Introduction
The association among obesity, insulin resistance, and type 2 diabetes mellitus is well documented,[1] and free fatty acids (FFAs) have been implicated as an important causative link among them.[2] An elevation of plasma FFAs has been reported to impair insulin action, to accelerate 汕-cell apoptosis, and might be a major risk factor for type 2 diabetes.[2, 3]
Almost 100 years ago, Williamson[4] showed that high-dose salicylate treatment reduces the severity of glycosuria in diabetic patients, and in 1957, Reid et al[5] further demonstrated that 10-14 days of aspirin treatment improves the results of oral glucose tolerance tests in diabetic patients. It has been reported recently that high-dose salicylate improves FFA-induced insulin resistance and 汕-cell dysfunction,[6, 7] but the mechanism remains uncertain. Previously we found that in insulin-resistant rats, the supplementation of sodium salicylate is associated with a reduction of plasma malondialdehyde (MDA), a marker of oxidative stress. To date, few studies have investigated the impact of salicylates on oxidative stress levels in animal models. While oxidative stress is associated with a wide variety of pathologies, including diabetes, cardiovascular disease, and cancer, diabetes mellitus is particularly strongly associated.[8] Thus, the objective of this study was to assess the impact of the anti-inflammatory drug sodium salicylate on insulin sensitivity and to explore the potential mechanism by which it improves hepatic and peripheral insulin resistance.
Methods
Animal models
Forty-eight normal male Wistar rats, weighing 230-260 g, were housed in the Department of Laboratory Animals, China Medical University (Shenyang, China). The rats were housed under controlled temperature (23 ⊥) and were exposed to a 12￣12-hour light-dark cycle with ad libitum access to water and standard rat chow. After 3-5 days of adaptation to the facility, the rats were anesthetized, and indwelling catheters were inserted as described previously.[9] The rats were allowed 3-4 days of postsurgical recovery before experiments.
Experimental design
The rats were fasted overnight for 14 hours and randomized to three groups, one of which received intralipid (20% intralipid+20 U/ml heparin, 5.5 µl/min; IH group, n=16), one was a saline-treated control (equal volume; SAL group, n=16), while another group received sodium salicylate (20% intralipid+20 U/ml heparin, 5.5 µl/min+sodium salicylate, 0.117 mg/kg per minute; IHS group, n=16). The duration of infusion in each group was 7 hours, and [6-3H] glucose (20 µCi, bolus+0.4 µCi/min infusion) was given during the last 2 hours of the experiment to assess endogenous glucose production (EGP) and glucose utilization (GU). Further, the rats were divided into 2 groups of 8 each: a basal infusion group and a hyperinsulinemic-euglycemic clamping group. Clamping was made to maintain blood glucose concentrations at 5.0 mmol/L during the last 2 hours, while steady-state human insulin infusion (5 mU/kg per minute) was given by infusing 20% glucose at a variable rate. Blood samples for testing glucose, insulin, FFAs, C-peptide, and [6-3H] glucose-specific activity were taken during the last 30 minutes. The total blood volume withdrawn was 3.0-3.3 ml during the basal experiment and 3.5-3.8 ml during the clamping experiment. After plasma separation, red blood cells diluted 1:1 in heparinized saline (4 U/ml) were reinfused into the rats. At the end of the experiment, liver and gastrocnemius samples were removed within 45 seconds of anesthetic injection while infusion was maintained through the jugular vein.
Laboratory methods
Plasma glucose was measured with the glucose oxygenase method (BIOSEN5030, Germany). Plasma radioactivity from [6-3H] glucose was determined after deproteinization with Ba(OH)2 and ZnSO4. The intra-assay coefficient of variation was 6.5%. Insulin and C-peptide levels were determined by radioimmunoassay (Beijing Furui Biological Engineering Co., China). The coefficients of variation were <8% and 10.5% respectively. Plasma FFA levels were measured using a colorimetric kit, MDA levels and glutathione peroxidase (GSH-PX) activity in the liver and muscle were also measured using colorimetric kits (Nanjing Jiancheng Institute of Bio-engineering, China).
Calculations
Glucose turnover (rate of appearance of glucose determined with [6-3H] glucose) was calculated using the steady-state formula.[10] Data were presented as average values in samples taken in the last 30 minutes of the experiment.
Statistical analysis
The data were expressed as mean㊣SD. All calculations were performed using the SPSS12.0 software package. Experimental results were analyzed using one-way ANOVA with a probability for type 1 error set at P<0.05.
Results
Plasma levels of FFAs, glucose, insulin, and C-peptide
IH elevated plasma FFA levels by 2-fold, and increased basal plasma insulin and C-peptide levels by 0.6 and 0.7-fold respectively. Plasma glucose levels were higher with IH vs. SAL infusion in the basal experiments but were maintained at 5.0 mmol/L during the clamping (Table). Sodium salicylate decreased FFAs slightly, significantly decreased basal plasma glucose level by 70%, and reduced basal plasma insulin and C-peptide levels by 39% and 32%, respectively (Table).
Hepatic glucose production (HGP)
In the basal state, IH increased HGP by 1.5-fold, while sodium salicylate decreased HGP by 16%. During the hyperinsulinemic-euglycemic clamping, HGP in the IH group was 2-fold that in the SAL group and the infusion of sodium salicylate resulted in a decrease of 20% in HGP.
Glucose utilization (GU)
Under basal state conditions, IH increased GU by 1.6-fold. GU was reduced by 20% with intralipid infusion during the clamping, as compared with that with SAL infusion. Sodium salicylate decreased GU by 18% in the basal state, and increased GU by 14% in clamping conditions, compared with the IH group.
MDA levels and GSH-PX activity in the liver and muscle
After intralipid infusion, MDA levels in the liver and muscle were increased by 2- and 4-fold, while GSH-PX activity decreased by 45% and 46%, respectively. Compared to the IH group, sodium salicylate treatment reduced MDA content in the liver and muscle by 63% and 66%, and elevated the GSH-PX activity by 35% and 37%, respectively (Figs. 1, 2).
Discussion
The elevation of plasma FFAs has been shown to impair insulin action and cause insulin resistance. Insulin resistance is a key etiological factor for type 2 diabetes mellitus. Additional 41 million people are prediabetic with a constellation of insulin resistance, hypertension, and dyslipidemia, which puts them at increased risk for cardiovascular morbidity and mortality.[11] Thus, there is an urgent need for effective interventions to prevent diabetes in insulin-resistant populations. In recent studies, the improvement of insulin resistance by anti-inflammatory salicylates has been investigated, but the molecular target remains uncertain. A better understanding of the mechanisms will be required to combat the epidemics of type 2 diabetes and cardiovascular diseases that are fueled by obesity-associated insulin resistance. In this study, the effects of FFAs on hepatic and skeletal muscle glucose metabolism were tested. In addition, we determined whether high-dose anti-inflammatory salicylates prevent FFA-induced alterations of insulin action and the biochemical mechanisms that underlie these effects.
In our animal model, IH elevated basal plasma FFAs to above the physiological range but within the FFA elevation seen in uncontrolled diabetes. The FFA levels in the clamping were lower than the basal FFA levels, which are consistent with the antilipolytic and FFA reesterification effects of insulin.[12] IH increased insulin and C-peptide levels in all groups, because of increased insulin secretion in the basal state and a decreased insulin clearance during the clamping.[13] Sodium salicylate down-regulated high FFA-induced endogenous insulin secretion, and decreased plasma glucose levels accordingly. Acetylsalicylic acid was reported to promote fatty acid oxidation and reduce the plasma FFA level.[14] However, we did not find a significant decrease of FFAs after sodium salicylate infusion, which may be due to the short infusion time.
Previous studies have reported that FFAs cause insulin resistance by increasing gluconeogenesis in the liver,[15] impairing the insulin-mediated suppression of HGP, and inhibiting insulin-stimulated glucose uptake in skeletal muscle.[16] The skeletal muscle is the major site for insulin-stimulated glucose disposal, and is the major target for peripheral insulin resistance.[17] Our study showed that FFAs induced hepatic insulin resistance by elevating HGP levels and induced peripheral insulin resistance by decreasing GU and metabolism. A 7-hour infusion of sodium salicylate resulted in significant improvements in insulin sensitivity, including a 20% decrease in HGP and a 15% increase in GU.
We found that IH increased MDA levels in the liver and skeletal muscle by 2- and 4-fold, and reduced the GSH-PX activity by 45% and 46%, respectively. MDA is a marker of oxidative stress, while GSH-PX reflects the capacity for elimination of free radicals. These results showed that FFAs are strongly associated with a persistent imbalance between the production of highly reactive molecular species and antioxidant defense.[18] It has been reported that the increased production of these active molecules or a reduced capacity for elimination causes abnormal changes in intracellular signaling and gene expression, ultimately resulting in a pathological situation that includes insulin resistance.[19] After administration of sodium salicylate, MDA levels in the liver and muscle decreased by 63% and 64%, and the GSH-PX activity increased by 35% and 37%, respectively. These results indicated that sodium salicylate significantly relieves the degree of oxidative stress in the liver and skeletal muscle. At the same time, it improved hepatic and peripheral insulin resistance by decreasing HGP and increasing GU. High doses of salicylate (4-10 g/d), including sodium salicylate and aspirin, have been used to treat inflammatory conditions such as rheumatic fever and rheumatoid arthritis. These high doses are thought to inhibit nuclear factor kappa B (NF-百B) and its upstream activator I百B kinase 汕 (IKK-汕), as opposed to working through cyclooxygenases, the classical targets of non-steroidal anti-inflammatory drugs.[20, 21] High doses of salicylates also lower blood glucose concentrations although their potential for treating diabetes has been all but forgotten by modern biomedical science. In this study, we found that the anti-inflammatory drug, sodium salicylate, relieved oxidative damage in the liver and skeletal muscle, and improved FFA-induced insulin resistance. Thus we presumed that sodium salicylate might inhibit IKK-汕- and NF-百B-mediated transcription, which in certain cells would enhance the production of low-level inflammatory cytokines, such as TNF-汐 and IL-6. It has been demonstrated that inflammatory cytokines increase the transcription and translation of reactive molecular species and activate some reactive molecular species.[22, 23] Ultimately, the anti-inflammatory drug sodium salicylate may improve insulin resistance through abating the degree of oxidative stress in the liver and skeletal muscle.
In summary, our data demonstrate that the short-term elevation of fatty acids induces insulin resistance by enhancing oxidative stress levels in the liver and skeletal muscle. Also, in this study we preliminarily assessed the efficacy of the anti-inflammatory drug sodium salicylate as a new treatment for insulin resistance. This effect was associated with at least one mechanism: Sodium salicylate reduced the degree of oxidative stress in the liver and skeletal muscle, and therefore improved hepatic and peripheral insulin resistance. IKK-汕 and NF-百B might provide a potential pathogenic link to oxidative stress.
Funding: This study was supported by a grant from the Bureau of Education of Liaoning Province, China (No. 20060999).
Ethical approval: Not needed.
Contributors: HB proposed the study and wrote the first draft. ZS analyzed the data. HP carried out the experiments. All authors contributed to the design and interpretation of the study and to further drafts. HP is the guarantor.
Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
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Received February 2, 2009
Accepted after revision November 7, 2009
