Up-regulation of the Ang II/AT1 receptor may compensate for the loss of gastric antrum ICC via the PI3k/Akt signaling pathway in STZ-induced diabetic mice

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Highlights

  • ICC number and mSCF were decreased in gastric antrum of STZ-induced diabetic mice.

  • Local RAS could be activated in the gastric antrum of STZ-induced diabetic mice.

  • Up-regulation of local RAS in gastric antrum may compensate for ICC loss in diabetic.

  • Ang II induced gastric smooth muscle cell proliferation and mSCF expression in vitro.

  • The effects of Ang II may be mediated by PI3k/Akt signaling pathway.

Abstract

The classic renin–angiotensin system (RAS) is a complex system in which angiotensin II (Ang II) has been identified as an important endogenous regulator that influences both smooth muscle contraction and cell growth. Although a local RAS is known to exist in the gastrointestinal tract, it is unclear whether Ang II is involved in the loss of gastric interstitial cells of Cajal (ICC) in diabetic mice. The present study was designed to investigate the effect of Ang II on ICC survival in streptozotocin (STZ)-induced diabetic mice. Western blot, immunofluorescence, isometric muscle recording, enzyme-linked immunosorbent assay (ELISA) and a cell counting kit-8 were used in this research. Our results demonstrate that the c-Kit and membrane-bound stem cell factor (mSCF) protein expression levels in gastric smooth muscle were decreased in STZ-induced diabetic mice. However, the angiotensin receptor type 1 (AT1R) expression levels in gastric smooth muscle and angiotensin-converting enzyme (ACE) expression levels in gastric mucosa were increased. The effect of Ang II on the tonic contraction of gastric smooth muscle was potentiated in diabetic mice, and the plasma Ang II level was enhanced. Ang II increased mSCF expression, cell proliferation, and Akt-Ser473 phosphorylation in cultured gastric smooth muscle cells (GSMCs). These effects were reduced by specific inhibitors ZD7155 (an AT1R antagonist) and LY294002 (a PI3-kinase inhibitor). Our results suggest that Ang II increases mSCF expression and cell proliferation in cultured GSMCs in a PI3K/Akt signaling-dependent manner. ACE and AT1R up-regulation in the stomach may help compensate for ICC loss in STZ-induced diabetic mice.

Introduction

Gastroparesis is a chronic disorder that is defined as the delayed gastric emptying of solids and liquids in the absence of physical obstruction (Parkman et al., 2004). Gastroparesis occurs in type 1 and type 2 diabetes mellitus patients (25%–55%) or can be idiopathic (Farrell and Keeffe, 1995, Kong et al., 1999, Parkman et al., 2011). Diabetic gastroparesis affects nutritional state, glycemic control and patient quality of life. Gastric motility requires interactions between smooth muscle, enteric and extrinsic autonomic nerves, and interstitial cells of Cajal (ICC) (Grover et al., 2011). Several factors contribute to gastroparesis, including acute hyperglycemia, dysregulation of gastrointestinal hormones, oxidative stress which induces the loss of neural nitric oxide synthase (nNOS) expression in the myenteric plexus and ICC network disturbances (Farrugia, 2008). Recent studies have been focused on the mechanism underlying ICC loss in diabetic gastroparesis.

ICC play critical roles in gastric motility, and damage to the ICC network is likely to contribute to gastropathy and gastroparesis (Vanormelingen et al., 2013). Stem cell factor (SCF)-Kit signaling is important for the maintenance of ICC phenotypes, proliferation, and differentiation (Torihashi et al., 1999). Membrane-bound SCF (mSCF) expressed by gastric smooth muscle cells (GSMCs) has been linked to smooth muscle myopathy and ICC depletion in murine diabetic gastroparesis. Exogenous SCF partially reverses the pathological changes in ICC in diabetic mice (Horváth et al., 2006).

With the exception of classical RAS, local RAS is present in various tissues, including kidney, brain, heart, and pancreas. Ang I can be converted to Ang II by angiotensin-converting enzyme (ACE) and then act on the angiotensin type 1 and 2 receptors (AT1R and AT2R) (de Gasparo et al., 2000, Touyz and Schiffrin, 2000). It is well known that angiotensin II (Ang II) is associated with increased gene expression and the production of growth factors, such as endothelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), and platelet activating factor (PAF) (Ohtsu et al., 2006, Pan et al., 2010). Therefore, we hypothesized that Ang II might be involved in the production of mSCF. Different signal transduction cascades have been implicated in Ang II-mediated cell growth and migration, such as the mitogen-activated protein kinase (MAPK) cascade and the phosphatidylinositol 3-kinase (PI3K) pathways (Chiou et al., 2011). It has been reported that hyperglycemia caused by streptozotocin (STZ) strongly increases AT1 receptor expression in rat kidney and bladder (Harrison-Bernard et al., 2002, Tobu et al., 2012), but reports detailing its actions in the gastrointestinal tracts of diabetic mice are lacking. Our previous study showed that Ang II is an excitatory regulator of gastric motility (Lu et al., 2011). The presence of Ang II receptors at various levels of the gastrointestinal tract has been demonstrated in human, guinea pig and rat (Ino et al., 2006, Wang et al., 2005, Ewert et al., 2006). Here, we investigate the role of ACE-Ang II-AT1R alterations in diabetic gastroparesis and the Ang II signaling pathways in the production of mSCF and ICC survival.

Section snippets

Ethics

This study was conducted strictly according to the recommendations of the Guide for the Care and Use of Laboratory Animals of the Science and Technology Commission of P.R.C. (STCC Publication No. 2, revised 1988). The protocol was approved by the Committee on the Ethics of Animal Experiments of Shanghai Jiaotong University School of Medicine (Permit Number: Hu 686-2009).

Animals and the STZ-induced diabetic mouse model

Adult male ICR mice aged 5 wks weighing 30 ± 2 g were used for this study. The mice were fasted overnight and randomly divided

Body weight and blood glucose

Mice were used at the 8th week after injection of STZ. The blood glucose levels of the diabetic mice (29.2 ± 1.4 mmol/L, n = 50) were approximately 3 times higher than those of control mice (6.8 ± 0.6 mmol/L, n = 50, p < 0.05). The body weight of diabetic mice (23 ± 1.4 g, n = 50) was significantly decreased in comparison to that of control mice (40 ± 1.2 g, n = 50, p < 0.05).

c-Kit and mSCF expression in ICC and gastric smooth muscles

The c-Kit was used as a marker of ICC to investigate whether ICC networks were disrupted in STZ-induced diabetic mice.

Discussion

It has been recognized that ICC are pacemaker cells that generate spontaneous electrical slow waves and mediate motor neurotransmission in the gastrointestinal tract (Farrugia, 2008). ICC loss is one of the histological findings in diabetic gastroparesis (Horváth et al., 2006, Ordög et al., 2000, Takayama et al., 2002). In our study, we confirmed that the number of ICC was dramatically decreased and that their networks were destroyed in STZ-induced diabetic mice (Fig. 1). ICC loss results from

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China, NSFS (13ZR1423100) and NSFC (31171107; 31071011 and 31271236) and 2014CB910303.

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