163 Hyperglycemia-Induced Hyperplasia of Interstitial Cells of Cajal (ICC) and ICC Stem Cells (ICC-SC) Is Associated With Accelerated Gastric Emptying in Obese Diabetic LeprDb/Db Mice

Background & Aims: ICC depletion is the most common cellular change in diabetic gastroparesis. However, in up to 22% of diabetic patients with gastric symptoms, gastric emptying (GE) is accelerated, rather than delayed. The fate of ICC in these patients is unclear. Previously we reported ICC and ICC-SC hyperplasia in hyperinsulinemic, diabetic Lepr mice. Here, we studied the relationship between GE of solids and ICC/ICC-SC numbers, and investigated the contribution of insulin/IGF1-dependent Kit ligand (Kitl) expression and hyperglycemia-induced ERK MAPK activation to ICC/ICC-SC hyperplasia. Methods: Lepr (n=32) and agesexand strain-matched controls (n=30) were studied 13-65 weeks after the onset of diabetes. ICC and ICC-SC were quantified by flow cytometry. Serum insulin was measured by enzyme immunoassay. Oxidative stress was determined by measuring serum malondialdehyde (MDA) and gastric 8OHdG. GE of solids was analyzed by 13C breath test. Gene expression was studied by qRT-PCR and WB. Effects of high glucose were investigated in murine gastric ICC primary cultures, a conditionally immortalized cell line derived from murine gastric ICC (ICL2A), and in an ICC-SC line isolated from the mouse stomach (2XSCS2F10). ERKMAPK signaling was evaluated byWB and pharmacological inhibition of MAPKK with PD98059. Cell proliferation and apoptosis were determined byMTS assay andCaspase Glo-3/7 assay, respectively. Results: Leprmice had significantly higher blood glucose (median[IQR]: 515[462;577] vs. 124[115;134] mg/dL; P,0.001) and serum insulin levels. Serum MDA and gastric 8OHdG were moderately increased (2.4and 2.2-fold, respectively, P,0.001). In Lepr mice, ICC and ICC-SC increased 2.0±0.1-fold (P,0.001) and 1.5±0.3-fold (P,0.05), respectively, and GE was accelerated (T1/2: 67±10 vs. 100±11 min; P,0.05). GE was similarly accelerated in Kit mice with generalized ICC hyperplasia due to an activating Kit mutation. In Lepr mice, total/soluble Kitl mRNA and Kitl protein were reduced. High glucose (500 mg/dL) significantly increased the numbers of primary ICC and stimulated proliferation and ERK MAPK phosphorylation in ICL2A and 2XSCS2F10 cells. PD98059 (20 μM) inhibited cell proliferation induced by high glucose without affecting basal proliferation. In contrast, osmotic stress induced by mannitol had no effect on ICC numbers, cell proliferation and ERK MAPK phosphorylation. ICC and ICC-SC were resistant to apoptosis induced by hyperglycemia at levels seen in Lepr mice. Conclusions: In the absence of major oxidative stress, hyperglycemia induces ICCSC and ICC hyperplasia via the ERK MAPK pathway even in the presence of reduced Kitl signaling. ICC hyperplasia, in turn, leads to accelerated GE and may contribute to gastric symptoms in a subset of diabetic patients. Grant support: NIH DK58185, DK68055.