Intraduodenal Liraglutide Regulates Hepatic Glucose Production:Aim At Gut-Brain-Liver Neuronal Axis

PART I DUODENAL GLP-1 RECEPTOR IS REQUIRED FOR LIRAGLUTIDE TO REGULATE HEPATIC GLUCOSE PRODUCTIONObjective:To investigate the effect of duodenal liraglutide on hepatic glucose production in rats feed by regular chow.Method:Male Sprague-Dawley (SD) rats (300-350g) were feed with regular chow. Construct the system of infusion into the duodenum. We divided rats into four groups according to the infusion of fluids. Saline group is the group infused with saline; GLP-1 group is the group infused with liraglutide; Ex-9 group is the group infused with Exendin Fragment 9-39; GLP-1+Ex-9 group is the group infused with liraglutide and Exendin Fragment 9-39. The insulin sensitivity and glucose metabolism were assessed by the Pancreatic (Basal Insulin)-Euglycemic Clamp technique using 3-[3H] glucose as a tracer.Results:During the steady-state of clamp, Glucose infusion rate (GIR) in GLP-1 group was significantly higher than that in Saline group (7.4+0.3 mg·kg-1·min-1 vs 2.2±0.3 mg·kg-1·min-1, P<0.01). The hepatic glucose production rate (HGP) was significantly lower in GLP-1 group than that in Saline group (10.2±0.5 mg·kg-1·min-1 vs 17.2±1.1 mg·kg-1·min-1,P<0.01). The suppression rate of hepatic glucose production in GLP-1 and Saline group was 48.7% and 18.8%, respectively. (P<0.01). Coinfusion of intraduodenal GLP-1 with Ex-9 attenuated the ability of duodenal GLP-1 to increase the glucose infusion rate and lower glucose production, while glucose uptake was comparable in all groups (P>0.05)Conclusion:Activation of duodenal GLP-1 receptor is necessary for duodenal liraglutide to increase insulin sensitivity and lower glucose production.PART Ⅱ INTRADUODENAL LIRAGLUTIDE SUPPRESSES GLUCOSE PRODUCTION BY ACTIVATING A GUT-BRAIN-LIVER AXISObjective:To investigate the neuronal network mechanism of duodenal liraglutide on hepatic glucose production.Method:Male Sprague-Dawley (SD) rats (300-350g) were feed with regular chow. Construct the system of infusion into the duodenum and micro infusion into the NTS within the fourth ventricle. Two groups of rats received hepatic branch vagotomy. Rats were divided into eight groups: Intraduodenal saline group (Saline group); Intraduodenal liraglutide group (GLP-1 group); Intraduodenal tetracaine group (tetracaine group), Intraduodenal tetracaine with liraglutide group (GLP-1+tetracaine); Intraduodenal saline with NTS infusion of MK-801(NTS MK-801group); Intraduodenal liraglutide with with NTS infusion of MK-801(GLP-1+NTS MK-801 group); Intraduodenal saline with hepatic branch vagotomy group (HVAG group); Intraduodenal liraglutide with hepatic branch vagotomy group (GLP-1+HVAG group). The insulin sensitivity and glucose metabolism were assessed by the Pancreatic (Basal Insulin)-Euglycemic Clamp technique using 3-[3H] glucose as a tracer. The expression of PEPCK, G6Pase, IR/P-IR, IRS/P-IRS and AKT/P-AKT proteins in liver was determined by Western blotting analysis.Results:Intraduodenal tetracaine alone had no effects on glucose kinetics but abolished the ability of intraduodenal GLP-1 to increase the glucose infusion rate and lower glucose production rate. This blockade effect independent of changes in plasma insulin and glucose levels. NTS MK-801 alone did not affect glucose kinetics during the clamps. However, NTS MK-801 fully abolished the ability of duodenal GLP-1 to increase the glucose infusion rate and lower glucose production required to maintain euglycemia. Intraduodenal GLP-1 failed to increase the glucose infusion rate and lower glucose production in rats received hepatic vagotomy. The interdict effect of hepatic vagotomy independent of changes in plasma insulin and glucose levels. Hepatic vagotomy alone had no effect on glucose metabolism. The PEPCK and G6Pase protein levels of liver in GLP-1 group were significantly lower than other groups (P<0.05), but the P-IR, P-IRS-land P-AKT levels were significantly higher than other groups (P<0.05).Conclusion:A Gut-Brain-Liver neuronal network is necessary for duodenal liraglutide to regulate hepatic glucose production.PART Ⅲ DUODENAL GLP-1 RECEPTOR IS REQUIRED FOR LIPID TO REGULATE HEPATIC GLUCOSE PRODUCTIONObjective:To investigate whether GLP-1 receptors in the gut are required for the duodenum-lipid regulate on glucose homeostasis.Method:Male Sprague-Dawley (SD) rats (300-350g) were feed with regular chow. Construct the system of infusion into the duodenum. According to the infusion of fluids,we divided rats into three groups: Intraduodenal Ex-9 group; Intraduodenal lipid group; Intraduodenal lipid with Ex-9 group. The insulin sensitivity and glucose metabolism were assessed by the Pancreatic (Basal Insulin)-Euglycemic Clamp technique using 3-[3H] glucose as a tracer.Results:During the steady-state of clamp, GIR in lipid group was significantly higher than that in Ex-9 group (6.1±0.3 mg·kg-1·min-1 vs 2.8±0.3 mg·kg-1·min-1, P<0.001). The hepatic glucose production rate (HGP) was significantly lower in lipid group than that in Ex-9 group (11.2±0.9 mg·kg-1·min-1 vs 15.9±0.6 mg·kg-1·min-1, P<0.01). The suppression rate of hepatic glucose production in lipid and Ex-9 group was 43.5% and 26.4%, respectively. (P<0.05). Coinfusion of intraduodenal lipid with Ex-9 attenuated the ability of duodenal lipid to increase the glucose infusion rate and lower glucose production, while glucose uptake was comparable in all groups (P>0.05)Conclusion:Activation of duodenal GLP-1 receptor is necessary for duodenal lipid to increase insulin sensitivity and lower glucose production.PART Ⅳ DUODENAL PKC-Δ IS REQUIRED FOR LIRAGLUTIDE TO REGULATE HEPATIC GLUCOSE PRODUCTIONObjective:To investigate the molecular mechanism in the duodenum of duodenal liraglutide on hepatic glucose metabolism.Method:Male Sprague-Dawley (SD) rats (300-350g) were feed with regular chow. Construct the system of infusion into the duodenum. According to the infusion of fluids,we divided rats into four groups: Intraduodenal saline group; Intraduodenal liraglutide group; Intraduodenal saline with PKC-δ specific inhibitor—Rottlerin group (ROT group); Intraduodenal liraglutide with Rottlerin group (GLP-1+ROT group). The insulin sensitivity and glucose metabolism were assessed by the Pancreatic (Basal Insulin)-Euglycemic Clamp technique using 3-[3H] glucose as a tracer.Results:During the steady-state of clamp, GIR in GLP-1+ROT group was significantly lower than that in GLP-1 group (3.2±0.5 mg·kg-1·min-1 vs 7.4±0.3 mg·kg-1·min-1, P<0.001). The hepatic glucose production rate (HGP) was significantly higher in GLP-1+ROT group than in GLP-1 group (16.8±1.0 mg·kg-1·min-1 vs 10.2±0.5 mg·kg-1·min-1, P<0.01). The suppression rate of hepatic glucose production in GLP-1+ROT and GLP-1 group was 22.9% and 48.7%, respectively. (P<0.01). Glucose uptake was comparable in all groups (P>0.05). Coinfusion of intraduodenal liraglutide with Rottlerin abolished the ability of duodenal liraglutide to increase the glucose infusion rate and lower glucose production.Conclusion:Activation of duodenal PKC-δ is necessary for duodenal liraglutide to increase insulin sensitivity and lower glucose production.