Inhibition of gluconeogenesis in primary hepatocytes by stromal cell-derived factor-1 (SDF-1) through a c-Src/Akt-dependent signaling pathway

Abstract

Hepatic gluconeogenesis is elevated in diabetes and a major contributor to hyperglycemia. Stromal cell-derived factor-1 (SDF-1) is a chemokine and an activator of Akt. In this study, we tested the hypothesis that SDF-1 suppresses hepatic gluconeogenesis through Akt. Our results from isolated primary hepatocytes show that SDF-1alpha and SDF-1beta inhibited glucose production via gluconeogenesis and reduced transcript levels of key gluconeogenic genes glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Additionally, SDF-1alpha and SDF-1beta both inhibited activation of the PEPCK promoter. In examining the mechanism by which SDF-1 inhibits gluconeogenesis, we found that SDF-1 promoted phosphorylation of Akt, FoxO1, and c-Src, but did not activate insulin receptor substrate-1-like insulin. Blockade of Akt activation by LY294002, FoxO1 translocation by constitutively nuclear FoxO1 mutant, or c-Src activation by the chemical inhibitor PP2, respectively, blunted SDF-1 suppression of gluconeogenesis. Finally, our results show that knocking down the level of SDF-1 receptor CXCR4 mRNA blocked SDF-1 suppression of gluconeogenesis. Together, our results demonstrate that SDF-1 is capable of inhibiting gluconeogenesis in primary hepatocytes through a signaling pathway distinct from the insulin signaling.

FIGURE 1.

Glucose production via gluconeogenesis is decreased by SDF-1α or -β in hepatocytes. Primary hepatocytes were isolated from mouse and cultured as described under “Materials and Methods.” A, measurement of glucose production. Cells were pretreated with either SDF-1 (α or β) or insulin (control) at indicated concentrations in serum-free Dulbecco's modified Eagle's medium for 1 h, and then treated for 2.5 h with cAMP (10 μm) and dexamethasome (Dex, 50 nm) in serum- and glucose-free Dulbecco's modified Eagle's medium supplemented with gluconeogenic substrate sodium lactate (2 mm) in the continuous presence or absence of SDF-11α or -1β. The glucose production via gluconeogenesis was determined, calculated, and normalized to protein concentrations as detailed under “Materials and Methods.” Results presented represent mean ± S.E. of three independent experiments, each in triplicate. B, measurement of LDH. Levels of LDH in the supernatants of the cell cultures described above were determined as detailed in “Materials and Methods” and compared with the positive control (1% Triton X-100). ^**, p < 0.01; and ^***, p < 0.001 versus cells treated with cAMP/dexamethasome alone.

FIGURE 2.

Inhibition of gluconeogenic gene expression by SDF-1. A, primary hepatocytes were pretreated with SDF-1α and -1β at the concentrations as noted for 1 h, followed by treatment with cAMP (10 μm) and dexamethasome (Dex, 50 nm) in the continuous presence or absence of SDF-1α or -1β for 2 h. Levels of G6Pase and PEPCK gene transcripts were evaluated with TaqMan Real-time reverse transcriptase-PCR. Results presented represent mean ± S.E. of two independent experiments, each in duplicate. ^*, p < 0.05; ^**, p < 0.01; and ^***, p < 0.001 compared with treatment with cAMP/dexamethasome alone. B, the plasmid containing the PEPCK promoter and a luciferase reporter gene was introduced into Hep1c1c7 cells via transient transfection with Lipofectamine 2000 for 24 h and then stimulated with cAMP (10 μm) and dexamethasome (Dex, 50 nm) for 4 h in the presence or absence of a pretreatment with SDF-1α,-1β, or insulin as noted. The promoter activity was measured by luciferase assays and normalized to protein concentrations. ^***, p < 0.001 compared with treatment with cAMP/dexamethasome alone.

FIGURE 3.

SDF-1 inhibits glucose production through phosphorylation of Akt. A, primary hepatocytes were treated with SDF-1α, SDF-1β, or insulin at noted concentrations for 15 min. Subsequently, levels of phospho-Akt (at serine 473 or serine 308) and total Akt were evaluated by immunoblottings with specific antibodies. B, PI 3-kinase inhibitor LY294002 prevented SDF-1β phosphorylation of Akt. Primary hepatocytes were pretreated with LY294002 for 30 min followed by SDF-1β treatment for 15 min. Akt phosphorylation at serine 473 was detected by immunoblotting with specific antisera. C, SDF-1β suppression of glucose production was prevented by LY294002. Primary hepatocytes were treated with 1 μm LY294002 for 1 h prior to the treatment with cAMP/dexamethasome in the presence or absence of SDF-1β for 2.5 h. Glucose production was subsequently evaluated as detailed under “Materials and Methods.” D, SDF-1β reduction in levels of key gluconeogenic gene transcripts was prevented by LY294002. Primary hepatocytes were pre-treated with 1 μm LY294002 for 1 h, and then treated with cAMP/dexamethasome for 4 h. Levels of PEPCK and G6Pase transcripts were measured by real-time PCR. Results represent three independent experiments. ^*, p < 0.05; ^**, p < 0.01; and ^***, p < 0.001 in comparison to the first lane.

FIGURE 4.

SDF-1 inhibition of glucose production is FoxO1-dependent. A, primary hepatocytes were treated with SDF-1α, SDF-1β, or insulin for 15 min. Levels of phospho-FoxO1 (at serine 256) and total FoxO1 were evaluated by immunoblottings with specific antibodies. The constitutively nuclear form of FoxO1 (FoxO1-ADA) or GFP encoded by adenoviruses (1–20 multiplicity of infection/well in 6-well plate) were introduced into isolated primary hepatocytes 36 h before the cells were treated with cAMP (10 μm)/dexamethasome (Dex) (50 nm) for 2.5 h in the presence of SDF-1β or insulin as noted. The expression level of the constitutively nuclear form of FoxO1 was determined by immunoblotting (B). Glucose production via gluconeogenesis was evaluated as detailed under “Materials and Methods” (C). Transcripts of G6Pase and PEPCK genes were quantified by TaqMan real-time PCR (D). Results represent three independent experiments. ^*, p < 0.05; and ^**, p < 0.01 in comparison to the first lane.

FIGURE 5.

SDF-1 suppresses hepatic gluconeogenesis through c-Src. Primary hepatocytes were treated with SDF-1 (α or β), macrophage inflammatory protein-1α (MIP) (negative control), or insulin (positive control) for 15 min. Levels of phospho-IRS-1 (at tyrosine 612) and total IRS (A), and phospho-c-Src (at tyrosine 416, activating) and total c-Src (B) were determined by immunoblottings with specific antibodies. NS, nonspecific bands. C, primary hepatocytes were pretreated with PP2 or PP3 (negative control) for 30 min, and then treated with cAMP (10 μm)/dexamethasome (Dex) (50 nm) in the continuous presence or absence of SDF-1β for 3 h. Sodium lactate (2 mm) was added to some cells as a substrate of gluconeogenesis. Glucose production via gluconeogenesis was quantified, calculated, and normalized to protein levels as detailed under “Materials and Methods.” Results represent mean ± S.E. of two independent experiments performed in triplicate. D, levels of PEPCK gene transcripts from these same cells were determined by real-time reverse transcriptase-PCR using specific TaqMan PCR probes. Results represent mean ± S.E. from two independent experiments performed in duplicate. ^*, p < 0.05 in comparison to the first lane. NS, non specific.

FIGURE 6.

Reduction of SDF-1 receptor (CXCR4) mRNA levels prevents SDF-1-induced suppression of hepatic gluconeogenesis. A, the cognate siRNA against CXCR4 (siCXCR4) or a scramble siRNA was introduced into primary hepatocytes by reverse transfection for 36 h as detailed under “Materials and Methods.” Levels of CXCR4 transcripts were evaluated by Real-time reverse transcriptase-PCR using the specific CXCR4 TaqMan PCR probe, and normalized to levels of β-actin transcripts. Cells were then treated with either SDF-1α (B) or SDF-1β (C) for 1 h prior to stimulation with cAMP (10 μ)/dexamethasome (Dex) (50 nm) in the continuous presence or absence of SDF-1 (α or β). Results represent mean ± S.E. of three independent experiments, each in triplicate. D, levels of PEPCK transcripts in cells that were similarly treated were evaluated using TaqMan real-time reverse transcriptase-PCR. Results represent mean ± S.E. of two independent experiments, each in triplicate.