An in vitro study of the effects of free fatty acids on insulin mediated glucose uptake and metabolism by myocytes and fibroblast derived adipocytes
The incidence of type 2 diabetes (T2D) is increasing at an alarming rate, especially in developing countries, including South Africa. In developing countries, changes in lifestyle, especially the increase in the consumption of a westernized diet, rich in fats and sugar, is associated with hyperlipidemia, obesity and development of T2D. Skeletal muscle and adipose tissues are the major tissues involved with post-prandial peripheral glucose disposal in response to insulin. Insulin resistance reduces the ability to clear glucose from the circulation resulting in hyperinsulinemia and hyperglycemia with the development of T2D. High levels of plasma free fatty acids (FFAs), particulary saturated FFAs such as palmitate, are associated with insulin resistance in muscle and adipose tissue. Monounsaturated FFAs, such as oleate and essential FFAs such as omega 3 and omega 6 are claimed to be beneficial and to improve insulin sensitivity. Aim This study aims to determine the in vitro effect of the free fatty acids (FFAs) palmitate, oleate, omega 3 and omega 6 on insulin-stimulated glucose metabolism in myocytes and adipocytes. Materials and methods Mouse C2C12 and rat L8 cells were differentiated into myocytes and myotubules by serum deprivation, while mouse 3T3-L1 fibroblasts were differentiated into adipocytes by culturing in 3-isobutyl-1-methylxanthine (IBMX), dexamenthasone and insulin. Myocytes and adpipocytes were cultured in DMEM containing 5.5 mM or 20 mM glucose supplemented with 0.75 mM of each of the respective FFAs (i.e. palmitate, oleate, omega 3 and omega 6) for 24 hours. Thereafter media was replaced with fresh media containing 0.75 mM of each of the FFAs with or without insulin stimulation in the last hour. Glucose uptake was measured using a 2-deoxy-[3H]-D-glucose method. Glycogen and glucose-6-phosphate (G6P) concentrations were determined using commercial kits. Glucose oxidation was measured by 14CO2 release from cells incubated with glucose D-[14C(U)]. Cell viability and mitochondrial dehydrogenase activity was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide method. Quantitative real-time PCR was used to measure messenger RNA levels of selected genes involved in the insulin signaling pathway. Results The different FFAs tested had varying effects in C2C12 and L8 myocytes, and 3T3-L1 adipocytes cultured in media containing 5.5 mM or 20 mM glucose. Palmitate decreased both basal and insulin-stimulated glucose uptake in C2C12 myocytes cultured in 5.5 mM and 20 mM glucose. Oleate decreased insulin stimulated glucose uptake in cells cultured in 5.5 mM glucose but had no effect at 20 mM glucose. The essential FFAs had no effect on glucose uptake. In L8 myocytes, palmitate decreased both basal and insulin-stimulated glucose uptake at 5.5 mM glucose, while only insulin-stimulated glucose uptake was reduced at 20 mM glucose. Neither oleate, omega 3 nor omega 6 affected basal glucose uptake in cells cultured in 5.5 mM or 20 mM glucose. However, at 20 mM glucose these three FFAs reduced insulin-stimulated glucose uptake. In contrast to myocytes, none of the FFAs affected basal or insulin-stimulated glucose uptake in 3T3-L1 adipocytes cultured at 5.5 mM glucose. However at 20 mM glucose only palmitate reduced both basal and insulin-stimulated. In terms of glucose metabolism, palmitate decreased basal and insulin-stimulated G6P concentrations in C2C12 myocytes cultured in 5.5 mM and 20 mM glucose. Oleate increased basal G6P concentrations at 5.5 mM glucose, but had no effect at 20 mM glucose. Omega 3 increased and omega 6 decreased basal G6P concentrations at both 5.5 mM and 20 mM glucose. At 5.5 mM glucose, oleate had no effect on insulin-stimulated G6P concentrations, while omega 3 and omega 6 decreased G6P concentrations. At 20 mM glucose these three FFAs increased insulin-stimulated G6P concentrations. In L8 myocytes palmitate, oleate and omega 3 reduced basal G6P concentrations, while omega 6 increased basal G6P concentrations when cultured in 5.5 mM glucose. At 20 mM glucose palmitate and oleate decresed both basal and insulin-stimulated G6P concentrations, while only insulin-stimulated G6P concentrations were decreased by omega 3 and omega 6. In 3T3-L1 adipocytes basal G6P was not affected by all the FFAs at 5.5 mM glucose. However, insulin-stimulated G6P was reduced by palmitate and omega 3. All of the FFAs increased basal G6P concentrations at 20 mM glucose in 3T3-L1 adipocytes. Only oleate decreased insulin-stimulated G6P at 20 mM glucose. In C2C12 myocytes none of the FFAs had an affect on basal or insulin-stimulated glycogen concentrations at 5.5 mM or 20 mM glucose. In L8 myocytes cultured in 5.5 mM glucose palmitate, oleate and omega 3 increased basal glycogen concentrations. Palmitate, oleate and omega 6 decreased insulin-stimulated glycogen concentrations. At 20 mM glucose, oleate, omega 3 and omega 6 increased basal glycogen concentrations while none of the FFAs had an effect on insulin stimuated glycogen concentration. In 3T3-L1 adipocytes cultured in 5.5 mM glucose only omega 3 and omega 6 reduced basal glycogen concentrations. None of the FFAs effected glycogen concentrations at 20 mM glucose. In C2C12 myocytes cultured in 5.5 mM glucose, none of the FFAs affected basal glucose oxidation. Palmitate decreased and oleate inceased insulin-stimulated glucose oxidation. In L8 myocytes only palmitate decreased basal glucose oxidation. Palmitate, oleate and omega 6 reduced insulin-stimulated glucose oxidation at 5.5 mM glucose. In 3T3-L1 adipocytes none of the FFAs affected basal glucose oxidation at 5.5 mM glucose. At 20 mM glucose all of the FFAs reduced both basal and insulin-stimulated glucose oxidation in all three cell lines used. In C2C12 myocytes, palmitate downregulated insulin-stimulated mRNA expression of Irs1, Pi3k and Glut4 although this was not statistically significant.
Diabetes -- South Africa, Ffree fatty acids, Insulin-stimulated glucose
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