No abstract

Palmitate has been shown to stimulate glucose transport, translocation of GLUT4 and insulin receptor autophosphorylation in isolated rat adipocytes (Biochem Biophys Res Commun 177:343-49, 1991). Here we further characterize the ability of short-term treatment with free fatty acids to stimulate glucose transport in isolated rat adipocytes and demonstrate that prolonged treatment induces insulin resistance. Treatment of adipocytes for 15 min with 1 mM myristate (14:0), palmitate (16:0), or stearate (18:0) stimulates glucose transport by 119 +/- 33, 89 +/- 29, and 114 +/- 30%, respectively. In contrast, oleate (cis 18:1), 1), elaidate (trans 18:1), and linoleate (cis 18:2) do not stimulate glucose transport. Palmitate stimulates glucose transport in a concentration-dependent manner, demonstrating saturation at 1 mM and half-maximal stimulation at 0.25-0.5 mM. Prolonged treatment (4 h) of rat adipocytes with 1 mM palmitate induces insulin resistance. After a 4-h preincubation with palmitate (1 mM), insulin stimulates glucose transport in rat adipocytes by 4.4-fold +/- 0.8, vs. 8.8-fold +/- 0.8 in controls (n = 3). Palmitate-induced resistant cells demonstrated a 40% inhibition in maximal insulin responsiveness with little change in insulin sensitivity. Insulin binding is only slightly decreased (8%) in palmitate-pretreated cells. These studies indicate that saturated fatty acids stimulate glucose transport acutely and on prolonged exposure induce insulin resistance via a post-insulin binding defect. The underlying molecular mechanisms of insulin resistance induced by prolonged treatment with saturated fatty acids may now be investigated using this unique cellular model.

Elevated serum-free fatty acid (FFA) levels induce insulin resistance in whole animals and humans. To understand the direct mechanism by which FFAs impact insulin-responsive tissue, we have used our previously developed in vitro model of long-chain saturated fatty acids (LCSFA)-induced insulin resistance in adipocytes. In addition to explanted rat adipocytes, we now demonstrate that overnight exposure of 3T3-L1 adipocytes to 1 mM individually of the LCSFA palmitate, myristate, and stearate, leads to an approximately 50% inhibition of insulin-induced glucose transport. Insulin resistance can be accomplished at 0.3 mM palmitate, which is within the range ofpalmitate found in diabetic and obese individuals. This inhibition was noted within 4 h of exposure to FFA, which is comparable to in vivo lipid infusion studies. Initial LCSFA-induced resistance is specific to glucose transport and does not affect insulin stimulation of glucose incorporation into glycogen. In 3T3-L1 adipocytes overexpressing the EGF receptor, LCSFA exposure also specifically inhibited EGF-induced GLUT4-mediated glucose transport, but not EGF-induced glycogen synthesis. We find that LCSFA treatment did not impair insulin stimulation of GLUT4 translocation or exofacial presentation on the cell surface as determined by trypsin accessibility. Our results suggest that the initial direct effect of elevated LCSFA is to impair activation of GLUT4 transporter activity and that this effect is specific for glucose transport.

Insulin-stimulated glucose transport in adipocytes is mediated by the insulin receptor. To ascertain whether a related receptor could also trigger this response, the epidermal growth factor (EGF) receptor (EGFR) was introduced into adipocytes. 3T3-L1 fibroblasts were infected by a retroviral construct encoding either the full-length (WT) or a carboxy-terminal truncated (c'973) human EGFR; truncation of the amino acids distal to 973 removes all autophosphorylation motifs. After selection and conversion to adipocytes, the level of EGFR expression was retained in infectant adipocytes (150,000 and 250,000/cell, respectively), but not in the parental 3T3-L1 adipocytes (< 5000/cell). WT and c'973 EGFR exhibited ligand-dependent tyrosine kinase activity and stimulated mitogen-activated protein kinase activity equivalently; neither phosphorylated insulin receptor substrate-1. WT EGFR, but not c'973 EGFR, underwent ligand-induced autophosphorylation. EGF did not stimulate tyrosine phosphorylation of the insulin receptor or insulin receptor substrate-1. EGF had a minimal effect on glucose transport by parental 3T3-L1 adipocytes. Glucose transport in the WT EGFR adipocytes was stimulated equivalently by insulin and EGF; exposure to insulin and EGF in combination did not result in augmented transport. Glucose transport in the c'973 EGFR adipocytes was stimulated by insulin, but not by EGF. GLUT4 was translocated to the plasma membrane to a similar extent in response to insulin or EGF in the WT EGFR adipocytes; only insulin caused a significant GLUT4 translocation in the parental or c'973 EGFR adipocytes. These data suggest that the insulin and EGF signaling pathways that lead to glucose transport converge in these adipocytes down-stream of the insulin receptor, and that activation of this pathway requires signaling motifs in the carboxy-terminus of the EGFR. This model system represents a novel approach with which to dissect signal transduction pathways in terminally differentiated adipocytes.

Energy restriction is associated with decreased plasma insulin and glucose concentrations, whereas long-chain saturated fatty acids (LCSFA) are strongly associated with insulin resistance. Our hypothesis is that energy restriction reduces LCSFA associated with plasma lipids in adult aging rats. Plasma LCSFA associated with triglycerides (TG), nonesterified fatty acids and phospholipids, as well as glucose, insulin, free fatty acids, TG and adipocyte glucose transport and insulin-sensitive glucose transporter (GLUT4) content were determined in aging, energy restricted [ER; 60% of ad libitum (AL) intake] and AL rats. In ER rats, plasma glucose concentrations were lower than in AL rats at each age. In contrast, body weight and plasma TG concentrations increased with age in both groups, but especially in the AL rats. In AL rats, combined LCSFA associated with plasma lipids was greater than in ER rats (P < 0.0001). Adipocyte insulin-stimulated glucose transport decreased in both groups with age but was most severe in AL rats, whereas GLUT4 was reduced only in AL rats. In ER rats it is possible that decreased plasma LCSFA contribute to reduced blood glucose concentrations as well as increased adipocyte GLUT4 compared with AL rats.