Volume 142, Issue 4, Pages 834-843.e3 (April 2012) Duodenal Activation of cAMP-Dependent Protein Kinase Induces Vagal Afferent Firing and Lowers Glucose Production in Rats  Brittany A. Rasmussen, Danna M. Breen, Ping Luo, Grace W.C. Cheung, Clair S. Yang, Biying Sun, Andrea Kokorovic, Weifang Rong, Tony K.T. Lam  Gastroenterology  Volume 142, Issue 4, Pages 834-843.e3 (April 2012) DOI: 10.1053/j.gastro.2011.12.053 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Duodenal PKA activation lowers glucose production. (A) Schematic representation of working hypothesis. Sp-cAMPS with or without H-89 or Rp-CAMPS (PKA inhibitors) was infused through a duodenal catheter. SAL, saline. (B) Experimental procedure and clamp protocol. SRIF, somatostatin. Venous and arterial catheters or duodenal catheters were implanted on day 1. To ensure a comparable postabsorptive state at the start of clamp experiments, all rats received a fixed portion of calories (∼56 kcal) the evening before the pancreatic (basal insulin) clamp studies. (C and D) During the pancreatic clamp, intraduodenal Sp-cAMPS infusion increased the glucose infusion rate (C, *P < .05 vs other groups) and decreased glucose production (D, *P < .05 vs other groups). In contrast, coinfusion with either H-89 or Rp-CAMPS abolished the effects of Sp-cAMPS on (C) glucose infusion rate and (D) glucose production. (E) Glucose uptake was unchanged in all groups. (F) Duodenal PKA activity was assessed using PepTag assay and quantified using ImageJ. Duodenal Sp-cAMPS infusion significantly increased the amount of phosphorylated A1 peptide versus nonphosphorylated A1 peptide (*P < .01 vs all groups). SAL, n = 10; Sp-cAMPS, n = 9; H-89 alone, n = 5; Sp-cAMPS + H-89, n = 6; Rp-CAMPS alone, n = 5; Sp-cAMPS + Rp-CAMPS, n = 5. Values are shown as mean ± SEM. Gastroenterology 2012 142, 834-843.e3DOI: (10.1053/j.gastro.2011.12.053) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Direct activation of duodenal PKA increases the spontaneous discharge rate of the mesenteric nerve and inhibits spinal afferent firing of the duodenum. (A) Schematic of working hypothesis. Sp-cAMPS with or without H-89 was infused through an ex vivo duodenal preparation containing a branch of mesenteric nerves. (B) Intraluminal infusion of Sp-cAMPS increased the spontaneous discharge rate of the duodenal mesenteric nerve. Nerve activity and discharge frequency are shown in the top and bottom panels, respectively. (C) Intraluminal infusion of Sp-cAMPS increased the spontaneous discharge rate of the mesenteric nerve, represented as normalized nerve activity (C, *P < .05 vs control). (D) The increase in mesenteric neuronal discharge was due to activation of PKA because intraluminal infusion of Sp-cAMPS with H-89 abolished the increase in afferent discharge. (E) Distention-evoked biphasic activation of the duodenal afferent nerves with and without intraduodenal Sp-cAMPS administration. Pressure increase and discharge frequency are shown in the top and bottom panels, respectively. Rectangle indicates activation of low-threshold mechanoreceptors. Arrow indicates activation of high-threshold mechanoreceptors. Intraduodenal Sp-cAMPS infusion inhibited the high-threshold mechanosensory responses. (F) Sp-cAMPS infusion inhibited the high-threshold mechanosensory responses, represented as a change in discharge rate in comparison to the basal discharge rate (F, **P < .05 vs control). Values are shown as mean ± SEM. n = 6 per group. Gastroenterology 2012 142, 834-843.e3DOI: (10.1053/j.gastro.2011.12.053) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Duodenal PKA activation lowers glucose production through a neuronal network. (A) Schematic of working hypothesis. Sp-cAMPS was coinfused with tetracaine through a duodenal catheter, which abolishes the neuronal innervation of the duodenum. SAL, saline. (B) Experimental procedure and clamp protocol. (C and D) Intraduodenal infusion of Sp-cAMPS increased the glucose infusion rate (C, *P < .01 vs all other groups) and decreased glucose production (D, *P < .01 vs all other groups). Rats receiving tetracaine infusion failed to respond to duodenal Sp-cAMPS to increase glucose infusion rate and lower glucose production. (E) Suppression of glucose production during the clamp expressed as the percentage decrease from basal (*P < .001 vs all groups). SAL, n = 10; Sp-cAMPS, n = 9; tetracaine, n = 5; Sp-cAMPS + tetracaine, n = 5. Values are shown as mean ± SEM. Gastroenterology 2012 142, 834-843.e3DOI: (10.1053/j.gastro.2011.12.053) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Duodenal PKA activation lowers glucose production through activation of the DVC NR1-containing NMDA receptor and hepatic innervation. (A) Schematic of working hypothesis. Sp-cAMPS was infused through a duodenal catheter. SAL, saline. Sp-cAMPS was also infused into rats that underwent hepatic vagotomy (HVAG). In 2 separate studies, the NMDA channel inhibitor MK-801 was infused into the DVC and an adenovirus expressing short hairpin RNA-NR1 versus mismatch was injected into the DVC to knock down expression of the NR1 subunit of the NMDA receptor. (B) Experimental procedure and clamp protocol. Stereotaxic surgeries were conducted 7 days before placing vascular and duodenal catheters. (C and D) During the pancreatic clamps, intraduodenal Sp-cAMPS infusion increased the glucose infusion rate (*P < .001 vs SAL, #P < .001) and decreased glucose production (*P < .001 vs SAL, #P < .01 vs SAL, respectively). Rats receiving DVC-MK-801 administration or HVAG failed to respond to duodenal Sp-cAMPS. (E) Suppression of glucose production during the clamp expressed as the percentage decrease from basal (*P < .001 vs SAL, #P < .01). SAL, n = 10; Sp-cAMPS, n = 9; DVC-MK-801, n = 5; Sp-cAMPS + DVC-MK-801, n = 5; DVC-shRNA NR1, n = 7; DVC-mismatch, n = 5; HVAG, n = 6; Sp-cAMPS + HVAG, n = 5. Values are shown as mean ± SEM. Gastroenterology 2012 142, 834-843.e3DOI: (10.1053/j.gastro.2011.12.053) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Duodenal CCK suppresses glucose production through PKA activation. (A) Schematic of working hypothesis. CCK-8 was infused with or without H-89 or Rp-CAMPS through a duodenal catheter. SAL, saline. (B) Experimental procedure and clamp protocol. (C and D) During the pancreatic clamp, intraduodenal CCK-8 infusion increased the glucose infusion rate (C, *P < .01 vs other groups) and decreased glucose production (D, *P < .05 vs other groups). In contrast, coinfusion with either H-89 or Rp-CAMPS abolished the effects of CCK-8 on (C) glucose infusion rate and (D) glucose production. (E) Suppression of glucose production during the clamp period expressed as the percentage decrease from basal (*P < .01 vs all groups). (F) Duodenal CCK-8 infusion significantly increased the amount of phosphorylated A1 peptide versus nonphosphorylated A1 peptide (F, *P < .05 vs all groups). SAL, n = 10; CCK-8, n = 8; H-89, n = 5; CCK-8 + H-89, n = 5; Rp-CAMPS, n = 5; CCK-8 + Rp-CAMPS, n = 5. Values are shown as mean ± SEM. Gastroenterology 2012 142, 834-843.e3DOI: (10.1053/j.gastro.2011.12.053) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Duodenal CCK fails to activate duodenal PKA and lower glucose production (GP) in response to high-fat feeding. (A) Schematic of working hypothesis. CCK-8 or Sp-cAMPS was infused through a duodenal catheter. (B) Experimental procedure and clamp protocol. On the second day, a group of rats was placed on a high-fat diet (HFD). (C) Intraduodenal CCK-8 decreased GP (C, *P < .05 vs other groups) in regular chow (RC)-fed rats. Rats placed on a HFD for 3 days failed to respond to intraduodenal CCK-8 infusion to decrease GP compared with rats fed RC. (D) Suppression of glucose production during the clamp period expressed as a percentage decrease from basal (D, *P < .01 vs other groups). (E) PKA activation in tissues taken after the clamp studies. Intraduodenal infusion of CCK-8 in HFD rats failed to increase PKA activity. Saline (SAL) RC, n = 10; SAL HFD, n = 5; CCK-8 RC, n = 8; CCK-8 HFD, n = 6. Values are shown as mean ± SEM. Gastroenterology 2012 142, 834-843.e3DOI: (10.1053/j.gastro.2011.12.053) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Duodenal Sp-cAMPS activates duodenal PKA activity and lowers glucose production in high-fat diet (HFD)-fed rats. (A) Intraduodenal Sp-cAMPS decreased glucose production (A, *P < .05 vs other groups) in rats fed regular chow (RC). Glucose production was reduced in rats fed HFD (A, #P < .01 vs other groups). (B) Suppression of glucose production during the clamp period expressed as a percentage decrease from basal (B, *P < .05 vs other groups, #P < .05). (C) Duodenal Sp-cAMPS infusion in rats fed HFD significantly increased the amount of phosphorylated A1 peptide versus nonphosphorylated A1 peptide (C, *P < .01 vs HFD SAL). SAL RC, n = 10; SAL HFD, n = 5; Sp-cAMPS RC, n = 9; Sp-cAMPS HFD, n = 9. Values are shown as mean ± SEM. Gastroenterology 2012 142, 834-843.e3DOI: (10.1053/j.gastro.2011.12.053) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 The rate of glucose uptake is not affected by any treatments. During the pancreatic clamp, glucose uptake was comparable in all groups. (a) Administration of intraduodenal SAL (n = 10), Sp-CAMPS (n = 9), tetracaine (n = 5), Sp-CAMPS + tetracaine (n = 5). (b) SAL (n = 10), Sp-CAMPS (n = 9), DVC-MK-801 (n = 5), Sp-CAMPS + DVC-MK-801 (n = 5), DVC-shRNA NR1 (n = 7), DVC-mistmatch (n = 5), HVAG (n = 6), Sp-CAMPS + HVAG (n = 5). (c) SAL (n = 10), CCK-8 (n = 8), H-89 (n = 5), CCK-8 + H-89 (n = 5), Rp-CAMPS (n = 5), CCK-8 + Rp-CAMPS (n = 5). Values are shown as mean + SEM. Gastroenterology 2012 142, 834-843.e3DOI: (10.1053/j.gastro.2011.12.053) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Duodenal Sp-CAMPS, but not CCK-8 infusion in HFD fed rats, increases the glucose infusion rate while glucose uptake is not altered. (a) Intraduodenal CCK-8 administration increased the glucose infusion rate (a, *P < .05 vs all groups) in rats fed with regular chow. Rats placed on a HFD for three days failed to respond to CCK-8 infusion to increase the glucose infusion rate. (b) Glucose uptake was unchanged in all groups. (c) Intraduodenal Sp-CAMPS administration increased the glucose infusion rate (c, *P < .05 vs all groups) in rats fed with regular chow. Intraduodenal Sp-CAMPS administration in rats fed a high fat diet for three days significantly increased the glucose infusion rate (c, #P < .05 vs all groups). (d) Glucose uptake remained unchanged in all groups. (e) CCK-A receptor expression was comparable in both regular chow and high fat diet fed rats. SAL RC (n = 10), SAL HFD (n = 5), Sp-CAMPS RC (n = 9), Sp-CAMPS HFD (n = 9). Values are shown as mean + SEM. Gastroenterology 2012 142, 834-843.e3DOI: (10.1053/j.gastro.2011.12.053) Copyright © 2012 AGA Institute Terms and Conditions