 10 ng total RNA as template. The mRNA levels were normalized using the 18S rRNA level in each sample. Values are means ± SE and are presented as a percentage of control (n = 3); open bars, saline; shaded bars, TNF-{alpha}. *P < 0.05 vs. controls.

 
To assess whether the observed changes in MTP expression in TNF-{alpha}-treated hepatocytes might be paralleled by changes in SREBP-1c, we also measured SREBP-1c mass and mRNA levels. Surprisingly, hepatocytes treated with TNF-{alpha} had a significantly decreased mass of mature and immature forms of SREBP-1c compared with control [Fig. 5A, 61.5 ± 9.3% (immature); 67.2 ± 5.3% (mature); P < 0.05]. Also mRNA expression of SREBP-1c decreased by 54 ± 5% (Fig. 5B, P < 0.05). TNF-{alpha} treatment did not affect apoB mRNA levels in hepatocytes (data not shown).


Figure 5
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  		Fig. 5. Effect of TNF-{alpha} on the mass and mRNA levels of sterol regulatory element-binding protein (SREBP)-1c in primary hamster hepatocytes. A: effects of TNF-{alpha} treatment on the mass of SREBP-1c in primary hepatocytes. Nuclear proteins were separated on 8% SDS-PAGE and transferred to nitrocellulose membrane, blocked in 5% milk, and incubated in SREBP-1c primary antibody (Santa Cruz Biotechnology) overnight at 4°C. After being washed, blots were probed with rabbit IgG secondary antibody. Proteins were detected by commercially available ECL kit, (Amersham Biosciences). B: MTP mRNA levels of 24-h TNF-{alpha} (10 ng/ml) treated hepatocytes at 37°C were determined with RT-PCR using cDNA made from 10 ng total RNA as template. The mRNA levels were normalized using the 18S rRNA level in each sample. Data are means ± SE (n = 3 for each group); open bars, saline; shaded bars, TNF-{alpha}. *P < 0.05 vs. control.

 
TNF-{alpha} stimulates apoB100 production in hepatocytes through p75 TNFR. To identify the receptor that mediates TNF-{alpha}-induced apoB100 overproduction, primary hamster hepatocytes were first incubated with specific blocking antibodies against TNFR-1 and TNFR-2. As shown in Fig. 6, only TNFR-2 antibody significantly blocked TNF-{alpha}-induced enhancement in both cellular (Fig. 6A) and secreted apoB100 (Fig. 6B) or VLDL-apoB100 secretion (Fig. 6C). No significant effects were observed with TNFR-1 antibody, suggesting that TNFR-2 is likely to mediate the stimulatory effect of TNF-{alpha} on apoB100 production. TNFR-1/2 antibodies had no effect on total apoB100 and VLDL-apoB100 secretion in normal/control hepatocytes without TNF-{alpha} stimulation (data not shown).


Figure 6
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  		Fig. 6. TNF receptor p75 mediates the effects of TNF-{alpha} on apoB100 and VLDL-apoB100 secretion. Anti-TNF receptor p75 antibody inhibits apoB100 oversecretion induced by TNF-{alpha}. The hepatocytes were pretreated with antibodies against the TNFR-1/2 (1:1,000) for 30 min and treated with TNF-{alpha} (10 ng/ml) for 60 min, followed by 2-h pulse labeling. VLDL fractions were isolated by ultracentrifugation as described. Labeled apoB was immunoprecipitated from VLDL fractions, subjected to SDS-PAGE, and analyzed by fluorography. Values given are representative of 2 independent experiments performed in duplicate. Data are means ± SE; open bars, saline; shaded bars, TNF-{alpha}; *P < 0.05 vs. control; **P < 0.05 vs. TNF-{alpha} alone.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Low-grade systemic inflammation is a prevalent feature of obesity and insulin resistance (12), and TNF-{alpha} may be a key mediator linking inflammation and dysregulation of lipid and glucose metabolism (6, 28). Development of insulin resistance is also known to be clearly associated with metabolic dyslipidemia (19, 51). However, mechanisms linking inflammatory cytokines with specific perturbations in hepatic lipoprotein metabolism have not been directly examined. In the present report, we found that TNF-{alpha} infusion over a 4-h period induced whole-body insulin resistance in hamsters leading to overproduction of VLDL-apoB100 in both fasting and postprandial states. TNF-{alpha} stimulated VLDL1-apoB100 production but had no effect on VLDL2 apoB production, which is consistent with the observation that acute insulin infusion inhibits the production of large buoyant VLDL1 particles (47). These abnormalities resemble those seen in type 2 DM in humans, in which increased VLDL1 production is the main abnormality of lipoprotein metabolism (2, 3, 24). Additionally, we observed that TNF-{alpha} significantly stimulates postprandial VLDL1-apoB100 and hypertriglyceridemia after a fat load. Recent evidence (63, 68) suggests that, in the postprandial state, the accumulation of apoB48 TRL may not only lead to the enhanced delivery of exogenous lipids to liver, which will result in an increased production of hepatic lipoproteins, but also delay in the clearance of hepatic TRLs by competing for clearance pathways. Therefore, the overproduction of postprandial TRL-apoB48 induced by TNF-{alpha} (49) may play an important role to stimulate postprandial VLDL production.

TNF-{alpha}-induced perturbations in insulin signaling have been well documented in insulin-responsive tissues such as muscle and adipose (7, 8, 33). Previously, TNF-{alpha} infusion has been shown to induce hepatic glucose output, despite hyperinsulinemia in rats (46), suggesting hepatic insulin resistance. However, a direct effect of TNF-{alpha} on hepatic insulin sensitivity has not been previously examined. In the hamster model, we examined the effects of TNF-{alpha} infusion on hepatic insulin signaling after insulin clamp. TNF-{alpha} infusion was found to alter the phosphorylation of a number of insulin-signaling molecules in the liver of hamster. Hotamisligil et al. (32) were the first to demonstrate that TNF-{alpha} lowers tissue insulin sensitivity by promoting serine phosphorylation of IRS-1, which in turn causes the serine phosphorylation of insulin receptor. This prevents the normal tyrosine phosphorylation of the insulin receptor in adipocytes and thus interferes with phosphorylation of Shc and the downstream activation in vascular smooth muscle cells (27). Our results suggest that TNF-{alpha} inhibits hepatic IR-β and IRS-1 tyrosine phosphorylation. In contrast, TNF-{alpha} markedly increased the phosphorylation of Ser307 IRS-1 and Shc. A previous study (26) has shown that TNF-{alpha} treatment, like insulin treatment, induces a transient phosphorylation of Shc expression in vascular smooth muscle cells, and pretreatment with TNF-{alpha} led to a transient suppression of insulin-induced tyrosine phosphorylation of Shc; after exposure to TNF-{alpha} for 90 min, insulin-stimulated Shc phosphorylation was restored. Furthermore, Li and Goldstein have shown that reducing IRS-1 serine phosphorylation results in increased IRS-1 tyrosine phosphorylation and decreased Shc tyrosine phosphorylation in liver cells (44). Therefore, it appears that Shc expression may differ in in vivo and in vitro conditions, as well as in different cell types. We also examined the impact of TNF-{alpha} infusion on phosphorylation of Ser473Akt, a key signaling factor downstream of phosphatidylinositol 3-kinase. In the liver from TNF-{alpha} infusion-treated hamsters, insulin-stimulated phosphorylation of Ser473Akt was significantly reduced, suggesting reduced Akt activity with TNF-{alpha}, most likely due to the observed decrease in tyrosine phosphorylation of upstream molecules of the insulin-signaling cascade. Our observations are in agreement with the findings that Akt phosphorylation was impaired in muscle and adipocytes of insulin-resistant diabetic subjects (42, 52).

We also observed that TNF-{alpha} markedly inhibited the phosphorylation of p38 MAPK, ERK1/2, and JNK. MAPKs are known to be affected by inflammation and have been implicated in the induction of insulin resistance (14, 22, 32) and diabetes-associated dyslipidemia (4). We initially expected that TNF-{alpha} infusion would lead to activation of molecules of the hepatic inflammatory signaling cascades such as JNK in the liver; however, hepatic JNK phosphorylation was found to be reduced. A previous study (29) reported that TNF-{alpha} induces insulin resistance in adipocytes and leads to a transient phosphorylation of JNK at 15 min; conversely, after TNF-{alpha} treatment for 4 h, the phosphorylation of JNK levels were significantly decreased, compared with the controls. Consequently, the regulation of phospho-JNK by TNF-{alpha} may be different under various experimental conditions. In our study, it appears that TNF-{alpha} may have initially induced pJNK, but after 4 h of treatment, JNK phosphorylation was actually decreased.

Increasing evidence (2, 59–61, 69) suggests that insulin regulates the assembly process of VLDL and impaired insulin signaling results in increased VLDL production. Our results suggest that TNF-{alpha} infusion induces whole-body insulin resistance and impairs hepatic insulin signaling, which might play key roles for downregulation of hepatic-VLDL apoB and VLDL-TG production (1). Insulin has been shown to acutely inhibit hepatic assembly and secretion of VLDL particles likely via mechanisms involving an increase in apoB degradation and a decrease in MTP expression (11, 45). In contrast to insulin, our ex vivo data suggest that TNF-{alpha} can markedly increase the intracellular stability of newly synthesized apoB100, suppressing its intracellular degradation and promoting its extracellular secretion. This stimulation of apoB secretion may be mediated by reduced insulin sensitivity of hepatocytes to the inhibitory effects of insulin on the VLDL assembly process. Present studies were not performed to examine the effect of TNF-{alpha} on different pathways involved in apoB degradation. Further experiments are necessary to investigate these mechanisms. Another important factor is likely to be MTP, which is rate limiting for the production of apoB-containing VLDL. MTP catalyzes the transfer of lipids to newly synthesized apoB within the endoplasmic reticulum, facilitating secretion of nascent lipoproteins (36). Hepatic expression of MTP is increased in the obese and hypertriglyceridemic rat (43) and in obese diabetic mice (5). Our previous studies have shown that an increased expression of MTP contributes to the overproduction of hepatic apoB100 lipoproteins from the fructose-fed hamster model of insulin resistance (9, 59); amelioration of hepatic insulin resistance in this model resulted in normalization of MTP expression and reduction of the overproduction of apoB100-containing lipoproteins (9). Furthermore, both cell and animal studies demonstrate that apoB secretion is decreased in a dose-dependent manner upon treatment with specific MTP inhibitors (37, 64). In this study, we observed that TNF-{alpha} treatment significantly increased hepatic MTP mass and mRNA. The increased MTP expression may potentially lead to upregulation of VLDL assembly and increase of apoB100 production.

Additionally, SREBP-1c, an important regulator of lipogenesis, seems to have a crucial role in the regulation of TG accumulation in the liver (65), and decisive evidence exists for the insulin-induced expression of SREBP-1c (20). Consistent with a previous study (53) that mRNA levels of SREBP-1c are decreased following TNF-{alpha} treatment of adipocytes, we found that hepatocytes treated with TNF-{alpha} had a significantly reduced mass and mRNA expression of SREBP-1c. Moreover, Shimomura et al. (55) reported that hepatic TG levels increased by 60% and SREBP-1c mRNA levels were reduced by 80% shortly (42 h) after a single high-dose streptozotocin injection. Interestingly, this appears to be in contrast to increased hepatic SREBP-1c mRNA levels in the livers of mice with type 2 DM (54) and of streptozotocin-treated mice with hyperglycemia (5). It is plausible that, although acute treatment with TNF-{alpha} reduces SREBP-1c mRNA, chronically increased circulating levels of TNF-{alpha} may be associated with increased SREBP-1c mRNA expression as observed in animal models of type 2 DM.

The activation of the TNF-{alpha}/TNFR pathway has been shown to be associated with several metabolic abnormalities, such as insulin resistance, increased hepatic VLDL secretion, and free fatty acid output by adipose tissue (30, 38). In the present study, we examined the effects of blocking TNFR function on apoB100-lipoprotein secretion in ex vivo experiments and observed that blocking of the TNFR-2 inhibited TNF-{alpha}-induced oversecretion of VLDL-apoB100 in radiolabeling experiments. In contrast, similar experiments with TNFR-1 demonstrated no significant effects. This is consistent with the observation that the adipose tissue of obese subjects exhibits increased expression of TNF-{alpha} and TNFR-2 but not TNFR-1 mRNA, which is accompanied by elevation in plasma soluble TNFR-2 (18, 31). Further experiments will still be necessary to elucidate the relative role of each receptor, as well as their respective action in regard to hepatic insulin resistance.

In summary, increased circulating TNF-{alpha} levels may interfere with hepatic insulin signal transduction and lead to significant overproduction of VLDL and hypertriglyceridemia. The stimulatory effects of TNF-{alpha} appear to be mediated via TNFR-2 and involve downregulation of SREBP-1c and upregulation of MTP, enhancing apoB100 protein stability, resulting in an increased rate of VLDL1 assembly and secretion. Further research is underway to examine the link between the inflammatory pathways induced by TNF-{alpha} and the process of VLDL1 assembly and secretion.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This project was supported by an operating grant from the Heart and Stroke Foundation of Ontario (T-4809) to K. Adeli and partly by Integrity Nutraceuticals International and the United States Department of Agriculture's Cooperative Research and Development Agreement NO. 58-3K95-7-1184.


   ACKNOWLEDGMENTS
 
We acknowledge Dr. Wei Qiu, Rita Kohen, Urban Joe, and Yates Allison for helpful discussions and comments on the manuscript, as well as Mark Naples, Elaine Xu, and Noella Bryden for their technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Adeli, Div. of Clinical Biochemistry, DPLM, Hospital for Sick Children, 555 Univ. Ave., Toronto, ON, Canada M5G 1X8 (e-mail: k.adeli{at}utoronto.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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