Carbon monoxide reverses diabetic gastroparesis in NOD mice - PubMed (original) (raw)

Carbon monoxide reverses diabetic gastroparesis in NOD mice

Purna C Kashyap et al. Am J Physiol Gastrointest Liver Physiol. 2010 Jun.

Abstract

Diabetic gastroparesis is associated with increased oxidative stress attributable to loss of upregulation of heme oxygenase-1 (HO1), with resultant damage to interstitial cells of Cajal and delayed gastric emptying. These changes can be reversed by induction of HO1. HO1 catalyzes the breakdown of heme into iron, biliverdin and, carbon monoxide (CO). The aim of this study was to determine whether inhalation of CO can mimic the protective effects of HO1. Nonobese diabetic (NOD) mice with delayed gastric emptying were treated with CO inhalation. Serum malondialdehyde was measured as a marker of oxidative stress. Gastric emptying of solids was measured using a [(13)C]octanoic acid breath test. Kit expression levels were determined in immunoblots of protein extracted from the external muscle layers of the gastric body and antrum. The effect of CO on oxidative stress and gastric emptying was also determined in the presence of HO activity inhibitor chromium mesoporphyrin. CO inhalation reduced oxidative stress, restored Kit expression and reversed delayed gastric emptying in diabetic NOD mice with delayed gastric emptying. CO inhalation maintained this effect in the presence of the HO activity inhibitor, chromium mesoporphyrin, also resulting in restoration of the delay in gastric emptying. CO inhalation mimics the protective effect of upregulation of HO1 and decreased oxidative stress, increased Kit expression, and restored delay in gastric emptying. This effect of CO was independent of HO activity, suggesting that its effects were downstream of HO1. CO represents a potential therapeutic option for treatment of diabetic gastroparesis.

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Figures

Fig. 1.

Fig. 1.

Cartoon of experimental plan for carbon monoxide (CO) treatment. Eight-week-old female nonobese diabetic (NOD)/ShiLtJ mice were received from Jackson laboratory, and 2 baseline gastric emptying readings were obtained for each mouse before development of diabetes. After onset of diabetes, gastric emptying (GE) was measured every week. Mice were considered to have delayed gastric emptying if two consecutive gastric emptying _t_1/2 values were greater than normal. Diabetic mice with delayed gastric emptying were assigned to 2 groups; one group (n = 5) was killed, and serum and gastric tissue was collected. The other group (n = 5) was assigned to receive CO inhalation 100 ppm for 6 h/day for a maximum period of 8 wk or less if the mouse had 2 consecutive normal gastric emptying _t_1/2. At the end of this period, mice were killed and gastric tissue and serum was collected.

Fig. 2.

Fig. 2.

Cartoon of experimental plan for CO inhalation with heme oxygenase (HO) inhibition. Eight-week-old female NOD/ShiLtJ mice were received from Jackson laboratory, and 2 baseline gastric emptying readings were obtained before the mice became diabetic. After development of diabetes, gastric emptying was measured weekly. After 2 wk of diabetes, mice either received daily intraperitoneal injections of chromium mesoporphyrin (CrMP), 3 μmol/kg per day dissolved in 0.25% ammonium hydroxide (n = 10), or daily intraperitoneal injections of 0.25% ammonium hydroxide (Vehicle; n = 5). All mice injected with CrMP developed delayed gastric emptying as confirmed by 2 consecutive gastric emptying readings; all mice injected with ammonium hydroxide alone had normal gastric emptying. Mice with delayed gastric emptying were either killed soon after development of delayed gastric emptying (n = 5), and serum was collected, or they were assigned to receive CO inhalation (100 ppm for 6 h/day) for a maximum period of 8 wk or less if the mouse had 2 consecutive normal gastric emptying _t_1/2 (n = 5). These mice continued to receive daily intraperitoneal injections of CrMP to continually inhibit HO activity. At the end of this period the mice were killed and serum was collected.

Fig. 3.

Fig. 3.

Reduced oxidative stress in mice treated with CO inhalation. Levels of oxidative stress as measured by serum malondialdehyde levels were measured from diabetic mice with delayed gastric emptying killed after development of delayed gastric emptying (untreated) and diabetic mice with delayed gastric emptying before CO inhalation and after CO inhalation. Open symbols represent individual values, whereas bars represent means ± SE, *P < 0.05, one-way ANOVA with Tukey's posttest.

Fig. 4.

Fig. 4.

Increased Kit expression in the stomach of mice treated with CO inhalation. Western blot analysis of Kit protein expression in the gastric body. A: representative images from Kit and GAPDH blots. B: relative protein expression (medians with interquartile ranges) obtained by densitometric analysis normalized to GAPDH. *P < 0.05, Mann-Whitney test. _X_-axis legends apply to both panels. Mr, relative molecular weight.

Fig. 5.

Fig. 5.

Gastric emptying normalizes following CO inhalation. Individual mean ± SE _t_1/2 values of gastric emptying for each mouse (open symbols) and the grouped data (bars) are shown. *P < 0.05, one-way ANOVA with Tukey's posttest. The 2 horizontal dashed lines indicate the normal range of gastric emptying.

Fig. 6.

Fig. 6.

Reduced oxidative stress in mice treated with CO inhalation in the presence of HO inhibition by CrMP. Serum levels of malondialdehyde were measured from CrMP-treated diabetic mice with delayed gastric emptying killed soon after development of delayed gastric emptying (untreated), with delayed gastric emptying before CO inhalation and after CO inhalation. Open symbols represent individual values, whereas bars represent means ± SE, *P < 0.05, one-way ANOVA with Tukey's posttest.

Fig. 7.

Fig. 7.

Gastric emptying normalizes following CO inhalation in the presence of HO inhibition by CrMP. Individual means ± SE _t_1/2 values of gastric emptying for each mouse (open symbols) and the grouped data (bars) are shown. *P < 0.05, one-way ANOVA with Tukey's posttest. The 2 horizontal dashed lines indicate the normal range of gastric emptying.

Fig. 8.

Fig. 8.

CrMP treatment inhibits HO activity. Individual values (open symbols) and means ± SE (bars) are shown. HO activity as measured by picomoles bilirubin per milligram of protein per hour. *P < 0.05, one-way ANOVA with Tukey's posttest.

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