Evidence coupling increased hexosamine biosynthesis pathway activity to membrane cholesterol toxicity and cortical filamentous actin derangement contributing to cellular insulin resistance

Abstract

Hyperinsulinemia is known to promote the progression/worsening of insulin resistance. Evidence reveals a hidden cost of hyperinsulinemia on plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP(2))-regulated filamentous actin (F-actin) structure, components critical to the normal operation of the insulin-regulated glucose transport system. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes PIP(2)/F-actin dysregulation and subsequent insulin resistance. Increased glycosylation events were detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nm insulin; 12 h) and in cells in which HBP activity was amplified by 2 mm glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP(2) and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP(2) corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP(2)/F-actin structure, pharmacological inhibition of the rate-limiting HBP enzyme [glutamine-fructose-6-phosphate amidotransferase (GFAT)] restored PIP(2)-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of GFAT was associated with a loss of detectable PM PIP(2) and insulin sensitivity. Even less invasive challenges with glucose, in the absence of insulin, also led to PIP(2)/F-actin dysregulation. Mechanistically we found that increased HBP activity increased PM cholesterol, the removal of which normalized PIP(2)/F-actin levels. Accordingly, these data suggest that glucose transporter-4 functionality, dependent on PIP(2) and/or F-actin status, can be critically compromised by inappropriate HBP activity. Furthermore, these data are consistent with the PM cholesterol accrual/toxicity as a mechanistic basis of the HBP-induced defects in PIP(2)/F-actin structure and impaired glucose transporter-4 regulation.

Fig. 1.

Insulin and GlcN induce similar changes in cellular O-linked glycosylation. After 36 h of incubation in DMEM containing 5.5 mm glucose, cells were left untreated (control) or treated overnight (12 h) with 5 nm insulin in the absence (12 h Ins.) or presence (12 h Ins. + DON) of DON. A subset of cells was treated with 2 mm glucosamine in the absence of insulin (12 h GlcN) for 12 h. Representative images of cells subjected to immunofluorescence (IF) microscopy with RL2 antibody (A) and quantitation (B) are shown. *, P < 0.05 vs. control; bar 1).

Fig. 2.

Acute insulin responsiveness is impaired similarly by hyperinsulinemia and GlcN. Cells were treated exactly as described in Fig. 1. After treatments, cells were washed, either left untreated (basal) or acutely (30 min) challenged with 100 nm insulin (30′ Ins.), and GLUT4 translocation (B) and glucose transport (A) were determined. Means (±se) from three to six independent experiments are shown. IF, Immunofluorescence. *, P < 0.05 vs. control; #, P < 0.05 vs. control 30′ Ins.).

Fig. 3.

Hyperinsulinemic and GlcN states induce a loss in PM PIP₂ detection and cortical F-actin. Representative immunofluorescent (IF) images of PIP₂ (panels 1–4) and WGA (panels 5–8) detected in PM sheets treated as described in the preceding figures are shown. Phalloidin stained F-actin and propidium iodide labeled nuclei in these cells (panels 9–12) are shown. Images are representative from eight to ten independent experiments.

Fig. 4.

Population-based LI-COR Odyssey analyses quantitate a loss in PM PIP₂ detection and cortical F-actin. For these analyses, PM sheets or cells on an entire 35-mm cell culture well were labeled as in Fig. 3 and the fluorescent signals in the entire well were quantitated using the LI-COR Odyssey system as described in Materials and Methods. PM PIP₂ (A) and F-actin (B) means ± se from three independent experiments are shown. *, P < 0.05 vs. control; #, P < 0.05 vs. 12 h Ins.).

Fig. 5.

PIP₂ add-back restores the GlcN-induced PIP₂ decrease and protects against GlcN-induced F-actin loss and insulin resistance. During the final 60 min of control or GlcN incubation, the medium was replaced with the same medium enriched with either histone H1 (A, panels 1, 2, 4, and 5; C and D, panels 1 and 2) or PIP₂/histone H1 (A, panels 3 and 6; C and D, panels 3). Representative immunofluorescent (IF) images of PM PIP₂ (A, panels 1–3) and WGA-stained PM (A, panels 4–6), and quantitation of PM PIP₂ (B) are shown. Phalloidin-stained F-actin and propidium iodide-stained nuclei in these cells (C), insulin-stimulated PM GLUT4 images (D), and GLUT4 quantitation (E) are shown. All microscope and camera settings were identical between groups and images and means ± se are from three to five independent experiments. *, P < 0.05 vs. control; #, P < 0.008 vs. 30′ Ins.).

Fig. 6.

Overexpression of GFAT reduces detectable cell-surface PIP₂ concomitant with a reduction in insulin-stimulated GLUT4-eGFP translocation. Differentiated 3T3-L1 adipocytes were electroporated with 50 μg GLUT4-eGFP cDNA and 200 μg GFAT cDNA empty vector or GFAT cDNA. The cells were allowed to recover for 16 h. Cells were subsequently incubated in serum-free medium for 2 h and then left untreated (basal, panels 1 and 2) or treated (30′ Ins., panels 3 and 4) with 100 nm insulin for 30 min. Cells were fixed, labeled for PIP₂, and subjected to confocal fluorescence microscopy. Representative images from three independent experiments are shown.

Fig. 7.

High glucose alone induces PIP₂/F-actin loss and insulin resistance in 3T3-L1 adipocytes that were cultured and differentiated in 5.5 mm glucose. Murine 3T3-L1 preadipocytes cultured and differentiated in DMEM containing 5.5 mm glucose were left untreated (5.5 mm Glu, panel 1) or treated overnight (16 h) with 25 mm glucose (25 mm Glu, panels 2 and 3) in the absence or presence of DON (panel 3). PM PIP₂ (A), F-actin (B), insulin-stimulated PM GLUT4 (C), and O-linked glycosylation (D) were determined and quantitated (E) as described in preceding figures. All microscope and camera settings were identical between groups, and representative images from three independent experiments are shown. Values are means ± se from three independent experiments. IF, Immunofluorescence. *, P < 0.05 vs. control).

Fig. 8.

Insulin- and glucosamine-treated cells display an increase in PM cholesterol, removal of which with βCD protects against PIP₂/F-actin loss and impaired insulin-stimulated glucose transport. PM cholesterol was determined in cells after 12 h Ins. and 12 h GlcN incubations in the absence or presence of DON as described in preceding figure legends (A). Cells were also exposed for 30 min to 2.5 mm βCD before PM cholesterol, PIP₂, and F-actin (B) or 2-DG transport (C) determinations. Values are means ± se from three to six independent experiments. *, P < 0.05 vs. control; #, P < 0.05 vs. 30′ Ins.-control).