Over-expression of NYGGF4 inhibits glucose transport in 3T3-L1 adipocytes via attenuated phosphorylation of IRS-1 and Akt

Abstract

Aim: NYGGF4 is a novel gene that is abundantly expressed in the adipose tissue of obese patients. The purpose of this study was to investigate the effects of NYGGF4 on basal and insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes and to understand the underlying mechanisms.

Methods: 3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1Myc/His B) or an NYGGF4 expression vector were differentiated into mature adipocytes. Glucose uptake was determined by measuring 2-deoxy-D-[3H]glucose uptake into the adipocytes. Immunoblotting was performed to detect the translocation of insulin-sensitive glucose transporter 4 (GLUT4). Immunoblotting also was used to measure the phosphorylation and total protein contents of insulin signaling proteins such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt, ERK1/2, p38, and JNK.

Results: NYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, p38, and JNK.

Conclusion: NYGGF4 regulates the functions of IRS-1 and Akt, decreases GLUT4 translocation and reduces glucose uptake in response to insulin. These observations highlight the potential role of NYGGF4 in glucose homeostasis and possibly in the pathogenesis of obesity.

Figure 1

Effect of NYGGF4 on glucose uptake. 3T3-L1 preadipocytes transfected with NYGGF4 or the empty vector (pcDNA3.1Myc/His B) were induced to differentiate. After serum starvation for 3 h, the cells were incubated with (black columns) or without (white columns) 100 nmol/L insulin for 30 min, followed by measurement of the 2-deoxy-D-[³H]glucose uptake. Values represent the means±SD from three independent experiments. ^fP=0.001<0.01 vs insulin-stimulated control (cells transfected with the empty vector).

Figure 2

Effect of NYGGF4 on GLUT4 translocation. 3T3-L1 preadipocytes transfected with NYGGF4 or the empty vector were induced to differentiate. Membrane proteins and total proteins were extracted from the differentiated cells incubated with or without 100 nmol/L insulin for 30 min. Immunoblotting was performed using antibodies against GLUT4. The results are representative of those obtained from three independent experiments.

Figure 3

Effect of NYGGF4 on insulin signaling transduction. 3T3-L1 preadipocytes transfected with NYGGF4 or the empty vector were induced to differentiate. After incubation with or without 100 nmol/L insulin for 30 min, the cell lysates were analyzed by SDS-PAGE, blotted onto a membrane, and then probed with antibodies against molecules involved in the insulin signal pathway. The results are representative of those obtained from three independent experiments. A–F represent the total protein concentration and phosphorylation level of AKT, ERK, p38, JNK, IRS-1, and IR, respectively.