Differential effects of palmitate and palmitoleate on insulin action and glucose utilization in rat L6 skeletal muscle cells

Abstract

An increase in circulating levels of specific NEFAs (non-esterified fatty acids) has been implicated in the pathogenesis of insulin resistance and impaired glucose disposal in skeletal muscle. In particular, elevation of SFAs (saturated fatty acids), such as palmitate, has been correlated with reduced insulin sensitivity, whereas an increase in certain MUFAs and PUFAs (mono- and poly-unsaturated fatty acids respectively) has been suggested to improve glycaemic control, although the underlying mechanisms remain unclear. In the present study, we compare the effects of palmitoleate (a MUFA) and palmitate (a SFA) on insulin action and glucose utilization in rat L6 skeletal muscle cells. Basal glucose uptake was enhanced approx. 2-fold following treatment of cells with palmitoleate. The MUFA-induced increase in glucose transport led to an associated rise in glucose oxidation and glycogen synthesis, which could not be attributed to activation of signalling proteins normally modulated by stimuli such as insulin, nutrients or cell stress. Moreover, although the MUFA-induced increase in glucose uptake was slow in onset, it was not dependent upon protein synthesis, but did, nevertheless, involve an increase in the plasma membrane abundance of GLUT1 and GLUT4. In contrast, palmitate caused a substantial reduction in insulin signalling and insulin-stimulated glucose transport, but was unable to antagonize the increase in transport elicited by palmitoleate. Our findings indicate that SFAs and MUFAs exert distinct effects upon insulin signalling and glucose uptake in L6 muscle cells and suggest that a diet enriched with MUFAs may facilitate uptake and utilization of glucose in normal and insulin-resistant skeletal muscle.

Figures

Figure 1. Effect of MUFAs/PUFAs on basal and insulin-stimulated glucose uptake in L6 myotubes

Uptake of 2-deoxyglucose was assayed in L6 myotubes following (A) incubation with 0.75 mM palmitoleate for the times indicated, (B) incubation with palmitoleate for 16 h at the concentrations indicated, (C) incubation with 0.75 mM of oleate, palmitoleate, linoleate or linoleneate for 16 h, and (D) incubation with 0.75 mM palmitate and/or 0.75 mM palmitoleate for 16 h, followed by incubation in the absence or presence of insulin (100 nM) during the last 30 min of incubation with the fatty acids. Controls were treated with vehicle alone. Values are expressed as a fold change relative to the untreated control (values are means±S.E.M. of three separate experiments each performed in triplicate). *P<0.05 compared with the control (vehicle only); N.S., non-significant change.

Figure 2. Uptake of oleate in L6 myotubes

(A) L6 myotubes were treated with 0.75 mM oleate containing 0.1 μCi/ml [1-14C]oleate for the indicated times. Radioactivity in medium (supernatant) and lysates (cells) was measured as described in the Experimental section. Values are means±S.E.M. from six experimental determinations. *P<0.05 compared with the 2 h group (cells); #P<0.05 compared with the 2 h group (supernatant). (B) Cells were treated either with 0.75 mM oleate for 16 h or the medium was refreshed with 0.75 mM oleate every 4 h over a period of 16 h. Cells were then assayed for glucose uptake. *P<0.05 compared with basal.

Figure 3. Effects of palmitoleate on glucose oxidation, glycogen synthesis and phosphorylation of GS in L6 myotubes

L6 myotubes were pre-incubated in the absence or presence of 0.75 mM palmitoleate (PO) and then the medium was replaced with HBS buffer containing 5 mM glucose (2 μCi/ml [U-14C]glucose) in the absence or presence of insulin (Ins). Subsequently, the culture medium was processed for glucose oxidation (A) and cells were lysed for determination of glycogen synthesis (B), as described in the Experimental section. In (C), L6 cells were treated either with 100 nM insulin for 15 min or with 0.75 mM palmitoleate for 16 h, then lysed and immunoblotted with phospho-specific antibodies against GSK3β Ser9, GS Ser641–645 or total GS. Immunoblots are representative of three separate experiments. The lower panel shows the quantification of the immunoblots from three separate experiments (values are means±S.E.M.) *P<0.05 compared with the control (vehicle only).

Figure 4. Effects of palmitate and palmitoleate on the phosphorylation/activation status of PKB, GSK3 and p70S6K in L6 myotubes

L6 myotubes were pre-incubated with 0.75 mM palmitoleate and/or palmitate for 16 h prior to incubation with either 100 nM insulin for 15 min. Whole-cell lysates were prepared and immunoblotted with phospho-specific antibodies against PKB Ser473 (p-PKB Ser473), GSK3β Ser9 (p-GSK3β Ser9) and p70S6K Thr389 (p-p70S6K Thr389), and an antibody against total PKB. Immunoblots from three separate experiments were quantified and are shown in the right-hand panels (values are means±S.E.M.).

Figure 5. Effect of protein kinase and protein synthesis inhibitors on palmitoleate-induced glucose uptake in L6 myotubes

L6 myotubes were pre-incubated in the absence or presence of (A) 100 nM wortmannin (Wort), 10 μM LY-294002 (LY), 100 nM rapamycin (Rap), 50 μM SB-415286 (SB4), 10 μM SB-203580 (SB2), 10 μM PD-98059 (PD), 5 μM cycloheximide (ChX) or 100 μM genistein (Gen), or (B) with either 0.1 or 1 μM Ro 31.8220, for 15 min prior to incubation with (+) or without (−) 0.75 mM palmitoleate (PO) for a further 4 h prior to assaying 2-deoxyglucose uptake. Values are means±S.E.M. of at least three separate experiments, each performed in triplicate. *Significant (P<0.05) increase compared with the appropriate untreated control; #significant inhibition (P<0.05) compared with palmitoleate treatment alone.

Figure 6. Effects of palmitoleate upon the kinetics of 2-deoxyglucose uptake in L6 myotubes

2-Deoxyglucose transport was assayed over a range of extracellular glucose concentrations following cell treatment with (●) or without (○) palmitoleate (0.75 mM for 16 h). The data were subsequently subjected to non-linear regression curve-fitting analysis using GraphPad Prism software to determine the Vmax and Km. Palmitoleate induced a significant (2.4-fold) increase in Vmax from 924±153 to 2193±142 pmol/min per mg of protein, whereas the concentration at which transport was half-maximal (Km) was not altered significantly from 470±90 μM (values are means±S.E.M. from three separate experiments conducted in triplicate at each hexose concentration). *P<0.05 compared with the appropriate untreated control for each hexose concentration.

Figure 7. Effect of palmitoleate on GLUT1 and GLUT4 plasma membrane abundance

(A) L6 myotubes were incubated with either 0.75 mM palmitoleate for 16 h or 100 nM insulin for 30 min (basal, control cells were treated with vehicle alone). Following this incubation period, cells were harvested and total cell or plasma membrane fractions were isolated as described in the Experimental section. Membrane fractions were loaded on to SDS/acrylamide gels and immunoblotted with antibodies against GLUT1, GLUT4 and Na+/K+-ATPase. Immunoblots are representative of three separate experiments. (B) GLUT1 and GLUT4 immunoreactive bands from plasma membrane fractions were quantified, and the abundance expressed relative to α1 subunit of Na+/K+-ATPase, a plasma membrane protein not affected by fatty acid treatment. Values (expressed as a ratio) represent means±S.E.M. of three separate experiments. *P<0.05 compared with the untreated control (basal). PO, palmitoleate; Ins, insulin.

Figure 8. Effect of palmitoleate on SNAT2 and Me-AIB uptake

(A) L6 myotubes were incubated with either 0.75 mM palmitoleate for 16 h or with 100 nM insulin for 30 min (basal, control cells were treated with vehicle alone). Following this incubation period, cells were harvested and total cell or plasma membrane fractions were isolated as described in the Experimental section. Membrane fractions were loaded on to SDS/acrylamide gels and immunoblotted with antibodies against SNAT2 and Na+/K+-ATPase. (B) SNAT2 immunoreactive bands from plasma membrane fractions were quantified and the abundance expressed relative to α1 subunit of Na+/K+-ATPase, a plasma membrane protein not affected by fatty acid treatment. Values (expressed as a ratio) are means±S.E.M. from two experiments. *P<0.05 compared with untreated control (basal). PO, palmitoleate; Ins, insulin. (C) L6 myotubes were incubated with 0.75 mM palmitoleate for various times and then assayed for Me-AIB uptake as described in the Experimental section. Values are means±S.E.M. of three separate experiments, each performed in triplicate. N.S., non-significant change.