Islet Endothelial Activation and Oxidative Stress Gene Expression Is Reduced by IL-1Ra Treatment in the Type 2 Diabetic GK Rat

Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra)

Expression of genes encoding factors involved in endothelium activation and inflammation in GK islets.

<p>Total RNA was extracted from freshly isolated islets of 2.5-month-old male Wistar and GK rats and quantitative RT-PCR was performed for the indicated genes and normalized to a housekeeping gene (rpL19 or Ef1a). Data are means±SEM of 5–6 different islet isolations per group except for caspase-1 and epoxide hydrolase-2 (n = 3).^*p<0.05 using Student's <i>t</i>-test.</p

Primer oligonucleotide sequences of selected genes.

<p>Primer oligonucleotide sequences of selected genes.</p

Proposed model illustrating the islet endothelial dysfunction and oxidative stress surrounding β cells in GK rats.

<p>Elevated glucose, FFA and possibly cytokines induce endothelial activation at the islet level by eliciting reactive oxygen species (ROS) production and cytokine/chemokine release by endothelial cells and vascular smooth muscle cells. Once released, chemokines (CXCL1, CCL2, CCL3) attract/retain immune cells (monocytes, neutrophils), which further induce ROS and cytokine production around β cells. In addition, cytokines as well as metabolic factors (high glucose and FFA) may act directly on β cells to increase intracellular cytokines and ROS production with consecutive antioxidant defense response. Antagonizing IL-1 by IL-1Ra may inhibit endothelial activation/dysfunction and subsequent immune cells attraction/activation. IL-1Ra may also blunt IL-1 signaling in β cells and subsequent ROS production and antioxidant defense response. CXCL1 (GRO1/KC, rodent equivalent of IL-8), CCL2 (MCP-1); CCL3 (MIP-1α); FFA (free fatty acids); IL-1β, interleukin-1β; IL-1Ra, interleukin-1 receptor antagonist; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α.</p

IL-1Ra treatment improves vascularization and reduces fibrosis in GK islets.

<p>Immunohistochemistry was performed for von Willebrand factor (VWF) (A) and fibronectin (B) in pancreas of young adult untreated Wistar and GK rats, and of s.c. saline- or IL-1Ra-treated-GK rats. The border of each islet is defined by the dashed line. As shown in panel A, VWF-labeled islets from untreated GK rats are extremely heterogeneous in terms of vascularization and extent of fibrosis, when compared to Wistar controls. In saline- and IL-1Ra-treated GK rats, immunolabeled islet area for VWF or fibronectin was quantified for each islet and expressed as to the corresponding islet surface (n = 3 GK rats for both treatment groups, n = 25–40 islets). Islets analyzed for quantification showed unchanged islet area between treatment groups. ^*p<0.05 using Student's <i>t</i>-test.</p

Metabolic data for 10-week-old control Wistar and diabetic GK male rats.

<p>Glucose, insulin, leptin, lipids, cytokine and chemokine levels were determined in serum. Alpha-tocopherol and homocysteine levels, and paraoxonase-1 (PON-1) activity were determined in plasma. Glutathione redox state (% of reduced glutathione (GSH)) and GSH content (Eq GSH) were determined in red blood cells (RBC). Glucose, insulin, leptin: n = 7 per group; lipids: n = 9 per group; cytokines/chemokines: n = 7 per group; α-tocopherol, glutathione redox state and GSH content (n = 7–13 per group) and homocysteine and PON-1: n = 7–8 per group. All parameters were assayed under fed conditions. FFA (free fatty acids); HDL: high density lipoproteins; GRO1/KC/CXCL1: rodent equivalent of IL-8; MCP-1/CCL2: monocyte/macrophage chemoattractant protein; MIP-1α/CCL3: macrophage inflammatory protein-1α; IL-6, interleukin-6. ^*p<0.05 versus age-matched Wistar group, as analyzed by Student's <i>t</i>-test.</p

IL-1Ra treatment reduces the expression of most of the selected genes for endothelial activation, oxidative stress, myeloid cells, and fibrosis in GK islets.

<p>Pancreatic islets were isolated from GK rats following 1-month-treatment with IL-1Ra by s.c. injections (GK saline n = 6, GK IL-1Ra (100 mg/kg/day), n = 5). For each animal, total RNA was extracted from isolated islets and quantitative RT-PCR was performed for the indicated genes, and expressed relative to GK saline. ^*p<0.05 using Student's <i>t</i>-test.</p