Oleic acid and peanut oil high in oleic acid reverse the inhibitory effect of insulin production of the inflammatory cytokine TNF-alpha both in vitro and in vivo systems

Abstract

Background: Chronic inflammation is a key player in pathogenesis. The inflammatory cytokine, tumor necrosis factor-alpha is a well known inflammatory protein, and has been a therapeutic target for the treatment of diseases such as Rheumatoid Arthritis and Crohn's Disease. Obesity is a well known risk factor for developing non-insulin dependent diabetes melitus. Adipose tissue has been shown to produce tumor necrosis factor-alpha, which has the ability to reduce insulin secretion and induce insulin resistance. Based on these observations, we sought to investigate the impact of unsaturated fatty acids such as oleic acid in the presence of TNF-alpha in terms of insulin production, the molecular mechanisms involved and the in vivo effect of a diet high in oleic acid on a mouse model of type II diabetes, KKAy.

Methods: The rat pancreatic beta cell line INS-1 was used as a cell biological model since it exhibits glucose dependent insulin secretion. Insulin production assessment was carried out using enzyme linked immunosorbent assay and cAMP quantification with competitive ELISA. Viability of TNF-alpha and oleic acid treated cells was evaluated using flow cytometry. PPAR-gamma translocation was assessed using a PPRE based ELISA system. In vivo studies were carried out on adult male KKAy mice and glucose levels were measured with a glucometer.

Results: Oleic acid and peanut oil high in oleic acid were able to enhance insulin production in INS-1. TNF-alpha inhibited insulin production but pre-treatment with oleic acid reversed this inhibitory effect. The viability status of INS-1 cells treated with TNF-alpha and oleic acid was not affected. Translocation of the peroxisome proliferator- activated receptor transcription factor to the nucleus was elevated in oleic acid treated cells. Finally, type II diabetic mice that were administered a high oleic acid diet derived from peanut oil, had decreased glucose levels compared to animals administered a high fat diet with no oleic acid.

Conclusion: Oleic acid was found to be effective in reversing the inhibitory effect in insulin production of the inflammatory cytokine TNF-alpha. This finding is consistent with the reported therapeutic characteristics of other monounsaturated and polyunsaturated fatty acids. Furthermore, a diet high in oleic acid, which can be easily achieved through consumption of peanuts and olive oil, can have a beneficial effect in type II diabetes and ultimately reverse the negative effects of inflammatory cytokines observed in obesity and non insulin dependent diabetes mellitus.

Figure 1

Insulin production by INS-1 cells treated oleic acid. (A) INS-1 cells were cultured (11 mM glucose) with varying concentrations of oleic acid for 18–24 hrs at 37°C. Shown is one representative experiment with triplicate values out of three independent experiments. *, p < 0.05 for untreated cells compared to cells treated with 10 μM and 5 μM oleic acid. (B) INS-1 cells were cultured (25 mM glucose) with varying concentrations of oleic acid for 18–24 hrs at 37°C. Shown is one representative experiment with triplicate values out of three independent experiments. *, p < 0.05 for untreated cells compared to cells treated with 10 μM and 5 μM oleic acid.

Figure 2

Increased insulin production by INS-1 cells treated with TNF-α and oleic acid. (A) INS-1 cells were pre-treated for two hours with varying concentrations of oleic acid in medium containing 11 mM glucose, followed by TNF-α (100 pg/mL) treatment for 18–24 hrs at 37°C. Shown is one representative experiment with triplicate values out of three independent experiments.*, p < 0.05 for cells treated with TNF-α compared to cells treated with TNF-α and 10 μM or 5 μM oleic acid. (B) Cells were pre-treated for two hours with varying concentrations of oleic acid in medium containing 25 mM glucose, followed by TNF-α (100 pg/mL) treatment for 18–24 hrs at 37°C. Shown is one representative experiment with triplicate values out of three independent experiments. *, p < 0.05 for cells treated with TNF-α compared to cells treated with TNF-α and 10 μM or 5 μM oleic acid.

Figure 3

TNF-α or oleic acid treatment has no apoptotic inducing effect on INS-1 cells. Cells were pre-treated with low (1 μM) and high (10 μM) oleic acid, low (100 pg/mL) and high (1,000 pg/mL) TNF-α or pre-treated for 2 hours with oleic acid (10 μM) and subsequently treated with TNF-α (100 pg/mL). Twenty-four hours later the apoptotic status of cells was assessed using flow cytometry (Annexin V/PI staining). Results are expressed as percent of apoptotic/necrotic cells (upper right and lower right quadrant). Shown is one representative experiment performed in duplicate out of three independent experiments.

Figure 4

(A) Oleic acid treatment does not increase intracellular cAMP in INS-1 cells. Cells were treated with oleic acid at 10 μM, 5 μM, 1 μM and 0.1 μM for 15 min. Cellular homogenates were subjected to cAMP assay. Shown is one representative experiment with triplicate values out of two independent experiments. No significant difference was observed between controls and treatments. (B) Oleic acid induces translocation of PPAR-γ in INS-1 cells. Cells were treated with oleic acid at 10 μM, 5 μM, 1 μM, TNF-α (100 pg/mL) or combination of TNF-α and oleic acid (10 μM) for 18–24 hrs. Nuclear extracts were subjected to PPAR-γ detection assay. Shown is one representative experiment with triplicate values out of three independent experiments. *, p < 0.05 for cells treated with oleic acid (10 μM) compared to control and combination of TNF-α (100 pg) with oleic acid (10 μM) compared to cells treated with TNF-α alone.

Figure 5

Fatty acid composition analysis of three peanut oil brands. One mL from each of the three oils was extracted twice with 1:1 (v/v) chloroform: methanol, 95% ethanol and 2:3 (v/v) ethanol: ether separately. Extracts were evaporated to dryness under nitrogen. The extracts were solubilized in 1.0 mL of 5% DMSO in phosphate buffered saline. Analysis was carried out by J. Leek Associates, Inc. Albany, GA, USA.

Figure 6

Robust decrease of blood glucose level in type type 2, but not in type 1 diabetic mice following 21 day peanut oil administration. Kunming mice were injected intraperitoneally with 40 mg/kg streptozocin to induce type1 diabetes. KKAy mice were maintained on a high fat diet for 10 days to induce type 2 diabetes. Once the mice were characterized to be diabetic, either type I or type II, 0.70 mL of peanut oil was administered by gavages to those mice for 21 days. Control mice were maintained on a standard diet and all other mice were maintained on a high fat diet. At the end of the experiments the fasting blood glucose levels were measured and expressed as mean ± SD (mM glucose) (p = 0.0004 between DM 2 and DM 2 + P. Oil; p = 0.0003 between control and DM 2 and p = 0.05 between control and DM 1).