Glucose-dependent insulinotropic polypeptide enhances adipocyte development and glucose uptake in part through Akt activation

Abstract

Background & aims: In addition to its role as the primary mediator of the enteroinsular axis, glucose-dependent insulinotropic polypeptide (GIP) may play a critical role in the development of obesity. The purpose of these studies was to characterize the effects of GIP and its receptor (GIPR) in adipocyte development and signaling.

Methods: Effects of GIP and GIPR on differentiated 3T3-L1 cells were analyzed using Western blot analysis, Oil-Red-O staining, cyclic adenosine monophosphate radioimmunoassay, immunofluorescence microscopy, and glucose uptake measurements.

Results: To determine whether GIP and GIPR are important components in adipocyte development, the expression profile of GIPR during differentiation was examined. GIPR protein expression was enhanced during the differentiation process, and coincubation with its ligand GIP augmented the expression of aP2, a fat cell marker. Conversely, the suppression of GIPR expression by a specific short hairpin RNA attenuated Oil-Red-O staining and aP2 expression, suggesting that the GIPR may play a critical role in adipocyte development. To investigate specific signaling components that may mediate the effects of GIP, we analyzed Akt, glucose transporter-4, and glucose uptake, all of which are modulated by insulin in fat cells. Like insulin, GIP induced the activation of Akt in a concentration-dependent manner, promoted membrane glucose transporter-4 accumulation, and enhanced [(3)H]-2-deoxyglucose uptake.

Conclusions: These studies provide further evidence for an important physiologic role for GIP in lipid homeostasis and possibly in the pathogenesis of obesity. Furthermore, our data indicate that the GIPR might represent a suitable target for the treatment of obesity.

Figure 1

GIPR differentiation. (A) Human and 3T3-L1 preadipocytes (Pre) were converted to differentiated fat cells (Dif). These samples, as well as isolated rat fat, were probed for GIPR protein using a polyclonal antibody. (B) 3T3-L1 preadipocytes were induced, and protein was extracted at the indicated time points for Western anaylsis of GIPR, phosphorylated-ERK1/2, PPARγ, and β-actin. Western blots representative of at least four independent experiments are depicted.

Figure 2

GIP enhancement of aP2 expression. 3T3-L1 cells were incubated with GIP, IBMX, dexamethasone, and insulin (1 – 0.01 μg/mL) for the first 4 days of the differentiation process. Cells were harvested on day 9 and protein extracted. Western blot analyses were performed with polyclonal aP2 and β-actin antibodies.

Figure 3

Effect of GIPR on preadipocyte differentiation. GIPR expression was repressed (GIPR-) by stably transfecting GIPR shRNA into 3T3-L1 preadipocytes. (A) Cells were harvested on day 0 (D0) and day 8 (D8), and the expression of GIPR, aP2, and β-actin protein was analyzed. (B) Oil-Red-O staining was performed on day 8 for parental 3T3-L1, negative control (GIPR+), and two GIPR shRNA (GIPR-) clones.

Figure 4

Effect of GIP on cAMP accumulation of in fat cells. Following a 10-min incubation under different conditions, cAMP extracted from both rat fat and differentiated 3T3-L1 cells was measured by radioimmunoassay. Basal signifies cAMP levels in the untreated sample sets. 1 μM isoproterenol (Iso) and 20 μM forskolin (Forsk) were used as positive controls. Similar results were obtained from at least two independent experiments. Data are expressed as the mean ±SE in pmol/mL.

Figure 5

Effects of GIP and insulin on Akt phosphorylation. (A) Rin 5AH cells (pancreatic islet β-cell line) were incubated in the presence of GIP (0.2 – 200 nM) and 10 nM insulin (Ins) for 10 min. Total protein lysates were extracted and probed for expression of the Ser 473 phosphorylated form of Akt (pAkt) and total β-actin. (B) Differentiated 3T3-L1 and human adipocytes were incubated with GIP (0.2 – 200 nM) and insulin (1 – 100 nM) for 10 min. Total protein lysates were extracted and probed for pAkt expression. (C) Differentiated 3T3-L1 adipocytes were initially treated for 30 min with 1 μM Wortmannin (Wort), followed by an additional 10-min incubation with 0.2 nM GIP, 2 nM GIP, or 10 nM insulin. Total protein lysates were extracted and probed for pAkt. Representative Western blots are depicted, and similar results were obtained from at least three independent experiments. (D) Negative control and GIPR repressed cells were treated with either 10 nM or insulin or GIP (0.02 – 20 nM) for 10 min and probed for pAkt and β-actin expression.

Figure 5

Effects of GIP and insulin on Akt phosphorylation. (A) Rin 5AH cells (pancreatic islet β-cell line) were incubated in the presence of GIP (0.2 – 200 nM) and 10 nM insulin (Ins) for 10 min. Total protein lysates were extracted and probed for expression of the Ser 473 phosphorylated form of Akt (pAkt) and total β-actin. (B) Differentiated 3T3-L1 and human adipocytes were incubated with GIP (0.2 – 200 nM) and insulin (1 – 100 nM) for 10 min. Total protein lysates were extracted and probed for pAkt expression. (C) Differentiated 3T3-L1 adipocytes were initially treated for 30 min with 1 μM Wortmannin (Wort), followed by an additional 10-min incubation with 0.2 nM GIP, 2 nM GIP, or 10 nM insulin. Total protein lysates were extracted and probed for pAkt. Representative Western blots are depicted, and similar results were obtained from at least three independent experiments. (D) Negative control and GIPR repressed cells were treated with either 10 nM or insulin or GIP (0.02 – 20 nM) for 10 min and probed for pAkt and β-actin expression.

Figure 6

Effects of GIP and insulin on the accumulation of GLUT-4 in the plasma membrane. (A) Differentiated 3T3-L1 adipocytes were incubated in the presence of either 20 nM GIP or 10 nM insulin with or without the GIP-specific antagonist ANTGIP (AG) for 1 h. Membrane fractions were extracted and probed for the accumulation of GLUT-4 protein. (B) 3T3-L1 preadipocytes were grown in chamber slides and differentiated, and probed for the accumulation of GLUT-4 protein. Cells were visualized using confocal laser scanning microscopy. Representative images are shown, and similar images were visualized in three additional independent experiments. (C) Differentiated 3T3-L1 adipocytes were pre-treated with 1 μM wortmannin for 30 min prior to additional incubation in the presence or absence of GIP. Fat cells were stained with polyclonal GLUT-4 primary and counterstained with FITC conjugated secondary antibodies.

Figure 6

Effects of GIP and insulin on the accumulation of GLUT-4 in the plasma membrane. (A) Differentiated 3T3-L1 adipocytes were incubated in the presence of either 20 nM GIP or 10 nM insulin with or without the GIP-specific antagonist ANTGIP (AG) for 1 h. Membrane fractions were extracted and probed for the accumulation of GLUT-4 protein. (B) 3T3-L1 preadipocytes were grown in chamber slides and differentiated, and probed for the accumulation of GLUT-4 protein. Cells were visualized using confocal laser scanning microscopy. Representative images are shown, and similar images were visualized in three additional independent experiments. (C) Differentiated 3T3-L1 adipocytes were pre-treated with 1 μM wortmannin for 30 min prior to additional incubation in the presence or absence of GIP. Fat cells were stained with polyclonal GLUT-4 primary and counterstained with FITC conjugated secondary antibodies.

Figure 7

Effects of GIP and insulin on [³H]-2-deoxyglucose uptake. Differentiated 3T3-L1 adipocytes were incubated under control conditions (U) or with 100 nM insulin, 20 nM GIP, or 20 nM GIP in the presence of ANTGIP (AG). Cells were lysed after the incorporation of [³H]-2-deoxyglucose, and equal amounts were counted in a liquid scintillation counter. Samples were assayed in duplicate. Data are expressed as nmol/mL per min (mean ±SE). * P ≤ 0.02 to U. ** P ≤ 0.02 to GIP.

Figure 8

Summary diagram. Overnutrition appears to increase GIP expression, which in turn enhances intestinal glucose absorption and insulin release. Our data suggest that GIP independently modulates signaling components that serve to further promote the storage of fat, thereby augmenting nutrient efficiency and potentially promoting obesity.