Prevention of proliferative changes of forestomach mucosa by blood glucose control with insulin in alloxan-induced diabetic rats

Abstract

Diabetes mellitus is one of the risk factors for carcinogenesis. Recently we reported that alloxan induces squamous cell carcinoma (SCC) with coincidental inflammation, bacteria/fungal infections, and a severe diabetic condition. The present study was conducted to examine the effects of blood glucose control with insulin on the proliferative changes of the forestomach in alloxan-induced diabetic rats. Male 15-week-old WBN/Kob rats were divided into a control group of non-treated rats with naturally occurring diabetes after 40 weeks of age (non-treated group), alloxan-induced diabetic rats (AL group), and alloxan-induced diabetic rats given insulin implant treatment (AL + In group). The animals were sacrificed at 90 weeks of age for histopathologic examination. The blood glucose and urinary glucose level of the AL + In group fluctuated variously from high to normal levels compared with a constantly high level of AL (for 75 weeks) as well as in the non-treated group (for 50 weeks). The mucosal hyperplasia in the forestomach developed in 88.2% of the AL group and 37.5% of the non-treated group, but in only 10.0% of the AL + In group. SCC was only detected in 23.5% of the AL group. Hyperplastic changes were constantly accompanied by inflammation and fungal/bacterial infections in the AL and non-treated groups, whereas inflammation and fungal infection were completely suppressed in the AL + In group. These findings demonstrate that blood glucose control suppressed neoplastic changes in alloxan-induced diabetic rats. We postulate that inflammation together with bacterial/fungal infections under prolonged severe diabetic conditions play a pivotal role in carcinogenesis.

Figure 1

Experimental design.

Figure 2

(above): Change of average blood glucose levels in alloxan (AL) (n = 17), alloxan plus insulin (AL + In) (n = 10), non‐treated groups (n = 16). Arrows showed the time of insulin treatment. (below): Change of average body weight in AL, AL + In and non‐treated groups.

Figure 3

Hyperplasia of squamous cell epithelium in forestomach. (a) Squamous cell hyperplasia of forestomach mucosa was completely suppressed in the alloxan plus insulin (AL + In) group (hematoxylin and eosin [H&E] stain). (b) Hyperplasia of mucosal squamous epithelium in rats from the alloxan (AL) group. Moderate hyperplastic changes were detected in the forestomach in the AL group. Neutrophils accumulated in mucosal epithelial layer (arrowhead), and varying numbers of lymphocytes, plasma cells and macrophages infiltrated throughout the entire submucosal layer (H&E stain). (c) Squamous cell carcinoma (SCC) in the forestomach in the AL group. Well‐differentiated SCC focally invaded the submucosal layer (H&E stain). Scale bars, 200 µm.

Figure 4

Immunohistochemistry for Candida albicans. All fungi of various forms such as filamentous, yeast and mycelial form were periodic acid‐Schiff (PAS)‐positive in ulcerative areas of mucosa lumen (PAS stain). Inset: Fungi positive for C. albicans antigen by immunohistochemical staining.

Figure 5

Immunohistochemistry for cyclooxygenase‐2 (COX‐2). (a) COX‐2 was positive in the cytoplasm of spindle mesenchymal cells and inflammatory mononuclear cells that infiltrated the lamina propria near the lumen and in superficial mucosal cells (arrow) in squamous cell hyperplasia. (b) COX‐2 was only weakly positive in the cytoplasm of spindle mesenchymal cells in the alloxan plus insulin (AL + In) group lumen. Scale bars, 50 µm.

Figure 6

Proliferating cell nuclear antigen (PCNA)‐positive cell indices of the forestomach squamous epithelium in each group. PCNA‐positive index of the alloxan plus insulin (AL + In) group was significantly lower than that of the alloxan (AL) group. Values represent the mean ± SD. *P < 0.005.