Orexins control intestinal glucose transport by distinct neuronal, endocrine, and direct epithelial pathways

Abstract

Objective: Orexins are neuropeptides involved in energy homeostasis. We investigated the effect of orexin A (OxA) and orexin B (OxB) on intestinal glucose transport in the rat.

Research design and methods and results: Injection of orexins led to a decrease in the blood glucose level in oral glucose tolerance tests (OGTTs). Effects of orexins on glucose entry were analyzed in Ussing chambers using the Na(+)-dependent increase in short-circuit current (Isc) to quantify jejunal glucose transport. The rapid and marked increase in Isc induced by luminal glucose was inhibited by 10 nmol/l OxA or OxB (53 and 59%, respectively). Response curves to OxA and OxB were not significantly different with half-maximal inhibitory concentrations at 0.9 and 0.4 nmol/l, respectively. On the one hand, OxA-induced inhibition of Isc was reduced by the neuronal blocker tetrodotoxin (TTX) and by a cholecystokinin (CCK) 2R antagonist, indicating involvement of neuronal and endocrine CCK-releasing cells. The OX(1)R antagonist SB334867 had no effect on OxA-induced inhibition, which is likely to occur via a neuronal and/or endocrine OX(2)R. On the other hand, SB334867 induced a significant right shift of the concentration-effect curve for OxB. This OxB-preferring OX(1)R pathway was not sensitive to TTX or to CCKR antagonists, suggesting that OxB may act directly on enterocytic OX(1)R. These distinct effects of OxA and OxB are consistent with the expression of OX(1)R and OX(2)R mRNA in the epithelial and nonepithelial tissues, respectively.

Conclusions: Our data delineate a new function for orexins as inhibitors of intestinal glucose absorption and provide a new basis for orexin-induced short-term control of energy homeostasis.

Figure 1. Oral glucose tolerance test (1 g/kg) in rats

Rats were injected i.p. with saline (control, ○), or with 55 μg/kg OxA (●) or OxB (◩) five min before they were challenged by oral administration of a 30% D-glucose solution. Results are presented as mean ± SEM. n= 6–10. *P<0.05. Area under the curve (insert) is expressed in arbitrary units (AU).

Figure 2. Effect of OxA and OxB on glucose-induced short-circuit current (Isc). A

Typical recording of Isc (in μA/cm²) across rat jejunum mounted in Ussing chamber. OxA (or OxB, not shown) diluted in saline was added serosally two minutes before luminal glucose challenge (10 mmol/l); maximal increase in Isc measured at plateau was taken as an index of SGLT-1 activity. B. Non cumulative dose-response curves to OxA (○) or OxB (□) in reducing glucose-induced Isc. number of tissues studied : 5–7.

Figure 3. Effect of OX₁R-antagonist SB334867 on inhibition of glucose-induced Isc triggered by OxA or OxB

(A) Non cumulative dose-effect curves for OxA alone (○) or in presence of 5 μmoles/l SB334867 (●) (B) Non cumulative dose-effect curves for OxB alone (□) or in presence of 5 μmoles/l SB334867 (■). No significant effect of the OX₁R antagonist was observed for OxA. By contrast, the antagonist markedly inhibited the inhibitory response induced by OxB at all concentrations, n = 5–7; *, P<0.05.

Figure 4. Effect of tetrodotoxin (A, C) and CCK receptor antagonists (B, D) on OxB-or OxA-induced inhibition of glucose transport

A: Non-cumulative dose-effect curves to OxB alone (□) or in presence (■) of tetrodotoxin (TTX, 5 μmoles/l). (B) Dose-effect curves to OxB alone (□) or in presence (◩) of CCK2R antagonist YM022 (1 nmoles/l) plus CCK1R antagonist L-364,718 (1 nmoles/l). (C) Non-cumulative dose-effect curves to OxA alone (○) or in presence (●) of TTX; *, P<0.05. (D) Dose-effect curves to OxA alone (○) or in presence (◐) of CCK2R antagonist. Number of tissues studied = 5–8

Figure 5. Effect of CCK receptor antagonists on OxA-induced inhibition of glucose transport

Inhibitory effect of OxA (10 nmoles/1) was studied in presence of CCK₁ receptor antagonist, L-364,718 (1 nmoles/l) or CCK₂ receptor antagonist, YM022 (1 nmoles/1). n=4–5 differents tissues. YM022 alone had no effect. *, P<0.05.

Figure 6. Effect of the OX₂R agonist Ala¹¹,D-Leu¹⁵OxB on inhibition of glucose transport

Inhibitory effect of OX₂R agonist (10 nmoles/l) was studied alone or in presence of TTX (5 μmoles/l) or in the presence of CCK2 receptor antagonist, YM022 (1 nmoles/l). n= 5–7; *, P<0.05 vs control.

Figure 7. RT-PCR analysis of OxR expression in epithelial and non-epithelial fractions of rat jejunal mucosa

OX₁R (top panel), OX₂R (middle panel) amplicon from CHO/OX₁R cells and CHO/OX₂R (positive controls) are shown in lane 1 and 3, respectively. Expression of OX₁R transcripts was found only in epithelial cells fractions (lane 5), whereas expression of OX₂R transcripts was found mainly in non-epithelial cells (lane 7) with a small positive signal in epithelial cell fraction (lane 5) which may be ascribed to the presence of enteroendocrine cells in the epithelial fraction. Bottom panel : expression of GADPH transcripts. Lane 2, 4, 6, 8 : negative controls of RT. Lane 9 : negative control of PCR.

Figure 8. Schematic drawing of inhibitory pathways involved in OxA and OxB inhibition of glucose absorption by enterocyte

SB334867 : specific antagonist of OX₁R; TTX : neuronal blocker tetrodotoxin; YM022 : CCK2 receptor antagonist.