Nitric oxide isoenzymes regulate lipopolysaccharide-enhanced insulin transport across the blood-brain barrier

Abstract

Insulin transported across the blood-brain barrier (BBB) has many effects within the central nervous system. Insulin transport is not static but altered by obesity and inflammation. Lipopolysaccharide (LPS), derived from the cell walls of Gram-negative bacteria, enhances insulin transport across the BBB but also releases nitric oxide (NO), which opposes LPS-enhanced insulin transport. Here we determined the role of NO synthase (NOS) in mediating the effects of LPS on insulin BBB transport. The activity of all three NOS isoenzymes was stimulated in vivo by LPS. Endothelial NOS and inducible NOS together mediated the LPS-enhanced transport of insulin, whereas neuronal NOS (nNOS) opposed LPS-enhanced insulin transport. This dual pattern of NOS action was found in most brain regions with the exception of the striatum, which did not respond to LPS, and the parietal cortex, hippocampus, and pons medulla, which did not respond to nNOS inhibition. In vitro studies of a brain endothelial cell (BEC) monolayer BBB model showed that LPS did not directly affect insulin transport, whereas NO inhibited insulin transport. This suggests that the stimulatory effect of LPS and NOS on insulin transport is mediated through cells of the neurovascular unit other than BECs. Protein and mRNA levels of the isoenzymes indicated that the effects of LPS are mainly posttranslational. In conclusion, LPS affects insulin transport across the BBB by modulating NOS isoenzyme activity. NO released by endothelial NOS and inducible NOS acts indirectly to stimulate insulin transport, whereas NO released by nNOS acts directly on BECs to inhibit insulin transport.

Figure 1

Effects of the nNOS inhibitor 7-NI (10 mg/kg) on LPS-induced increase in transport of I-Ins across the BBB. LR was used as the control injection. The first ip injection (LR or 7-NI) was followed 30 min later by the second ip injection (LR or LPS). The injection sequence was repeated twice (total of three sets of injections) and mice were studied 4 h after the last injection (28 h after the first injection). LPS caused a significant increase (*, P < 0.05) and 7-NI caused a further increase (**, P < 0.01) in I-Ins transport (n = 9/group).

Figure 2

Effects of AG (30 mg/kg) plus L-NIO (10 mg/kg), inhibitors of iNOS and eNOS, respectively, on the LPS-induced increase in I-Ins transport across the BBB. The two NOS inhibitors in combination prevented LPS from enhancing I-Ins transport (**, P < 0.01, n = 4–7/group).

Figure 3

Effects of l-arginine on LPS and 7-NI. l-arginine enhanced the LPS-induced increase in I-Ins transport, but addition of 7-NI did not result in a further statistically significant increase in transport. *, P < 0.05; **, P < 0.01 (n = 9–10/group).

Figure 4

Effects of l-arginine (L-arg) on LPS and AG + L-NIO. These eNOS and iNOS inhibitors countered the increase in I-Ins transport induced by LPS + l-arginine. *, P < 0.05; **, P < 0.01 (n = 7–9/group).

Figure 5

Effects of LPS on mRNA and protein levels of NOS isoenzymes. Left-hand panels show effects on mRNA (n = 3–4/group) as measured by RT-PCR, and right-hand panels show effects on protein levels (n = 6–8/group) as measured with Western blots. **, P < 0.01; *, P < 0.05; +, 0.05 < P < 0.10.

Figure 6

Brain region-specific effects of LPS and the nNOS inhibitor 7-NI. Most regions of the brain as represented by the olfactory bulb [F(3,21) = 20.1, P < 0.01] and hypothalamus [F(3,21) = 17.2, P < 0.01] had increases in I-Ins transport induced by LPS and 7-NI + LPS. Hippocampus [F(3,19) = 4.04, P < 0.05] and pons medulla [F(3,24) = 10.1, P < 0.01] had uptakes of I-Ins that were not further stimulated by 7-NI in the presence of LPS. *, P < 0.05; **, P < 0.01 (n = 7–8/group).

Figure 7

LPS in the presence of lymphocytes enhanced the transport of I-In across MBECs in an in vitro model of the BBB. Results not shown showed that LPS in the absence of lymphoctyes had no effect on I-Ins transport. The increase was inhibited by unlabeled insulin, showing that it was the saturable transport of I-Ins that was enhanced. All groups were different from all the others at P < 0.001; only the two most relevant differences are indicated by ** (P < 0.01, n = 5/group).

Figure 8

The nitric oxide donor SNAP inhibited insulin transport across the MBECs of the in vitro model of the BBB. SNAP was added to a concentration of either 30 or 150 μm to either the luminal or abluminal side of the MBECs and the luminal to abluminal transport of insulin assessed. *, P < 0.05; **, P < 0.01, n = 9–11/group.

Figure 9

SNAP did not disrupt the BBB. Upper panel shows that SNAP did not affect TEER when added either to the luminal (L) or abluminal (A) chambers. Bottom panel shows SNAP did not affect permeability to albumin. Together, these measures indicate that neither paracellular nor transcytotic disruption of the BBB occurred (n = 9–11/group).

Figure 10

Working model of the relations among LPS, NOS isoenzymes, l-arginine, and insulin transport across the BBB. LPS stimulates all three NOS isoenzymes at cells other than the BEC. Nitric oxide (NO) generated from nNOS acts directly on the BECs comprising the BBB to inhibit insulin transport. Nitric oxide generated from eNOS and iNOS acts indirectly on the BBB to stimulate insulin transport. The stimulatory effect of eNOS/iNOS is enhanced by l-arginine, the substrate for NOS.