Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

Abstract

Glucagon-like peptide 1 (GLP-1) is a naturally occurring peptide secreted by the L cells of the small intestine. GLP-1 functions as an incretin and stimulates glucose-mediated insulin production by pancreatic beta cells. In this study, we demonstrate that exendin-4/GLP-1 has a cognate receptor on human hepatocytes and that exendin-4 has a direct effect on the reduction of hepatic steatosis in the absence of insulin. Both glucagon-like peptide 1 receptor (GLP/R) messenger RNA and protein were detected on primary human hepatocytes, and receptor was internalized in the presence of GLP-1. Exendin-4 increased the phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK-1), AKT, and protein kinase C zeta (PKC-zeta) in HepG2 and Huh7 cells. Small interfering RNA against GLP-1R abolished the effects on PDK-1 and PKC-zeta. Treatment with exendin-4 quantitatively reduced triglyceride stores compared with control-treated cells.

Conclusion: This is the first report that the G protein-coupled receptor GLP-1R is present on human hepatocytes. Furthermore, it appears that exendin-4 has the same beneficial effects in vitro as those seen in our previously published in vivo study in ob/ob mice, directly reducing hepatocyte steatosis. Future use for human nonalcoholic fatty liver disease, either in combination with dietary manipulation or other pharmacotherapy, may be a significant advance in treatment of this common form of liver disease.

Figures

Figure 1. Identification of GLP-1R on hepatocytes

A Primary human hepatocyte and HuH7 cells show the presence of GLP-1R by western blot analysis. Brain lysate was used as a positive control. B: Bioluminescence analysis for GPCRs demonstrates the presence of GLP-1R on HuH7 cells. Bars show % increase in bioluminescence in GLP-1R as compared with no- primary-antibody treatment. (Means ± SE; *p < 0.05 compared with control no- primary antibody monolayer). The experiment was repeated three times in triplicate.

Figure 2. GLP-1R is present on the plasma membrane and was internalized upon agonist stimulation

A Monolayers were stimulated with GLP or Exendin-4 for 5 min, 15 min, 30 min. (10 nM) as described in MATERIALS AND METHODS. Bars show % decrease in bioluminescence compared with unstimulated (no ligand) monolayer (means ± SE; *p < 0.05 compared with unstimulated monolayer). The experiment was repeated three times and compared with untreated controls and pre-immune serum treated controls. B: Cell fractionation of HuH7 monolayers was performed as described in MATERIALS AND METHODS. Samples from membrane (M), cytoplasm(C) and nuclear (N) fractions were subjected to Western blot analysis using anti-GLP-1R antibody (1:500). Blots were also probed for Na+-K+-ATPase, Lamin A/C and β-actin to confirm equal protein loading. The results are representative of 2 independent experiments. C: Confocal imaging of GLP-1R was performed on filter grown monolayers of HuH-7 cells. Cells were stained with rabbit polyclonal antibody against GLP-1R (1:200) followed by Alexa Fluor secondary antibody. Rhodamine (blue arrow) was used to stain the cytoskeleton. In 1) GLP-1R (yellow arrow) is seen localized to the membrane and in 2) upon agonist stimulation GLP-1R is decreased from the membrane.

Figure 3. Reduction of steatosis on exposure to Exendin-4

A HuH7 cells were treated with palmitic acid ( 400uM/l) and oleic acid ( 400uM/l) for 12 h under insulin-free conditions; and subsequently exposed to Exendin-4 (20nM) for 6 h. Figure 3a) shows Oil red O staining of HuH7 cells treated with FFA and Exendin-4. A marked increase in Oil red O stained droplets (red) are visible in the cells treated with FFA as compared with the non treated cells. On exposure to Exendin4 there is a significant loss of fat droplets (40X). B: Triglyceride assay was performed on HuH7 cell lysate after treatment with palmitic and oleic acid followed by exposure to Exendin-4 as described in MATERIALS AND METHODS. Bars show % increase in TG content and then % decrease on treatment with Exendin-4. (Means ± SE; *p < 0.05 compared with untreated steatotic cells). The experiment was repeated three times in triplicate and compared with FFA exposed and non Exendin4 treated controls. C.: HepG2 cells were grown in either control media or methionine-choline deficient (MCD) media. Cells were then treated with Exendin-4 for 24 h. Following treatment, intracellular lipids (polar and neutral) were stained with Nile Red (NR) (0.5μg/ml). Flow cytometry was performed as described in Materials and Methods. 3T3L1 cells were served as a positive control. The figure is representative of three independent experiments.

Figure 4. GLP-1R signals through key insulin signal transduction elements

HuH-7 cells were treated with GLP/Exendin-4 (10nM) following the time course indicated: 5, 15, 30, 60, 90 and 120 m and Western blot was performed. β-actin was used as loading control. A: Phosphorylation of PDK was induced. B–C: AKT phosphorylation was also increased in a time dependent manner as was the phosphorylation of PKC-ζ. All data presented are representative of the mean ± SE of at least 3 experiments *p<0.05 vs. basal or untreated.

Figure 5. Knockdown of GLP-1R by siRNA

HuH7 cells were transfected with siRNA (at 30nM) against GlP-1R, and Western blot analysis with β-actin serving as loading control was performed. A) Knockdown was achieved as compared to control with 30nM siGLP-1R. B) Transfected HuH7 cells were treated with Exendin-4 (10nM) for 60 min. siRNA GLP-1R abolished the Exendin-4 mediated-effects on PDK-1 and PKC-ζ. (B and C, respectively). These studies represent multiple independent experiments. (*p<.05 vs. control).

Figure 6. Proposed GLP-1R signal transduction scheme

In our previous work we demonstrated that GLP-1 or Exendin-4 increased cAMP production. Here we propose that the GLP-1 action shares key downstream components of the insulin signaling pathway, including PKCζ, which has been shown to be a key factor in NAFLD.