Fig 1.
Flow diagram for atropine study.
The flow diagram is for a crossover study in humans with impaired glucose tolerance and was designed so that each subject would receive all 8 graded glucose infusions. The same peptide(s) was administered during 2 successive visits- once with saline and once with atropine. First, the order of the peptide infusions was randomized. Second, the order of saline or atropine administration was randomized for each peptide. For example, if the subject was randomized to first receive Xen alone, this person would receive Xen in study visits 1 and 2. The atropine and saline infusions would then be randomized to visit 1 and 2. Eight subjects received all 8 graded glucose infusions. One subject received 6 infusions but did not receive either infusion with Xen alone. One subject received only the 2 infusions with GIP plus Xen.
Fig 2.
The graded glucose infusion protocol.
The glucose infusion rate was increased in a step wise fashion every 40 minutes starting at time zero as shown in red. The primed-constant infusion of each peptide(s) was started at time zero and is shown in yellow. Note that GIP and Xen were each infused at the same rate when administered together. The primed-constant infusion of atropine (or saline) was initiated 30 minutes before the start of the graded glucose infusion as shown in blue. All infusions were terminated 240 minutes after the glucose infusion was initiated.
Table 1.
Subject demographics and clinical characteristics.
Fig 3.
Peptide levels during graded glucose infusions.
Subjects were administered 8 different graded glucose infusions, each on a separate day. Each visit was separated by at least 2 weeks. Glucose and peptides were infused from 0 min to 240 min and atropine (or saline control) was infused from -30 to 240 min as shown in Fig 2. Steady state levels of immunoreactive-GIP (IR-GIP; panel A) and immunoreactive-Xen (IR-Xen; panel B) were measured during infusion with albumin alone (Alb), Xen alone, GIP alone, and the combination of GIP plus Xen (G+X). Each peptide was measured during infusion of atropine or the saline control. Because of limiting sample volumes, GIP and Xen were measured only in the 80 or 240 minute samples, respectively. Values represent group means ± SEM.
Fig 4.
Atropine increases resting heart rate.
Resting heart rate was measured at the indicated times before, during and after the graded glucose infusion (GGI) with Alb alone (Panel A), Xen alone (Panel B), GIP alone (Panel C), and GIP plus Xen (G+X; Panel D). Atropine or saline infusion was started 30 minutes before the graded glucose infusion. Note that atropine or GIP increased resting heart and values were normalized approximately 90 minutes after the graded glucose infusions were terminated. After 240-minutes, heart rates increase in the subjects administered saline instead of atropine because they are no longer confined to the bed. Values represent group means ± SEM.
Fig 5.
Atropine inhibits pancreatic polypeptide release.
Panels A and B. Pancreatic polypeptide (PP) levels were measured at the indicated times before and during graded glucose infusions (GGIs) in the presence of the indicated peptide(s) and with infusion of saline (Panel A) or atropine (Panel B). Total (Panel C) and incremental (Panel D) AUCs from 0–240 minutes were calculated from data in panels A and B. Incremental and total AUCs were determined for each individual and values represent group means ± SEM. Significance was determined using the mixed effects model. p values for each peptide versus albumin alone are indicated within charts in Panels C and D. p values for the effects of atropine for each peptide infusate (or albumin control) are indicated below Panels C and D. The p values for infusate effects were calculated for all 8 treatments. Note that atropine infusion completely blocks all pancreatic polypeptide responses.
Fig 6.
Xen amplifies the effects of GIP on ISRs.
Graded glucose infusions (GGIs) with the saline control (Panels A-C) or atropine infusion (Panels D-F) were conducted. Plasma glucose (Panels A and D) and ISRs (Panels B and E) are shown for the indicated times before and during GGIs. The glucose infusion rate (GIR) at each 40 minute step is shown in white. Note that plasma glucose levels increase progressively even though glucose was administered in a step-wise fashion. Data from panels A and B are re-graphed in panel C whereas data from panels D and E are re-graphed in panel F. Symbols and error bars are eliminated in panels C and F for clarity. The rapid and transient increases in ISR in response to GIP and GIP plus Xen from 0–40 minutes (Panels B and E) are reflected by the initial spikes in ISRs (Panels C and F) that occur in the absence of a significant increase in plasma glucose levels. Values represent group means ± SEM.
Fig 7.
Atropine does not inhibit effects of peptides on ISRs.
Incremental (Panels A and C) and total (Panels B and D) AUCs were calculated using data shown in Fig 6. Baseline values (average from -50 to -30 min) were subtracted to calculate incremental AUCs. Outcomes were determined for each individual and values represent group means ± SEM. Significance was determined using the mixed effects model. Panels A and B: Data are for 0–40 min (Panels A and B). Panels C and D: Data are for 40–240 min. Values were calculated from the 40 to 240 ISR (i)AUCs for each individual divided by the 40 to 240 glucose (i)AUCs for the same individual. Values represent group means ± SEM.
Fig 8.
Xen amplifies the rapid and transient response to GIP via a muscarinic receptor-independent mechanism.
Panels A and B: Plasma glucagon levels were measured at the indicated times during graded glucose infusions (GGIs) with the indicated peptide(s) and with the saline infusion (Panel A) or atropine infusion (Panel B). The glucose infusion rate (GIR) for each 40 minute step is shown in white. Note the rapid and transient increase in glucagon levels during peptide infusions only from 0 to 40 minutes. Panels C and D: Plasma glucagon levels during graded glucose infusions with saline (Panel C) or atropine (Panel D) infusions were plotted as a function of plasma glucose levels. Panels E-H: Plasma glucagon and glucose levels were determined during graded glucose infusions with Albumin alone (Panel E), Xen alone (Panel F), GIP alone (Panel G), and the combination of GIP plus Xen (Panel H). Plasma glucagon versus glucose values during infusion of saline or atropine are shown for each peptide(s). Note that atropine had little effect on the rapid and transient (0 to 40 min) or on the glucose-regulated (40–240 min) glucagon responses regardless of the peptide administered.
Fig 9.
Atropine does not inhibit the glucagon response to peptides.
Data from Fig 8A and 8B representing the rapid and transient responses (0 to 40 minutes) were replotted. Panel A: Note that compared to albumin alone, the increase in plasma glucagon is highly significant during infusion of GIP+Xen from 5 to 40 minutes. Red #, *, **, and *** represent p <0.03, <0.01, <0.001, and <0.0001, respectively compared to albumin alone. The yellow + sign indicates a p value <0.07 for GIP alone compared to albumin alone. Panels B-E. Data from graded glucose infusions with albumin alone (Panel B), Xen alone (Panel C), GIP alone (Panel D) and GIP+Xen (Panel E) are shown for infusions with (red squares) and without (blue circles) atropine. Compared to the saline control, atropine had no statistically significant effect on the glucagon response to any of the 4 peptide treatments.