Inhibition of apoB secretion from HepG2 cells by insulin is amplified by naringenin, independent of the insulin receptor

Abstract

Hepatic overproduction of apolipoprotein B (apoB)-containing lipoproteins is characteristic of the dyslipidemia associated with insulin resistance. Recently, we demonstrated that the flavonoid naringenin, like insulin, decreased apoB secretion from HepG2 cells by activation of both the phosphoinositide-3-kinase (PI3-K) pathway and the mitogen-activated protein kinase/extracellular-regulated kinase (MAPK(erk)) pathway. In the present study, we determined whether naringenin-induced signaling required the insulin receptor (IR) and sensitized the cell to the effects of insulin, and whether the kinetics of apoB assembly and secretion in cells exposed to naringenin were similar to those of insulin. Immunoblot analysis revealed that insulin stimulated maximal phosphorylation of IR and IR substrate-1 after 10 min, whereas naringenin did not affect either at any time point up to 60 min. The combination of naringenin and submaximal concentrations of insulin potentiated extracellular-regulated kinase 1/2 activation and enhanced upregulation of the LDL receptor, downregulation of microsomal triglyceride transfer protein expression, and inhibition of apoB-100 secretion. Multicompartmental modeling of apoB pulse-chase studies revealed that attenuation of secreted radiolabeled apoB in naringenin- or insulin-treated cells was similar under lipoprotein-deficient or oleate-stimulated conditions. Naringenin and insulin both stimulated intracellular apoB degradation via a kinetically defined rapid pathway. Therefore, naringenin, like insulin, inhibits apoB secretion through activation of both PI3-K and MAPK(erk) signaling, resulting in similar kinetics of apoB secretion. However, the mechanism for naringenin-induced signaling is independent of the IR. Naringenin represents a possible strategy for reduction of hepatic apoB secretion, particularly in the setting of insulin resistance.

Fig. 1.

Multicompartmental modeling of apolipoprotein B-100 (apoB-100) synthesis and secretion from HepG2 cells. A schematic representation of the assembly and secretion of apoB-100-containing lipoproteins from hepatocytes (A). Secretion of apoB-100-containing lipoproteins requires the assembly of apoB, triglyceride (TG), free cholesterol, cholesteryl ester (CE), and phospholipid (PL) within the endoplasmic reticulum (ER). Microsomal triglyceride transfer protein (MTP) is an absolute requirement for apoB secretion, facilitating the transfer of TG, CE, and PL to apoB as well as accretion of ER luminal TG for subsequent transfer to apoB. Assembly and secretion of apoB-100 involves a) apoB mRNA transcription and translation, b) translocation of apoB-100 across the ER membrane, and either c) association of apoB-100 with core and surface lipid via MTP and transport through the secretory pathway into plasma, or d) intracellular degradation via the cytoplasmic proteasome or luminal proteases. A diagram of the multicompartmental kinetic model used for analysis of apoB secretion and intracellular degradation (B). Compartments 1 to 5 and 7 are within the HepG2 cell. Compartment 6 represents apoB in the culture media. Compartments 1 and 2 represent an intracellular pool of tracer and a delay compartment to allow for apoB synthesis after introduction of the tracer, respectively. Compartment 3 represents newly synthesized apoB. A parameter termed “Init” calculated by the model represents the amount of radioactive apoB-100 entering the system (initially compartment 3) required to achieve the tracer curve for total apoB (cell plus media) measured experimentally. ApoB-100 from compartment 3 may be transferred to compartment 4 and subsequently secreted. From compartment 4, apoB passes through a delay, compartment 5, before secretion into the media, compartment 6. ApoB may be degraded directly from compartment 3 by a rapid degradation pathway. A second, more slowly turning over pool of apoB destined for degradation is represented by compartment 7. The shaded compartments represent the compartments containing apoB radioactivity that were determined experimentally.

Fig. 2.

Naringenin does not signal through the insulin receptor (IR) or insulin receptor substrate-1 (IRS-1). HepG2 cells were incubated with DMSO (C), insulin (I) (100 nM) or naringenin (N) (100 μM) for 10, 20, 30, or 60 min in serum-free media. Cell lysates were immunoprecipitated with an anti-IR (β-subunit) (A) or anti-IRS-1 (B) antibody. Half of each sample was run on one of two separate 10% gels, transferred to a membrane, and then probed with either an anti-phospho-tyrosine antibody (p-Tyr) (A and B), an anti-IR (β-subunit) (A), or anti-IRS-1 (B) antibody. C: For IRS-1, the level of activation was determined by expressing the amount of phosphorylated protein relative to total protein. Values are the mean ± SEM and are presented as the ratio of the phosphorylated form to total protein (relative to DMSO control). *P < 0.05 compared with control.

Fig. 3.

Naringenin and insulin dose-dependently increase extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation. HepG2 cells in serum-free media were treated with DMSO (control) and 12.5, 25, 50, 100, or 200 μM naringenin for 30 min (A) or DMSO and 12.5, 25, 50, 100, or 200 nM insulin for 15 min (B). Total and phosphorylated ERK1/2 were measured by immunoblot analysis and presented as a ratio of the phosphorylated form to total ERK1/2 protein (relative to DMSO control). Values are the mean ± SEM for six experiments. Values with different letters are significantly different at P < 0.05.

Fig. 4.

Addition of naringenin to insulin enhances and prolongs ERK1/2 activation in HepG2 cells. HepG2 cells in serum-free media were incubated with DMSO, naringenin (closed triangle), insulin (closed square), or insulin and naringenin added simultaneously (open diamond) from 0 to 60 min. Total and phosphorylated ERK1/2 for incubations with 25 μM naringenin alone, 25 nM insulin alone, and insulin (25 nM) plus naringenin (25 μM) (A) and 100 μM naringenin alone, 100 nM insulin alone, and 100 μM naringenin plus 25 nM insulin (B) were measured by immunoblotting. C: HepG2 cells in serum-free media were pretreated for 30 min with DMSO or naringenin (100 μM) followed by the addition of 25 nM insulin for a subsequent 30 min (closed diamond). Cells were also treated with insulin alone (at both 25 nM, open squares and 100 nM, closed squares) or 100 μM naringenin alone (closed triangles) from 0 to 60 min. Total and phosphorylated ERK1/2 were measured by immunoblot analysis and presented as a ratio of phosphorylated form to total ERK1/2 protein (relative to DMSO control). Mean incremental area under the curve (AUC) ± SEM are presented for four experiments. *P < 0.05 for simultaneous addition of naringenin (25 μM) plus insulin (25 nM) compared with naringenin alone (25 μM) and insulin alone (25 nM). **P < 0.05 for simultaneous addition of 100 μM naringenin plus 25 nM insulin compared with naringenin alone (25 and 100 μM), insulin alone (25 and 100 nM), and the simultaneous addition of naringenin (25 μM) and insulin (25 nM). ^†P < 0.05 for naringenin (100 μM) (0–60 min) plus insulin (25 nM) (30–60 min) compared with insulin alone (25 nM) and naringenin alone (100 μM).

Fig. 5.

Naringenin sensitizes HepG2 cells to the effects of insulin on mRNA expression of both MTP and the LDLr and apoB-100 secretion. Cells were pretreated with or without naringenin (100 μM) for 30 min before being treated with 25 nM or 100 nM insulin for a combined time of 6 h (cross-hatched bars). Insulin alone (25 nM or 100 nM, striped bars) for 6 h and naringenin alone (100 μM, solid bars) are also shown. MTP (A) or LDLr (B) expression was determined by quantitative real-time RT-PCR and normalized to GAPDH. Values are the mean ± SEM for five experiments. C: HepG2 cells were incubated in LPDS media for 24 h in the presence of DMSO, naringenin alone, insulin alone, or the combination of naringenin plus insulin at the indicated concentrations. Media was collected and apoB-100 measured by immunoblotting. D: HepG2 cells in LPDS media were preincubated for 30 min with DMSO, the MEK1/2 inhibitor UO126 (10 μM), or its inactive isoform UO124 (10 μM) followed by a 23.5 h incubation with naringenin alone, insulin alone, or the combination of naringenin plus insulin at the indicated concentrations. Media apoB-100 was measured by immunoblotting. Values are the mean ± SEM for five experiments. Values with different letters are significantly different at P < 0.05.

Fig. 6.

Naringenin and insulin similarly inhibit the secretion of apoB-100 from HepG2 cells through a kinetically defined rapid intracellular degradation pathway. HepG2 cells were preincubated for 24 h in lipoprotein-deficient serum (LPDS) with naringenin (100 μM, triangles), insulin (100 nM, squares), or DMSO (control, circles). Cells were then incubated for a further 20 min in the absence (A, C, E) or presence (B, D, F) of 0.1 mM oleic acid prior to pulse labeling (10 min). Cells were then chased for a further 120 min in the presence of their respective treatments in LPDS or oleate-enriched media. Intracellular apoB-100 radioactivity is shown in panels A and B, apoB-100 radioactivity secreted into the media is shown in panels C and D, and total apoB-100 radioactivity (determined as the sum of apoB-100 in the media and in the cell) is shown in panels E and F. The symbols in each graph represent apoB-100 radioactivity measured experimentally and are expressed as the mean ± SEM for four experiments (LPDS) and five experiments (oleate). The curves in each graph are fits to the experimental data obtained from analyses using the multicompartmental model shown in Fig. 1.