Heme oxygenase-1 protects interstitial cells of Cajal from oxidative stress and reverses diabetic gastroparesis

Abstract

Background & aims: Diabetic gastroparesis (delayed gastric emptying) is a well-recognized complication of diabetes that causes considerable morbidity and makes glucose control difficult. Interstitial cells of Cajal, which express the receptor tyrosine kinase Kit, are required for normal gastric emptying. We proposed that Kit expression is lost during diabetic gastroparesis due to increased levels of oxidative stress caused by low levels of heme oxygenase-1 (HO-1), an important cytoprotective molecule against oxidative injury.

Methods: Gastric emptying was measured in nonobese diabetic mice and correlated with levels of HO-1 expression and activity. Endogenous HO-1 activity was increased by administration of hemin and inhibited by chromium mesoporphyrin.

Results: In early stages of diabetes, HO-1 was up-regulated in gastric macrophages and remained up-regulated in all mice that were resistant to development of delayed gastric emptying. In contrast, HO-1 did not remain up-regulated in all the mice that developed delayed gastric emptying; expression of Kit and neuronal nitric oxide synthase decreased markedly in these mice. Loss of HO-1 up-regulation increased levels of reactive oxygen species. Induction of HO-1 by hemin decreased reactive oxygen species, rapidly restored Kit and neuronal nitric oxide synthase expression, and completely normalized gastric emptying in all mice. Inhibition of HO-1 activity in mice with normal gastric emptying caused a loss of Kit expression and development of diabetic gastroparesis.

Conclusions: Induction of the HO-1 pathway prevents and reverses cellular changes that lead to development of gastrointestinal complications of diabetes. Reagents that induce this pathway might therefore be developed as therapeutics.

Figure 1

Glucose and β-hydroxybutyrate levels and gastric emptying. Panels A and B shows the glucose and β-hydroxybutyrate levels in the different groups of mice. Boxes are medians with IQRs. Bars represent means ± SEMs while the symbols represent individual mice. * P <0.01, ** P <0.001, Kruskal-Wallis test with Dunn’s post-test for glucose levels, * P < 0.001, one-way ANOVA with Tukey’s post-test for β-hydroxybutyrate levels (n = 9 for non-diabetic controls and n = 6 for others). Panel C shows average gastric emptying curves for each group (solid lines with circles are controls, dashed lines with squares are diabetic animals). Panel D shows individual mean± SEM T_1/2 values foreach mouse and the grouped data. * P < 0.0001, Paired t-Test. Gastric emptying was accelerated after 2 weeks of diabetes as previously shown and delayed in the mice assigned to the delayed gastric emptying group. The two horizontal dashed lines indicate normal range of gastric emptying.

Figure 2

Western blot analysis of nNOS and Kit protein expression in the gastric body. Panel A shows representative images from nNOS, Kit and GAPDH blots. Predicted molecular weights are shown by arrows. Panel B shows relative protein expression (medians with IQRs) obtained by densitometric analysis normalized to GAPDH. nNOS protein expression was significantly decreased at 4–5 weeks and 10 weeks as well as in the mice with delayed gastric emptying. Kit expression was decreased only in the mice that developed delayed gastric emptying. Wilcoxon matched pairs test, * P < 0.05 (n=6). X axis legends apply to both panels.

Figure 3

Western blot analysis of nNOS, Kit protein from the gastric antrum. Panel A shows representative images and panel B the relative protein expression. Similar results were obtained as in the body with decreased nNOS protein expression after 2 weeks of diabetes and Kit expression was decreased only in the mice that developed delayed gastric emptying. Wilcoxon matched pairs test, * P < 0.05 (n=5). X axis legends apply to both panels.

Figure 4

Levels of oxidative stress as measured by serum malondialdehyde levels. Serum levels of malondialdehyde were measured from non-diabetic controls, diabetic NOD mice after 10 weeks of diabetes (mice resistant to development of delayed gastric emptying) and diabetic NOD mice with delayed gastric emptying. Bars are means ± SEM, one-way ANOVA with Tukey’s post-test, * P < 0.05, ** P < 0.001 (n = 9 for non-diabetic controls and n = 6 for the others).

Figure 5

Western blot analysis of HO1 protein from mouse gastric body and antrum using the same tissues as in Figures 2 and 3. Panel A shows representative HO1 immunoblots from gastric body and relative protein expression normalized to GAPDH from same blots as in Fig 2 (medians with IQRs). Panel B shows the corresponding blots and graphs from the antrum. HO1 protein expression was significantly increased in all mice at 4–5 weeks (and 2 weeks in the antrum) and in mice resistant to the development of diabetic gastroparesis (10 weeks of diabetes). However HO1 expression dropped back to baseline in all mice with delayed gastric emptying. Wilcoxon matched pairs test, * P < 0.05 (n=6).

Figure 6

Hemin treatment reversed delayed gastric emptying. Panel A shows time course of mean T_1/2 values before and after development of delayed gastric emptying (week 0) and after treatment with hemin (□-started 2 weeks after development of delayed gastric emptying) or vehicle (○). Panel A also shows the mean kinetics of gastric emptying taken from the last gastric emptying for each mouse in each group (dotted line: vehicle; solid line: hemin). Panel B shows T_1/2 values (means ± SEM) for the last gastric emptying for each mouse in each group (○ vehicle, □ hemin). Paired t-test, * P < 0.05 (n=5). Panel C shows HO activity (individual values and mean ± SEM, Paired t-test, * P <0.005, n=5). Panels D and E show representative blots and relative protein expression (median with IQRs) for Kit and nNOS (Wilcoxon matched pairs test, * P < 0.05, n=5). Panel F shows malondialdehyde levels (mean ± SEM. Paired t-test, * P <0.0005, (n=5). Hemin treatment returned Kit expression and nNOS expression to normal values, reduced oxidative stress and normalized gastric emptying in all mice treated. (DGE = delayed gastric emptying).

Figure 7

CrMP treatment resulted in delayed gastric emptying. CrMP treatment was started 2 weeks after development of diabetes. The layout of this figure is similar to figure 5. CrMP treatment decreased Kit expression, increased oxidative stress and delayed gastric emptying in all mice treated. nNOS expression was not changed by CrMP.

Figure 8

HO1 staining from whole mount tissues obtained from the gastric body. Panel A shows double-labeled whole mount tissues with antibodies to HO1 (red) and to F4/80 (green) from non-diabetic mice and panel B from diabetic mice resistant to development of delayed gastric emptying. The bottom images are the merged images. There was little HO1 expression in the non-diabetic mice. HO1 was up-regulated in the diabetic mice resistant to delayed gastric emptying and most of the HO1 co-localized in macrophages (F4/80 positive cells). Most but not all macrophages expressed HO1. Scale bar 100 μm for all images.