Volume 141, Issue 5, Pages 1720-1727 (November 2011) Duodenal Mucosal Protein Kinase C-δ Regulates Glucose Production in Rats  Andrea Kokorovic, Grace W.C. Cheung, Danna M. Breen, Madhu Chari, Carol K.L. Lam, Tony K.T. Lam  Gastroenterology  Volume 141, Issue 5, Pages 1720-1727 (November 2011) DOI: 10.1053/j.gastro.2011.06.042 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 (A) During a fasting-refeeding protocol, rats administered intraduodenal saline (n = 10) had increased plasma glucose levels for 20 minutes following refeeding. In a group of rats receiving intraduodenal BIM (n = 8), the plasma levels also rose, with a significantly increased plasma glucose level at 20 minutes following refeeding, as compared with saline control. *P < .01 vs saline. (B) Food intake during refeeding was similar in both treatment groups within 20 minutes. (C) Rats receiving intraduodenal lipid infusion (n = 6) had an increased glucose infusion rate during the clamp compared with saline controls (n = 7), and such an increase in glucose infusion rate was negated when BIM was coadministered with lipid (n = 7). BIM alone, n = 5. *P < .001 vs other groups. (D) Intraduodenal lipid infusion lowers glucose production, and this glucose production-lowering effect was negated when lipid was coadministered with BIM. *P < .001 vs all other groups. Gastroenterology 2011 141, 1720-1727DOI: (10.1053/j.gastro.2011.06.042) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 (A) Immunohistochemical stain of PKC-δ (left panel) and the corresponding secondary antibody alone (right panel) in the rat duodenum. PKC-δ was strongly expressed in both the duodenal mucosal and muscle layers. Minimal staining was observed with the secondary antibody alone. Nuclear stain is blue, and PKC-δ is stained green. Intraduodenal OAG infusion (n = 5) increased duodenal PKC-δ activity in the mucosal layer (B) but not in the underlying smooth muscle layer (C) compared with saline control (n = 5). *P < .01 vs saline. During the clamps, intraduodenal infusion of OAG (n = 7) increased (D) glucose infusion rate and lowered (E) glucose production as compared with saline control (n = 7) (*P < .001 vs saline). Gastroenterology 2011 141, 1720-1727DOI: (10.1053/j.gastro.2011.06.042) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 (A) In rats undergoing a basal pancreatic insulin clamp in conjunction with intraduodenal OAG infusion (n = 7), the glucose infusion rate was increased. This effect was abolished when OAG was coadministered with BIM (n = 6) or rottlerin (n = 6). The intraduodenal lipid effect of increased glucose infusion rate was also abolished with the coinfusion of BIM (n = 7) or rottlerin (n = 7). Saline, n = 7; BIM alone, n = 5; rottlerin alone, n = 5. *P < .001 vs all other groups. (B) Glucose production during the clamps. *P < .001 vs all other groups. (C) Immunohistochemical stain of LacZ (top panel) and the corresponding secondary antibody alone (bottom panel) in rat duodenum and viral injection/clamp protocol. LacZ was observed in the mucosal and muscle layers of the duodenum. Minimal staining was observed with the secondary antibody alone. Nuclear stain is blue, and LacZ is stained green. (D) In rats that were injected with Ad DN PKC-δ (n = 5) for 3 days, the duodenal mucosal PKC-δ activity was decreased compared with the Ad LacZ controls (n = 5) (*P < .05). In rats undergoing the clamps, intraduodenal administration of OAG increased the glucose infusion rate in Ad LacZ–injected rats (n = 7) but failed to do so in the AdV DN PKC-δ–injected rats (n = 7). Saline + Ad LacZ, n = 5; saline + Ad DN PKC-δ, n = 5. *P < .001 vs all other groups. (E) Glucose production during the clamps. *P < .001 vs all other groups. (F) In rats undergoing the clamps, intraduodenal administration of lipid increased the glucose infusion rate in AdV LacZ–injected rats (n = 6) but failed to do so in the AdV DN PKC-δ–injected rats (n = 7). Saline + Ad LacZ, n = 5; saline + Ad DN PKC-δ, n = 5. *P < .001 vs all other groups. Glucose production during the clamps, *P < .001 vs all other groups. ROT, rottlerin. Gastroenterology 2011 141, 1720-1727DOI: (10.1053/j.gastro.2011.06.042) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 (A) Schematic of working hypothesis. Accumulation of LCFA in the mucosal layer of the duodenum activates PKC-δ to trigger a gut-brain-liver neuronal axis to lower glucose production (GP). (B) In rats undergoing the clamps in conjunction with intraduodenal OAG infusion (n = 7), the induction of glucose infusion rate was negated when OAG was coinfused with intraduodenal tetracaine (n = 6) or NTS NMDA receptor blocker MK-801 (n = 5) and was performed in rats with hepatic vagotomy (HVAG) (n = 5). Tetracaine alone, n = 5; NTS MK-801 alone, n = 4; HVAG alone, n = 4. *P < .001 vs all other groups. (C) Glucose production during the clamps. *P < .001 vs all other groups. Gastroenterology 2011 141, 1720-1727DOI: (10.1053/j.gastro.2011.06.042) Copyright © 2011 AGA Institute Terms and Conditions