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1 Department of Physiology,
2 Institute of Medical Science and
3 Department of Medicine, University of Toronto, 1 King’s College Circle, Toronto, Ontario, M5S 1A8, Canada
(Correspondence should be addressed to A Giacca; Email: adria.giacca{at}utoronto.ca)
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Abstract |
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B
kinase ß (IKKß ), which can impair insulin signaling directly via serine phosphorylation of insulin receptor substrates (IRS) and/or indirectly via induction of transcription of proinflammatory mediators, has been implicated in free fatty acid (FFA)-induced insulin resistance in skeletal muscle. However, it is unclear whether liver IKKß activation plays a causal role in hepatic insulin resistance caused by acutely elevated FFA. In the present study, we wished to test the hypothesis that sodium salicylate, which inhibits IKKß , prevents hepatic insulin resistance caused by short-term elevation of FFA. To do this, overnight-fasted Wistar rats were subject to 7-h i.v. infusion of either saline or Intralipid plus 20 U/ml heparin (IH; triglyceride emulsion that elevates FFA levels in vivo) with or without salicylate. Hyperinsulinemic–euglycemic clamp with tracer infusion was performed to assess insulin-induced stimulation of peripheral glucose utilization and suppression of endogenous glucose production (EGP). Infusion of IH markedly decreased (P < 0.05) insulin-induced stimulation of peripheral glucose utilization and suppression of EGP, which were completely prevented by salicylate co-infusion. Furthermore, salicylate reversed IH-induced 1) decrease in I B content; 2) increase in serine phosphorylation of IRS-1 (Ser 307) and IRS-2 (Ser 233); 3) decrease in tyrosine phosphorylation of IRS-1 and IRS-2; and 4) decrease in serine 473-phosphorylated Akt in the liver. These results demonstrate that inhibition of IKKß prevents FFA-induced impairment of hepatic insulin signaling, thus implicating IKKß as a causal mediator of hepatic insulin resistance caused by acutely elevated plasma FFA. |
Introduction |
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Recent studies have demonstrated that FFA cause insulin resistance in the skeletal muscle mainly via inhibition of tyrosine phosphorylation of insulin receptor substrate (IRS)-1 (Kim et al. 2001, 2004b, Yu et al. 2002), which is a critical step in insulin signal transduction. This process is likely mediated by the phosphorylation of serine residues on IRS-1 by certain serine kinases, such as protein kinase C (PKC), inhibitor of I B kinase ß (IKKß ), and c-Jun NH2-terminal kinase1 (JNK 1). Shulman (Kim et al. 2001, 2004b, Yu et al. 2002) and others (Itani et al. 2000, 2002, Yuan et al. 2001, Boden et al. 2005) have implicated accumulation of intramyocellular lipid metabolites (e.g., diacylglycerol, long-chain fatty acyl CoA) and activation of serine kinases PKC (isoforms ß and 
in humans, and 
and 
in rodents) and IKKß as potentially causal events in the pathway of FFA-induced insulin resistance in skeletal muscle. In particular, it has been shown that mice lacking PKC- are protected from insulin resistance caused by a short-term lipid infusion (Kim et al. 2004b). Furthermore, treatment with high-dose sodium salicylate, an IKKß inhibitor, or IKKß-deficiency prevents fat-induced insulin resistance in rodent skeletal muscle (Kim et al. 2001).
Recently, treatment of primary mouse hepatocytes with saturated fatty acids was found to activate JNK in association with increased IRS-1 serine 307 phosphorylation and decreased insulin-stimulated Akt activation, which were absent in hepatocytes isolated from JNK– /– mice (Solinas et al. 2006). However, potentially causal roles of serine kinases PKC, IKKß , and JNK1 in FFA-induced hepatic insulin resistance have not been extensively investigated in in vivo models. Our laboratory has previously established an association between PKC- membrane translocation, a marker of its activation, and FFA-induced hepatic insulin resistance (Lam et al. 2002). This association has recently been confirmed by Boden et al. (2005), who, in the same study, also linked hepatic IKKß activation with FFA-induced hepatic insulin resistance. However, it has not been clearly shown whether IKKß causes hepatic insulin resistance induced by short-term elevation of circulating FFA. While Arkan et al. (2005) have demonstrated that hepatocyte-specific IKKß knockout mice are protected from hepatic insulin resistance caused by high-fat feeding or genetically induced obesity, both of these models exhibit chronically elevated FFA levels, which may cause insulin resistance via a different mechanism than short-term FFA elevation. Thus, in the present study, we wished to test the hypothesis that IKKß activation is causally involved in the mechanism of hepatic insulin resistance induced by acute FFA elevation. To do this, we used a rat model of short-term (7 h) i.v. lipid infusion with or without co-infusion of sodium salicylate to examine whether inhibition of IKKß results in restoration of hepatic insulin action via prevention of FFA-induced impairment of hepatic insulin signaling.
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Materials and Methods |
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Female Wistar rats (Charles River, Quebec, Canada) weighing 250–300 g were used for experiments. The rats were housed in the University of Toronto’s Department of Comparative Medicine. They were exposed to a 12 h light:12 h darkness cycle and were fed rat chow (Teklad Global # 2018; Harland Teklad Global Diets, Madison, WI, USA) and water ad libitum. The Animal Care Committee of the University of Toronto approved all procedures.
Surgery
After 3–5 days of adaptation to the facility, rats were anesthetized with isoflurane, and indwelling catheters were inserted into the right internal jugular vein for infusion and the left carotid artery for blood sampling, as previously described (Lam et al. 2002). Briefly, polyethylene (PE) catheters (PE-50; Cay Adams, Boston, MA, USA), each extended with a segment of silastic tubing (length 3 cm and 0.58 mm in internal diameter; Dow Corning, Midland, MI, USA), were used. The venous catheter was inserted to the level of the right atrium and the arterial catheter was advanced to the level of the aortic arch. Both catheters were tunneled subcutaneously and exteriorized. The catheters were filled with a mixture of 60% polyvinylpyrrolidone and heparin (1000 U/ml) to maintain patency and were closed with a metal pin. The rats were allowed for a minimum of 3 days to recover from the surgery before experiments.
Experimental design
After overnight fasting (10–12 h), the rats (n = 6–8/group) were subject to a 7-h i.v. infusion of either saline (SAL), Intralipid plus heparin (IH; 20% Intralipid plus 20 U/ml heparin at 5.5 µ l/min), IH plus sodium salicylate (SS; 7 mg/kg bolus plus 0.117 mg/kg per min), or sodium salicylate alone. The dose of sodium salicylate (Sigma–Aldrich) used in the study was derived from a previous in vivo study by Kim et al. (2001) in which SS treatment was shown to prevent FFA-induced insulin resistance in skeletal muscle. After 3 h of infusion, [3-3H] glucose (Perkin-Elmer, Boston, MA, USA) was initiated (8 µ Ci bolus followed by constant infusion at 0.15 µ Ci/min) to assess peripheral glucose utilization and endogenous glucose production (EGP). Hyperinsulinemic–euglycemic clamp was performed with tracer infusion during the last 2 h of the 7-h infusion period to assess hepatic and peripheral insulin sensitivity. During 30 min preceding the clamp (‘basal period’), measurements were taken at 10-min interval for plasma glucose, insulin, FFA, and [3-3H] glucose-specific activity. At the onset of the clamp, an infusion of porcine insulin at 5 mU/kg per min, resulting in plasma insulin levels in the postprandial range, was initiated. To maintain euglycemia during insulin infusion, a variable infusion of 20% glucose was given through the jugular catheter and adjusted according to glycemic determinations every 5 min. The glucose infusate was radiolabeled with 48 µ Ci/g [3-3H] glucose to maintain plasma glucose-specific activity constant. The total blood volume withdrawn was ~3.8 ml. After plasma separation, red blood cells were diluted at 1:1 ratio in heparinized saline (4 U/ml) and re-infused into the rats. At the end of the experiments, the rats were anesthetized with i.v. administration of ketamine:xylazine: acepromazine cocktail (87:1.7:0.4 mg/ml), immediately after which liver, soleus muscle, and white adipose tissue samples were freeze clamped with pre-cooled aluminum tongs, while infusions were maintained through the jugular vein.
Plasma assays
Plasma glucose was measured with a Beckman GlucoseAnalyzer II (Beckman, Fullerton, CA, USA). Plasma radioactivity from [3-3H] glucose was determined after deproteinization with Ba(OH)2 and ZnSO4, and subsequent evaporation to dryness. Aliquots of the [3-3H] glucose and of the tritiated glucose infusate were assayed together with the plasma samples (Lam et al. 2002). Insulin levels in plasma were determined by RIAs using kits specific for rat insulin (but with 100% cross-reactivity with porcine insulin used for infusion), as previously described (Lam et al. 2002). Plasma FFA levels were measured using a colorimetric kit from Wako Industrials (Richmond, VA, USA), as previously described (Lam et al. 2002).
Immunoprecipitation and western blot analysis
Liver samples (150 mg) were homogenized by hand-held glass homogenizer in buffer A (50 mM Tris–HCl (pH 7.5); 10 mM EGTA; 2 mM EDTA; 1 mM NaHCO3; 5 mM MgCl2; 1 mM Na3VO4; 1 mM NaF; 1 µ g/ml aprotinin, leupeptin, and pepstatin; 0.1 mM phenylmethylsulfonyl fluoride; and 1 µ M microcystin). The homogenates were centrifuged at 100 000 g for 1 h at 4 °C, and the supernatants were retained as the cytosolic fraction. The pellet was resuspended in buffer B (buffer A1% Triton X-100), homogenized by passing through a 23-gauge needle thrice, incubated for 15 min on ice, and centrifuged at 100 000 g for 1 h at 4 °C. The supernatant provided the solubilized membrane fraction. The purity of the cytosolic and membrane fractions was previously assessed by assaying glucose-6-phosphate dehydrogenase (Sigma) and 5'-nucleotidase activities (Sigma) respectively (Lam et al. 2002). The results showed that the index of purity of both fractions were > 90%. Homogenization of muscle (Wong et al. 2006) and fat (Beard et al. 2006) samples was performed as described previously. The protein concentration in all samples was determined by the detergent-compatible modified Lowry microassay, using a standardized assay kit.
Fifty micrograms of protein in liver, muscle, or fat samples were mixed with equal volumes of 3sample-loading buffer (6.86 M urea, 4.29% SDS, 300 mM dithiothreitol, and 43 mM Tris–HCl (pH 6.8)) and left at room temperature for 30 min. The mixture was then vortexed and subjected to SDS-PAGE (10% polyacrylamide). Following electrophoretic separation, protein was transferred to polyvinylidene fluoride membranes. The membranes were then incubated for 1 h at room temperature in Tris-buffered saline–Tween (TBST) containing 5% nonfat dried milk (pH 7.4). After the blocking step, membranes were incubated overnight in TBST plus 5% milk containing affinity-purified polyclonal antibody specific for I B (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a concentration of 1:2000 (note that hepatic I B content was measured in the cytosolic fraction). The same procedure was used to determine serine-phosphorylated and total IRS-1 and IRS-2 in the cytosolic fraction of liver and serine-phosphorylated IRS-1 in the soleus muscle and fat. Antibodies against IRS-1, Serine 307 IRS-1, IRS-2 (Upstate, Temecula, CA, USA), and Serine 233 IRS-2 (Labvision, Fremont, CA, USA) were added to the western membranes at dilutions of 1/500, 1/1000, 1/500, and 1/500 respectively.
For detection of tyrosine phosphorylation of IRS in the liver, proteins were extracted from rat liver tissue as previously described (Zinker et al. 2002). Liver lysates containing equal amounts of protein (1 mg) were subject to immunoprecipitation overnight at 4 °C with agarose-conjugated anti-IRS-1 antibody (Upstate) or with anti-IRS-2 antibody (Upstate) followed by 2-h incubation with Protein A/G PLUS-Agarose immunoprecipitation reagent (Santa Cruz) at room temperature. Immune complexes were collected by brief centrifugation (12 000 g) and washed four times with ice-cold PBS. The equivalent amount of protein samples was then resuspended in 1Laemmli sample buffer (2% SDS, 10% glycerol, 62.5 mM Tris (pH 6.8), 0.1% bromophenol blue, and 5% ß-mercaptoethanol), boiled for 5 min, and separated by SDS-PAGE (10% polyacrylamide) under reducing conditions, as described above. This was followed by immunoblotting with anti-phosphotyrosine antibody (Santa Cruz; 1/1000 dilution). Note that serine 473-phosphorylated Akt (antibody purchased from Cell Signaling (Beverly, MA, USA); 1/1000 dilution) was measured by western blotting using the same liver lysates that were used for immunoprecipitation of IRS-1 and IRS-2.
After washing thrice with TBST, membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Santa Cruz). The membranes were then washed several times with double-distilled water and developed using ECL reagent (Amersham Biosciences). The bands obtained from immunoblotting were quantified by scanning laser densitometry.
Calculations
Glucose turnover (rate of appearance of glucose determined with [3-3H] glucose) was calculated using Steele’s non-steady-state equation (Altszuler et al. 1956), taking into account the extra tracer infused with the glucose infusate (Finegood et al. 1987). In the basal state, the total rate of glucose appearance corresponds to the EGP. During the clamp, EGP was calculated by subtracting the exogenous glucose infusion rate from the total rate of glucose appearance. At steady state, glucose disappearance corresponds to the rate of glucose appearance and at euglycemia glucose disappearance corresponds to tissue glucose utilization as renal glucose clearance is zero. Data are presented as average values of the samples that were taken during the 30 min preceding the clamp and during the last 30 min of the clamp.
Statistical analysis
One-way ANOVA followed by Tukey’s post hoc test was used to compare differences between treatment groups for the following parameters: whole body insulin sensitivity, I B content, serine-phosphorylated IRS-1 and IRS-2, tyrosine-phosphorylated IRS-1 and IRS-2, total IRS-1 and IRS-2, and serine 473-phosphorylated Akt. For plasma glucose, FFAs, and insulin as well as EGP and peripheral glucose utilization, we used two-way ANOVA with Tukey’s post hoc test in order to compare results between treatment groups and, if necessary, to compare basal and clamp data within each group. Statistical calculations were performed using Statistica software (Statistical Analysis System, Cary, NC, USA). Significance was accepted at a P < 0.05.
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Results |
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shows plasma glucose, FFA, and insulin levels during the basal period and hyperinsulinemic–euglycemic clamp. Plasma glucose levels were not different between treatments during the basal period or the clamp. Plasma FFA levels that were measured during the basal period increased by ~100% in the IH and IH plus SS groups compared with the SAL group due to IH infusion (P < 0.01). As expected, plasma FFA levels were lower during the hyperinsulinemic clamp than during the basal period in all treatment groups due to the anti-lipolytic and fat esterification effects of insulin. Plasma insulin levels were not different between groups during the basal period and were markedly elevated during the clamp compared with the basal period in all treatment groups due to insulin infusion.
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). Salicylate co-infusion with IH prevented the IH-induced decrease in GIR (P < 0.01 versus IH). Salicylate alone did not have any effect.
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and 3A). During the clamp, we were able to maintain fluctuations in plasma glucose-specific activity from basal at low levels (SAL, 8%; IH, – 8%; IHSS, – 21%; SS, – 4%; average percentage difference between basal and clamp), which prevents significant errors in calculation of glucose production (Fisher et al. 1996). IH infusion decreased insulin-stimulated increase in peripheral glucose utilization during the clamp when compared with SAL infusion (P < 0.05; Fig. 2B). This IH-induced decrease was completely abolished by salicylate co-infusion (P < 0.05 versus IH). The insulin-stimulated increase in peripheral glucose utilization in the SS group did not differ from the SAL group.
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). Co-infusion of salicylate prevented IH-induced decrease in suppression of EGP during the clamp (P < 0.05 versus IH). Salicylate alone did not have any effect.
In order to assess the effect of salicylate on IKKß activity in the liver, we measured hepatic I B content (Fig. 4A), which is a marker of IKKß activation as I B upon phosphorylation by IKKß is targeted for degradation. IH infusion decreased hepatic I B levels (P < 0.05), which were restored to control levels by salicylate co-infusion. Salicylate alone did not have any effect on hepatic I B levels. IH infusion also decreased I B content in the soleus muscle (P < 0.05) and salicylate co-infusion reversed the IH-induced decrease in I B content (P < 0.05 versus IH; Fig. 4B). Again, salicylate alone did not have any effect on soleus muscle I B levels. In adipose tissue, however, IH infusion did not change I B content (Fig. 4C).
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; P < 0.05) and serine 233 phosphorylation of IRS-2 (Fig. 5B; P < 0.05) in the liver. Co-infusion of salicylate, however, completely reversed these effects of IH (P < 0.05 versus IH). IH infusion, however, did not change total IRS-1 and IRS-2 levels (data not shown) and salicylate alone did not have any effect. Furthermore, IH infusion dramatically increased serine 307 phosphorylation of IRS-1 in the soleus muscle (P < 0.01), which was completely reversed by salicylate co-infusion (P < 0.01 versus IH; Fig. 5C). IH did not alter serine 307 phosphorylation of IRS-1 in the adipose tissue (Fig. 5D).
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) and IRS-2 (Fig. 6B; both P < 0.05) and that salicylate co-infusion prevented the IH-induced decreases (both P < 0.05 versus IH).
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; P < 0.05 versus IH).
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Discussion |
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B (NF B) system, in various animal models of insulin resistance (Kim et al. 2001, Yuan et al. 2001, Itani et al. 2002, Arkan et al. 2005, Boden et al. 2005, Cai et al. 2005). However, it is unclear whether IKKß activation is causally involved in the impairment of hepatic insulin action caused by short-term elevation of FFA. Therefore, the present study in rats was performed to investigate whether inhibition of IKKß activity provides protection from FFA-induced hepatic insulin resistance. We demonstrate that treatment with high-dose sodium salicylate completely prevents hepatic insulin resistance caused by short-term lipid infusion in association with prevention of impairment of hepatic insulin signaling. As expected, i.v. infusion of IH markedly elevated plasma FFA levels. IH is a triglyceride emulsion containing heparin that is broken down to non-esterified fatty acids and glycerol in vivo by lipoprotein lipase. It is thus possible that glycerol derived from triglycerides by itself affects EGP measured in the present study; however, we have shown in a previous study (Lam et al. 2002) that glycerol infusion (5 mg/kg per min) resulting in plasma glycerol levels similar to that achieved by 7 h of IH infusion has no effect on EGP when compared with saline infusion either in the basal fasting state or during the hyperinsulinemic–euglycemic clamp.
Infusion rate of exogenous glucose during the clamp is an indication of whole body insulin sensitivity. Numerous studies (Boden 1996, Boden et al. 2001, 2005, Kim et al. 2001, 2004b, Lam et al. 2002, 2003, Yu et al. 2002) have shown that lipid infusion causes whole body insulin resistance and our results are consistent with these studies. The whole body insulin resistance caused by IH infusion was completely reversed with salicylate co-infusion, suggesting that the site of salicylate’s effect includes both liver and peripheral tissues. Using infusion of [3-3H] tracer, which enabled us to separately assess hepatic and peripheral insulin sensitivity, we show that IH infusion causes both hepatic and peripheral insulin resistance, as indicated by decreases in insulin-induced suppression of EGP from basal and in insulin-stimulated peripheral glucose utilization respectively. These results are in agreement with our previous findings (Lam et al. 2002). With salicylate co-infusion, IH-induced insulin resistance in both liver and periphery was completely prevented.
Consistent with the effect of salicylate to prevent IH-induced decrease in peripheral glucose utilization during the clamp, salicylate co-infusion prevented IH-induced decrease in I B content in parallel with reversal of IH-induced increase in serine 307 phosphorylation of IRS-1 in the skeletal muscle. In the adipose tissue, IH infusion did not alter I B content or serine 307 phosphorylation of IRS-1. Although FFA have been shown to decrease glucose uptake in association with the activation of IKKß and increased serine 307 phosphorylation of IRS-1 in 3T3-L1 adipocytes (Gao et al. 2004),the differencesin the experimental models used likely explains the discrepancies in the results. It is noteworthy that, in Kim’s study (Kim et al. 2001), short-term lipid infusion did not alter insulin-stimulated glucose uptake in white adipose tissue, which is in keeping with our findings. These results indicate that liver and skeletal muscle were selectively targeted by salicylate and that prevention of FFA-induced peripheral insulin resistance by salicylate is associated with the reversal of serine phosphorylation of IRS-1 in skeletal muscle.
As far aswe know, this study is the first to show that salicylate is effective in restoring hepatic insulin sensitivity in a model of acute FFA elevation. While recent studies (Yuan et al. 2001, Arkan et al. 2005) have demonstrated that IKKß deficiency is protective against insulin resistance caused by high-fat feeding or genetically induced obesity in rodents, there is an important distinction in the experimental model of insulin resistance used between these studies and our present study. High-fat feeding and genetically induced obesity usually result in chronically elevated FFA levels, which may induce insulin resistance via a different mechanism than acute FFA elevation. For instance, in high-fat-fed animals, inhibition of NF B appears to be sufficient to restore insulin sensitivity without inhibition of IKKß activity (Cai et al. 2005), suggesting that the insulin resistance is secondary to transcriptional effects of NF B, which may not be true after short-term lipid infusion. Furthermore, these models are associated with increased release of adipocyte-derived factors other than FFA, such as pro-inflammatory cytokines, tumor necrosis factor (TNF ), interleukin-1ß , and interleukin-6 (Hotamisligil & Spiegelman 1994, Rotter et al. 2003, Kim et al. 2004a, He et al. 2006, Jager et al. 2007) which are known to induce insulin resistance. Accordingly, in these studies, insulin resistance on which IKKß inhibition was shown to have a protective effect cannot be attributed only to FFA. Instead, the model of insulin resistance we used is similar to that used by Kim et al. (2001) in their investigation of salicylate’s effect on fat-induced insulin resistance in rat skeletal muscle. In this study, however, a high rate of insulin infusion (60 pmol/kg per min) used during the clamp completely suppressed EGP in all groups, potentially masking the effect of salicylate to prevent FFA-induced hepatic insulin resistance. Using a much lower rate of insulin infusion (30 pmol/kg per min) and maintaining plasma glucose-specific activity constant to avoid underestimation of glucose production during the clamp (Finegood et al. 1988), we were able to reveal the ability of salicylate to prevent hepatic insulin resistance caused by FFA.
Numerous recent studies (Kim et al. 2001, Arkan et al. 2005, Boden et al. 2005, Cai et al. 2005) have implicated activation of intracellular inflammatory pathway in fat-induced insulin resistance, although it is not clear whether this defect is due to the direct inhibitory effect of IKKß on insulin signaling or its indirect effect to promote NF B-mediated production of pro-inflammatory cytokines. Since short-term fat infusion can activate IKKß (Kim et al. 2001, Boden et al. 2005) and IKKß can directly phosphorylate serine residues of IRS-1 (Gao et al. 2002), which prevents tyrosine phosphorylation of IRS and thus insulin signaling (Peraldi & Spiegelman 1998), it is likely that salicylate prevents fat-induced hepatic insulin resistance directly through prevention of IKKß activity. In support of this notion, we found that salicylate completely prevents IH-induced decrease in hepatic I B content, which indicates increased IKKß activityas I B uponphosphorylation by IKKß is targeted fordegradation. Interestingly, salicylate alone had no effect on hepatic I B content in the absence of elevated FFA, presumably because the dose of salicylate we used was not sufficiently high to suppress IKKß activity beyond its baseline levels.
The preventive effect of salicylate on IH-induced IKKß activation occurred in association with prevention of IH-induced increase in serine 307 phosphorylation and decrease in tyrosine phosphorylation of IRS-1 in the liver (Figs 5A and 6A, respectively). While there are numerous serine residues on IRS that are associated with impaired insulin signaling, serine 307 appears to be a critical site (Birnbaum 2001). For instance, TNF , by triggering activation of JNK1, has been shown to increase serine phosphorylation of IRS-1 at 307 site, which decreases tyrosine phosphorylation of IRS-1 and thus impedes insulin signaling (Aguirre et al. 2000). It was recently shown that FFA cause insulin resistance through IKKß- and JNK1-medicated serine (307) phosphorylation of IRS-1 in 3T3-L1 adipocytes (Gao et al. 2004). Furthermore, serine 307 phosphorylation of IRS-1 caused by short-term fat infusion was associated with decreased tyrosine phosphorylation of IRS-1 and impairment of insulin signaling in rat skeletal muscle, although the serine kinase responsible was not identified (Yu et al. 2002). Our results show that salicylate also prevents IH-induced increase in serine 233 phosphorylation of IRS-2 (Fig. 5B), which has been linked to insulin resistance (Scioscia et al. 2006). Although it is still unknown whether serine 233 phosphorylation interferes with tyrosine phosphorylation of IRS-2, our finding of concomitant decrease in tyrosine phosphorylation of IRS-2 indicates that these processes may be linked.
In addition to defects in tyrosine phosphorylation of IRS, IH infusion was found to reduce serine 473 phosphorylation of Akt, which was prevented with salicylate co-infusion. Taken together, our results suggest that the effect of salicylate to restore hepatic insulin sensitivity is closely associated with its reversal offat-induced impairment of hepatic insulin signaling.
Although our findings confirm the ability of high-dose salicylate to inhibit IKKß , we cannot exclude the possibility that salicylate prevents IKKß-induced transcription of various pro-inflammatory cytokines, thereby inhibiting their autocrine effect to impair hepatic insulin signaling. However, relatively short exposure to elevated FFA may preclude IKKß-induced transcription of pro-inflammatory genes. Furthermore, while salicylate is a relatively weak inhibitor of cyclooxygenase (COX) due to a lack of acetyl group that is required for deactivation of the enzyme, it is nonetheless possible that salicylate had some effect in inhibiting COX-mediated prostaglandin production. However, given that neither a treatment with non-steroidal anti-inflammatory drugs, which are robust inhibitors of COX, nor COX deficiency prevented insulin resistance in Fao hepatoma cells and in obese mice (Yuan et al. 2001) respectively, it is unlikely that potentially inhibitory effect of salicylate on prostaglandin played a role in the present study.
In summary, the present study demonstrates that 1) a high-dose treatment with sodium salicylate prevents hepatic insulin resistance caused by short-term elevation of plasma FFA and 2) the effect of salicylate to restore hepatic insulin sensitivity occurs in association with prevention of FFA-induced hepatic IKKß activation and corresponding impairment of hepatic insulin signaling. The findings of this study suggest that IKKß is a causal mediator of hepatic insulin resistance induced by acutely elevated plasma FFA. Therefore, hepatic IKKß represents a potential therapeutic target to prevent or treat fat-induced hepatic insulin resistance and associated diseases.
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Acknowledgements |
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Received in final form 6 August 2007
Accepted 30 August 2007
Made available online as an Accepted Preprint 30 August 2007
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