High glucose and/or high insulin affects HIF-1 signaling by regulating AIP1 in human umbilical vein endothelial cells

Highlights

• The first demonstration of a rapid increment in AIP1 synthesis in cells exposed to high glucose.

• Insulin partially reverses the effect of high glucose on the expression of AIP1 and HIF-1α signaling.

• Evidence of a possible relationship between AIP1 and HIF-1α signaling under high glucose.

• AIP1 downregulation reduces apoptosis and promotes cell proliferation and migration along with tubule formation under high glucose.

• AIP1 might be a key molecule involved in disordered signaling and impaired function of cells in diabetes.

Abstract

Objective

The objective of this study was to explore the effects of high glucose/high insulin on AIP1 expression in HUVECs and the possible regulation of HIF-1α signaling by AIP1.

Methods

We investigated the expression of AIP1 and HIF-1α signaling in HUVECs at the levels of mRNA and protein following exposure to 30 mmol/L glucose (high glucose), 1 nmol/L insulin (high insulin), and the combination of the two (high glucose/high insulin). We detected changes in HIF-1α and VEGF expression with AIP1 siRNA interference by real-time PCR and western blotting. The CCK8 cell proliferation assay, the scratch/wound-healing assay, and flow cytometry were used to assess cell proliferation, migration and apoptosis, respectively. Matrigel was used to perform a tubule formation assay.

Results

Compared with 5.5 mmol/L glucose alone (control), high glucose, high insulin, and the combination of high glucose + high insulin increased AIP1 expression at 24 h at the mRNA and protein levels. High glucose, high insulin, and high glucose + high insulin decreased HIF-1α expression at the mRNA and protein levels. AIP1 knockdown significantly increased HIF-1α and VEGF expression at both the mRNA and protein levels in HUVECs under high glucose conditions. In the presence of high insulin, the effect of high glucose on target gene expression was altered. The downregulation of AIP1 promoted cell proliferation, migration, and tubule formation, and it decreased apoptosis.

Conclusions

High glucose increases AIP1 expression and decreases the expression of HIF-1α and downstream molecules. Decreased HIF-1α signaling may be regulated by increased AIP1 under high glucose.

Introduction

Diabetes mellitus is a chronic disease threatening the health of individuals worldwide [1]. Researchers have attempted to elucidate the molecular mechanism of impaired functioning of cells during diabetes and to identify novel targeted therapies. Apoptosis signal-regulating kinase 1-interacting (ASK1-interacting) protein-1 (AIP1), also known as DAB2-interacting protein (DAB2IP), which is a member of the Ras GTPase-activating protein family, has been implicated in cell growth inhibition and apoptosis [2], [3]. AIP1, via its Ras GTPase-activating protein (Ras-GAP) activity, inhibits Ras-mediated cell survival signaling, resulting in cell growth inhibition [4]. However, AIP1 functions as a positive regulator of apoptosis by mediating activation of the apoptotic kinase apoptosis signal-regulating kinase 1 (ASK1) [3]. Consistent with its role as an inhibitor of cell survival and growth, AIP1 expression is often downregulated in various human cancers [2], [5], [6]. Recently, it has been reported that AIP1 is recruited to the VEGFR2-PI3K complex, binding to both VEGFR2 and PI3K p85 during a late phase of the VEGF response, and that this activity leads to the inhibition of VEGFR2 signaling. Considering the critical role of VEGFR2 signaling in angiogenesis, the regulation of VEGFR2 activity/activation may represent an important mechanism for the control of angiogenesis [7]. Studies have suggested that AIP1 may function as a tumor suppressor gene. However, the effects of high glucose and insulin on AIP1 gene expression are still unknown.

Hypoxia-inducible factor-1 (HIF-1), which is a key transcription factor, is known to promote tissue vascularization by activating the transcription of genes encoding angiogenic factors [8]. HIF-1 is a heterodimer composed of the rate-limiting factor, HIF-1α, and constitutively expressed HIF-1β [9], [10]. During hypoxia, the regulatory subunit of the HIF-1 heterodimer, HIF-1α, is stabilized through a decrease in the activity of prolyl hydroxylases that target the protein for degradation [11]. The regulation of HIF-1α can also occur independent of the oxygen level in the environment. Under normoxia, HIF-1α can be activated by the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR or mitogen-activated protein kinase (MAPK) pathways initiated by growth factors, cytokines, and other signaling molecules [12], [13], [14]. Studies have demonstrated that PI3K/Akt signaling is required for VEGF expression through HIF-1 in response to growth factor stimulation and oncogene activation [12], [15]. Previous studies have shown that glucose and hyperglycemia impair the stability and function of HIF-1α [16], [17]. However, the molecular mechanisms underlying this impairment in HIF-1 signaling and its relation to AIP1 regulation have not been elucidated.

To define the roles of AIP1 and HIF-1 signaling in diabetes, we detected changes in AIP1 expression and HIF-1 signaling in cells exposed to high glucose, high insulin, and the combination of the two (high glucose + high insulin), which simulate conditions observed during the progression of type 2 diabetes. Therefore, we hypothesized that the inhibition of HIF-1 signaling activity in human umbilical vein endothelial cells observed in diabetes is a result of the regulation of AIP1.