Inducible Nitric Oxide Regulates Na-Glucose Co-transport in a Spontaneous SAMP1/YitFc Mouse Model of Chronic Ileitis

Abstract

In mammalian small intestine, glucose is primarily absorbed via Na-dependent glucose co-transporter (SGLT1) on the brush border membrane (BBM) of absorptive villus cells. Malabsorption of nutrients (e.g., glucose) leads to malnutrition, a common symptom of inflammatory bowel disease (IBD), where the mucosa is characterized by chronic inflammation. Inducible nitric oxide (iNO) is known to be elevated in IBD mucosa. SAMP1/YitFc (SAMP1) mouse is a spontaneous model of chronic ileitis that develops lesions in its terminal ileum, very similar to human IBD. How SGLT1 may be affected in SAMP1 model of chronic ileitis is unknown. Ten-week-old SAMP1 mice with AKR mice as control were treated with N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) to inhibit iNO production. Intracellular NO levels were found to be increased in villus cells from SAMP1 mice. Moreover, SGLT1 and Na^+/K⁺-ATPase activities and BBM SGLT1 expression were significantly decreased. However, L-NIL treatment reduced the intracellular iNO production, and reversed both downregulated SGLT1 and Na⁺/K⁺-ATPase activities in SAMP1 mice. Inhibition of iNO by L-NIL treatment also significantly reversed the BBM SGLT1 protein expression in SAMP1 mice. L-NIL reversed the inflammation mediated downregulation of SGLT1 activity by restoring the BBM SGLT1 expression. Thus, regulation of SGLT1 in chronic ileitis is likely mediated by iNO.

Keywords: L-NIL; SAMP1/YitFc; SGLT1; inducible nitric oxide; inflammatory bowel disease.

Figure 1

Representative example of cross section of the ileum of AKR and SAMP1 mouse stained with H&E. (A) AKR mouse intestine. (B) SAMP1 mouse intestine demonstrating crypt hypertrophy, villus blunting, and increased intra-epithelial lymphocytes characteristic of inflammatory bowel disease (IBD). Scale bar 100 µm.

Figure 2

Physiological consequences of chronic intestinal inflammation in SAMP1 mouse. (A) Nitric oxide (NO): Mucosal levels of NO are significantly increased in SAMP1 mouse intestine. (B) Na-glucose co-transport (SGLT1): Na-dependent and phlorizin-sensitive ³H-OMG uptake is significantly decreased in villus cells from the SAMP1 intestine. (C) Na⁺/K⁺-ATPase: The activity of Na⁺/K⁺-ATPase, which provides the favorable Na gradient for SGLT1, is significantly diminished in SAMP1 mice intestine.

Figure 3

Effect of N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) on intracellular NO levels. NO level was significantly increased in ileal villus cells of SAMP1 mice compared to control AKR mice. L-NIL treatment reversed the stimulation of intracellular NO levels in SAMP1 mice, and had no effect on NO levels in villus cells from AKR mice.

Figure 4

Effect of L-NIL treatment on Na-glucose co-transport in intact villus cells. Na-dependent ³H-OMG uptake that was significantly decreased during spontaneous ileitis in SAMP1 mice was reversed back to normal by L-NIL treatment. However, Na-glucose co-transport was unchanged in ileal villus cells isolated from AKR mice treated with L-NIL compared to the untreated control mice.

Figure 5

Effect of L-NIL treatment on Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity which was significantly reduced in SAMP1 mice intestinal villus cells was reversed back to normal levels by L-NIL treatment. L-NIL treatment did not change Na⁺/K⁺-ATPase activity in AKR mice intestinal villus cells.

Figure 6

Effect of L-NIL treatment on Na-glucose co-transport in BBMV. Na-dependent ³H-OMG uptake was significantly decreased in ileal villus cell BBMV from SAMP1 mice. L-NIL treatment reversed the decreased BBM SGLT1 activity in SAMP1 mice to normal levels. Villus BBM Na-dependent glucose co-transport activity in villus cells from L-NIL treated and untreated AKR mice intestine remained unchanged.

Figure 7

RT-qPCR analysis of SGLT1 mRNA expression. SGLT1 mRNA expression levels were significantly inhibited in SAMP1 mice villus cells compared with control AKR mice. L-NIL treatment reversed the inhibition of SGLT1 mRNA expression levels in SAMP1 mice to normal levels. However, SGLT1 mRNA expression levels remained unchanged between AKR mice untreated and treated with L-NIL.

Figure 8

Effect of L-NIL on villus cell SGLT1 protein expression in SAMP1 mouse intestine. The panel on the top is the representative blot that shows that the villus cell SGLT1 expression in SAMP1 mice was significantly reduced, which was reversed back to normal levels by L-NIL treatment. SGLT1 levels remained unaltered by L-NIL treatment in AKR mice. Densitometric analysis as shown in the bottom panel confirmed these findings.

Figure 9

Effect of L-NIL on villus cell BBM SGLT1 protein expression during chronic ileitis. The panel on the top is the representative blot that shows that the BBM levels of SGLT1 expression in SAMP1 mice was significantly reduced, and was reversed back to normal levels by L-NIL treatment. BBM SGLT1 levels remained unaltered by L-NIL treatment in AKR mice. Densitometric analysis as shown in the bottom panel confirmed these findings.

Figure 10

Immunofluorescence of SGLT1 in SAMP1 mice intestine. Images captured at 10X magnification. (A) SGLT1 in AKR mouse intestine, demonstrating its abundance in the BBM of villus cells lining the villus. (B) SAMP1 mouse intestine demonstrating the diminished SGLT1 in the BBM of villus cells lining the villus. (C) SGLT1 in L-NIL treated AKR mouse intestine, demonstrating that SGLT1 expression is unchanged in the BBM of villus cells. (D) L-NIL treated SAMP1 mouse intestine demonstrating the restoration of diminished SGLT1 in the BBM of villus cells. (E) Quantitation of SGLT1 in AKR and SAMP1 mice intestine, demonstrating that L-NIL treatment reverses the inhibition of SGLT1 in SAMP1 mice.