A role for phospholipase C activity in GLUT4-mediated glucose transport.

Overexpression of surrogate receptors [epidermal growth factor (EGF) receptor (EGFR) and platelet-derived growth factor receptor] in adipocytes has demonstrated that multiple signaling pathways may lead to GLUT4-mediated glucose uptake. These implicated pathways function independently of IRS-1 phosphorylation and PI3-kinase activation. In addition, we previously demonstrated that EGFR tyrosyl autophosphorylation is required to stimulate GLUT4-mediated glucose transport in 3T3-L1 adipocytes. This observation suggests that signaling molecules that are dependent on EGFR autophosphorylation, such as phospholipase C (PLC), may lie in the signaling pathway to glucose transport. As PLC has been implicated in glucose transport by several clinical and basic mechanistic studies, we investigated whether EGFR signaling may promote glucose transport via modulation of PLC activity. Activation of EGFR overexpressing 3T3-L1 adipocytes leads to a 3.4 +/- 1.2-fold stimulation of PLC activity over basal levels vs. only 1.06 +/- 0.01-fold stimulation by insulin. Pharmacological inhibition of PLC by 50 microM U73122 reduced phosphoinositide accumulation by 79.2 +/- 16.9% and resulted in a concomitant 56.0 +/- 12.7% decrease in EGF-induced glucose transport. This inhibition of glucose transport by U73122 was specific, because the inactive congener, U73343, failed to block EGF-induced glucose transport. Despite the low levels of insulin-induced PLC activity, insulin-stimulated glucose transport activity was similarly inhibited by U73122 (55.9 +/- 13.1% inhibition). Inhibition of PLC activation did not impair either EGF- or insulin-induced activation of glycogen synthase or incorporation of glucose into lipid, supporting the hypothesis that both EGF- and insulin-induced glucose disposal can be independent of GLUT4-mediated glucose transport. The diminution of glucose transport secondary to inhibition of PLC activity was reflected by a decrease in GLUT4 translocation to the plasma membrane upon either EGF or insulin stimulation. These results are consistent with either a permissive or an active role for PLC activity in the translocation of GLUT4 to the plasma membrane.

Fatty acid-induced insulin resistance in adipocytes.

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.

Epidermal growth factor induces glucose storage in transgenic 3T3-L1 adipocytes overexpressing epidermal growth factor receptors.

3T3-L1 adipocytes represent an established physiological model for studying glucose uptake and storage. Overexpression of epidermal growth factor (EGF) receptors in these cells (200,000-250,000 receptors per cell) confers EGF-inducible GLUT4-mediated glucose uptake (17). We now report that EGF receptor (EGFR)-mediated signals can induce incorporation of glucose into glycogen and lipids in these cells. Incorporation into lipids was stimulated to similar levels by insulin or EGF in adipocytes expressing full-length (wild type) EGFR (2.05 +/- 0.26-fold for insulin vs. 2.28 +/- 0.15-fold for EGF). EGF induced incorporation into glycogen at roughly 60% of the level of insulin (4.53 +/- 0.57-fold for insulin vs. 2.76 +/- 0.25-fold for EGF); this corresponded with similarly lower levels of glycogen synthase activation by EGF relative to insulin stimulation. EGFR kinase activity was required for induced storage because a kinase-inactive (M721) EGFR failed to stimulate glucose incorporation into glycogen or lipids. EGFRs that lack all or part of the unique EGFR COOH-terminal tail induced glucose incorporation at levels similar to that stimulated by full-length (wild type) EGFR. Thus, domains in the COOH-terminal tail of the EGFR, which are necessary for stimulating glucose transport, are not required for signaling EGF-induced glucose storage. EGF-induced glucose storage did not require de novo protein synthesis, suggesting that EGFR signaling uses existing pathways in the adipocytes. These data demonstrate that signaling pathways for EGFR-mediated glucose storage and GLUT4-mediated glucose transport diverge at the receptor level. Thus, EGF-induced glucose storage can be achieved in the absence of induced GLUT4-mediated glucose transport.