Linearity of -cell response across the metabolic spectrum and to pharmacology: insights from a graded glucose infusion-based investigation series

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1 Am J Physiol Endocrinol Metab 310: E865 E873, First published April 12, 2016; doi: /ajpendo CALL FOR PAPERS Islet Biology Linearity of -cell response across the metabolic spectrum and to pharmacology: insights from a graded glucose infusion-based investigation series Sudha S. Shankar, 1 R. Ravi Shankar, 1 Lori A. Mixson, 1 Deborah L. Miller, 1 Christopher Chung, 1 Caroline Cilissen, 2 Chan R. Beals, 1 S. Aubrey Stoch, 1 Helmut O. Steinberg, 1 and David E. Kelley 1 1 Merck & Company, Inc., Kenilworth, New Jersey; and 2 Merck Sharp & Dohme Corp. (Europe), Brussels, Belgium Submitted 28 December 2015; accepted in final form 5 April 2016 Shankar SS, Shankar RR, Mixson LA, Miller DL, Chung C, Cilissen C, Beals CR, Stoch SA, Steinberg HO, Kelley DE. Linearity of -cell response across the metabolic spectrum and to pharmacology: insights from a graded glucose infusion-based investigation series. Am J Physiol Endocrinol Metab 310: E865 E873, First published April 12, 2016; doi: /ajpendo The graded glucose infusion (GGI) examines insulin secretory response patterns to continuously escalating glycemia. The current study series sought to more fully appraise its performance characteristics. Key questions addressed were comparison of the GGI to the hyperglycemic clamp (HGC), comparison of insulin secretory response patterns across three volunteer populations known to differ in -cell function (healthy nonobese, obese nondiabetic, and type 2 diabetic), and characterization of effects of known insulin secretagogues in the context of a GGI. Insulin secretory response was measured as changes in insulin, C-peptide, insulin secretion rates (ISR), and ratio of ISR to prevailing glucose (ISR/G). The GGI correlated well with the HGC (r 0.72 for ISR/G, P 0.01). The insulin secretory response in type 2 diabetes (T2DM) was significantly blunted (P 0.001), whereas it was significantly increased in obese nondiabetics compared with healthy nonobese (P 0.001). Finally, robust (P over placebo) pharmacological effects were observed in T2DM and healthy nonobese volunteers. Collectively, the findings of this investigational series bolster confidence that the GGI has solid attributes for assessing insulin secretory response to glucose across populations and pharmacology. Notably, the coupling of insulin secretory response to glycemic changes was distinctly and uniformly linear across populations and in the context of insulin secretagogues. (Clinical Trial Registration Nos. NCT , NCT , NCT ) graded glucose infusion; hyperglycemic clamp; nonobese; obese; diabetes; exenatide; liraglutide THE ABILITY TO APPROPRIATELY MODULATE insulin secretion in response to acute changes in prevailing glucose is the sine qua non of -cell health (26, 29, 30). Glucose responsiveness of -cells is diminished in individuals with type 2 diabetes mellitus (T2DM) (22), yet few clinical investigations describe the pattern of this impairment across a dynamic range of hyperglycemic stimulation. Instead, most extant clinical investigation literature centers upon insulin secretory response to a single level of hyperglycemic challenge (e.g., a hyperglycemic clamp) (10) or measured insulin secretion to a bolus glucose Address for reprint requests and other correspondence: S. S. Shankar, Lilly Research Laboratories, Indianapolis, IN ( sudhashankar@comcast.net). challenge (e.g., an IVGTT) (1). The intrinsic property of the -cell to sense and respond to changes in circulating glucose is perhaps best described using the dynamic relationship between insulin secretion and glucose. Insulin secretory responses using data from an oral glucose or mixed-meal challenge have been examined in relation to the range of glycemia attained during that particular challenge (11, 12). Isolating and delineating -cell response to glycemia alone, across a broad range of hyperglycemia, independent of incretin hormone responses evoked during nutrient ingestion, should enhance understanding of the impaired -cell function in T2DM and the nature of -cell compensation in obesity. A platform that focuses on the dynamic aspects of -cell response to changing hyperglycemia will be particularly valuable in appraising the therapeutic impact of pharmacological and nonpharmacological interventions. Several methodologies have been developed for the measurement of insulin secretory capacity. Among these, the hyperglycemic clamp (HGC) is regarded as the gold standard for isolating -cell response to glucose, independent of modulating endogenous incretins (9, 18, 24, 27, 35). However, the HGC requires technical expertise to rapidly achieve and maintain a stable hyperglycemic target, usually at one or two discrete levels of hyperglycemia (9, 28, 31, 35), thereby limiting its ability to assess the responsiveness of the -cell to changing glycemia. Given the recent surge in glucose-dependent insulin secretagogues as therapies for T2DM, there is a need to characterize not only the amplitude but also the pattern of changes in insulin secretion across a dynamic range of hyperglycemia. In contrast to the HGC, the graded glucose infusion (GGI) methodology uses stepwise increments of glucose infusion, producing a range of experimentally induced hyperglycemia (8, 32) that allows for the assessment of -cell responsiveness to changes in prevailing glycemia. Use of the GGI as a tool for measurement of -cell function was pioneered by Polonsky and colleagues (7, 16, 17, 20, 32). These investigators and others have used the GGI to describe -cell function in various phenotypes of dysregulated glucoseinsulin homeostasis as well as in pertinent genotype-phenotype relationships that influence -cell function (4 7, 16, 17, 20, 32). Brandt et al. (3) and Kjems et al. (22) have evaluated the effects of exogenous glucagon-like peptide-1 (GLP-1) in GGIbased experiments. However, despite these important prior applications, the GGI in general remains less characterized than the HGC for quantitative estimation of glucose-dependent /16 Copyright 2016 the American Physiological Society E865

2 E866 insulin secretion (GDIS). Notably, there are no reports of a direct comparison of the GGI against the gold standard HGC. The current series of investigations was undertaken to address these gaps, first by calibrating the GGI against the HGC in a head-to-head setting. This was followed by GGI-based experiments in healthy nonobese, obese nondiabetic, and T2DM subjects, undertaken to compare the insulin secretory response to a dynamic range of hyperglycemia across these three groups. Last, we undertook platform-building studies to use the GGI in conjunction with acute pharmacological stimulation in both healthy nonobese subjects and in those with T2DM. Across this range of studies, a standardized protocol for the GGI and a consistent set of analyses were followed. In addition to the findings pertinent to each study, there emerged an intriguing overarching observation that the pattern of -cell responsiveness to a dynamic range of hyperglycemia is linear in each of the groups that was studied (Clinical Trial Registration Nos. NCT , NCT , and NCT ). MATERIALS AND METHODS Three separate trials were conducted in three separate populations, healthy nonobese, overweight and obese nondiabetic, and type 2 diabetic (T2DM) volunteers. Study Subjects and Trial Design Trial I: nonobese healthy volunteers (NOB). The purpose of this randomized, double-blind, placebo-controlled trial was to calibrate the GGI against the HGC as a tool for measurement of insulin secretion in a head-to-head setting and to characterize -cell response in a GGI in response to exogenous glucose alone as well as in response to coadministration of exogenous glucose and the GLP-1 analog exenatide. After meeting study entry criteria, 16 healthy nonobese, nondiabetic male volunteers with no family history of diabetes were randomized to one of two treatment groups [exenatide 5 g (NOB- EXE) or placebo (NOB-PBO)]. To maintain stable exenatide exposures throughout each procedure, the study drug was administered as two subcutaneous injections, one dose 90 min prior to initiation of the HGC or GGI and the second dose 120 min later. Following randomization to treatment, every subject underwent each procedure (a GGI and a HGC) on separate occasions 14 days apart in a crossover design with the same pharmacological treatment for both procedures. Procedural details are described below. Trial II: overweight and obese nondiabetic volunteers (OB). The purpose of Trial II was to characterize -cell response to exogenous glucose alone in a GGI in a cohort of 12 otherwise healthy overweight and obese nondiabetic male volunteers with no family history of diabetes. Study entry criteria included BMI of 27 kg/m 2 and 35 kg/m 2 and weight 70 kg, blood pressure 160/95 mmhg, fasting glucose between 65 and 95 mg/dl, with lipid parameters and routine safety parameters within normal limits, and not on any routine medications. After meeting study entry criteria, subjects underwent a single GGI procedure. Trial III: T2DM volunteers. The purpose of this randomized, double-blind, placebo-controlled trial with a crossover design was to characterize -cell response in T2DM in a GGI in response to exogenous glucose alone as well as in response to coadministration of exogenous glucose and a single dose of the GLP-1 analog liraglutide. All 12 subjects had physician-diagnosed T2DM and were naïve to GLP-1 analogs and DPP4-inhibitor therapies. Three subjects were on lifestyle measures only, nine on metformin, and of these, fourwere on metformin and sulfonylureas. All background antihyperglycemic therapy was held for 24 h prior to the GGI in each period. Study entry criteria included BMI 38 kg/m 2, blood pressure 160/95 mmhg, Hb A1C 9.0%, fasting glucose not greater than 300 mg/dl, with lipid parameters and routine safety parameters within normal limits. After meeting study entry criteria, 12 male subjects with T2DM were randomized to one of two treatments [0.6 mg of liraglutide (T2DM- LIRA), or placebo (T2DM-PBO)]. Following randomization, subjects underwent a GGI at 9 h following a single subcutaneous dose of liraglutide or placebo (approximate T max of liraglutide), and crossed over after a 7-day washout. Procedures The trial in the NOB was conducted at the University of Brussels, Belgium, after receiving approval from the Ethics Committee Universitair Ziekenhuis Brussels, and the trials in the OB and T2DM were conducted at PRA International in Zuidlaren, The Netherlands after receiving approval from the Independent Ethics Committee of the Stichting Beoordeling Ethiek Bio-Medisch Onderzoek in The Netherlands. All volunteers gave written informed consent. Subjects were instructed to maintain their usual dietary and physical activity habits during the trial and were required to refrain from alcohol and vigorous exercise for 24 h and from tobacco for 12 h prior to the study procedures. All subjects were admitted to the Clinical Research Unit in the evening prior to the study and fed a standardized meal. Hyperglycemic Clamp Subjects underwent a HGC as per the protocol, using the Andres method (13). Following an overnight fast, a 120-min HGC was initiated to achieve target glucose of 200 mg/dl. At steady state of the clamp (SS: 90 to 120 min), blood glucose levels varied by 5%, and glucose infusion rates (GIR) varied by 10%. Graded Glucose Infusion Following an overnight fast and baseline evaluations, a 160-min GGI with 20% dextrose was initiated with the GIR set at 2, 4, 6, and 12 mg kg 1 min 1, each infusion period lasting 40 min. The GIRs were chosen so as to match/suppress hepatic glucose output at onset, progressing in a stepwise manner up to a peak infusion rate that would provide a submaximal glycemic stimulus estimated to match the level and duration of glycemia and GIR in the HGC in NOB-PBO. In the T2DM, the highest GIR was limited to 10 mg kg 1 min 1 to avoid risk of excessive hyperglycemia. The T2DM underwent an overnight infusion of intravenous insulin to enable achievement of similar baseline glucose levels in both treatment groups prior to the GGI, with the insulin infusion turned off for a period before initiation of the GGI to avoid suppression of endogenous insulin secretion. Sample Analysis Central laboratory glucose was assayed using the Roche Modular platform using Roche reagents (interassay CV: %, intra-assay CV: %). Insulin and C-peptide were performed in the NOB using the Siemens Centaur XP using reagents from Siemens (interassay CV of % and intra-assay CV of % for insulin; interassay CV of % and intra-assay CV of % for C-peptide) and in the OB and T2DM using the Immulite 2000 system with reagents from Siemens (Insulin has an interassay CV of % and an intra-assay CV of %; C-peptide has an interassay CV of % and an intra-assay CV of %). Bedside glucose was assayed using a Yellow Spring Instruments apparatus by the glucokinase method. Plasma total, LDL, and HDL cholesterol, as well as triglyceride levels, were measured at baseline. Quantitative and Statistical Methods Estimation of insulin secretion rates (ISR) was done by deconvolution of circulating C-peptide measurements, using the methods of Eaton et al. (15) and Van Cauter et al. (34). Key end points. HGC. HGC included traditional parameters such as glucose (SSPG), insulin (SSPI), and C-peptide (SSPC) concentrations,

3 as well as GIR, estimated as the time-weighted average (TWA) during SS. The TWA provides the average concentration over a prespecified time interval and is calculated as the area under the curve (AUC) divided by the length of the time interval. In addition, ISR and the ratio of ISR to glucose (ISR/G) at SS were computed. GGI. GGI included circulating glucose, insulin, and C-peptide concentrations measured during the GGI, estimated as the TWA (TWA min and TWA min) and maximum glycemic excursion (G max). ISR and ISR/G were also computed. All parameters were estimated or computed for the entire duration of the GGI and during the period of highest GIR alone (period of final 40 min). Head-to-head comparison of GGI and HGC. To provide a direct, head-to-head quantitative comparison between the HGC and the GGI, the correlation between the same measure of GDIS (ISR/G) as measured in the HGC (at SS) and GGI (at the highest GIR) in the same subjects was assessed using a Spearman rank correlation coefficient. -Cell glucose sensing and responsiveness in the GGI. The slopes of the regression of ISR on G (slope ISR vs. G) and of the regression of change from baseline in ISR on change from baseline in prevailing glucose (slope ISR vs. G) throughout the GGI were computed, to provide indexes of -cell glucose sensing and responsiveness. Statistical Methods All analyses, with the exception of slope ISR vs. G were conducted as a change from baseline. All concentration end points and ISR were analyzed on the log scale and back-transformed to the original scale and reported as a mean and 90% confidence interval. Slope ISR vs. G as well as slope ISR vs. G were analyzed on the raw scale. A linear mixed-effects model was used to compare the effects of exenatide 5 g to placebo in the healthy volunteers undergoing both a GGI and HGC. The model included fixed effects for treatment and period as well as a random effect for subject. An unstructured covariance structure was assumed to account for the within-subject correlation unless convergence issues were found. In those cases, a compound symmetric covariance structure was used. A similar linear mixed-effects model was used for the comparison of GDIS effects in placebo-treated nonobese (NOB-PBO), overweight and obese (OB- PBO), and T2DM (T2DM-PBO) subjects undergoing the GGI for the between-metabolic-group comparisons. For these analyses, the model included a fixed effect for population and a random effect for subject. All comparisons were based on a one-sided t-test at 0.05 level. RESULTS Head-to-Head Comparison of GGI and HGC in Nonobese Healthy Subjects This first set of results describes -cell response observed in a HGC and separately in a GGI to glucose alone and in the presence of the GLP-1 analog exenatide, followed by a headto-head comparison of GDIS effects in a HGC and GGI. Both procedures were generally well tolerated. Baseline characteristics of the subjects are listed in Table 1. GDIS effects of glucose alone in HGC and GGI. In NOB- PBO, in response to a hyperglycemic stimulus alone during the HGC, plasma glucose rose within minutes and remained at target glycemia throughout steady-state conditions. In response to the hyperglycemic stimulus, there were brisk and significant increases in insulin, C-peptide, ISR, ISR/G, and GIR at steady state and throughout the procedure (Tables 1 and 2). In the GGI conducted in NOB-PBO, plasma glucose rose progressively with each of the stepwise increases in GIR, accompanied by significant (P 0.001) responses in insulin, C-peptide, ISR, and ISR/G (Table 1 and Fig. 1). At the highest Table 1. Baseline characteristics of healthy nonobese subjects NOB-PBO NOB-EXE E867 Parameter Mean SD Mean SD BMI, kg/m Age, yr Systolic BP, mmhg Diastolic BP, mmhg Fasting plasma glucose, mg/dl Fasting serum insulin, IU/ml Fasting C-peptide, ng/ml ISR, ng/min ISR/G BMI, body mass index; BP, blood pressure; ISR, insulin secretion rates; NOB-EXE, nonobese healthy exenatide-treated subjects; NOB-PBO, nonobese healthy placebo-treated subjects; ISR/G, ISR-to-glucose ratio. GIR of the GGI (12 mg kg 1 min 1 ), plasma glucose rose to a maximum glycemic excursion (G max ) over 200 mg/dl, accompanied by significant (P 0.001) increases in insulin, C-peptide, ISR, and ISR/G relative to baseline (see Table 4). GDIS effects of exenatide in HGC and GGI. Baseline clinical characteristics between treatment groups were similar (Table 1). One subject undergoing the HGC procedure experienced emesis that was rated as drug related (exenatide 5 g); this subject was discontinued from the study and is not included in the data presented. The background corrected TWA (95% CI) exenatide concentration measured during both procedures was (113.3, 142.5) pg/ml for exenatide 5 g. During the HGC, in the NOB-EXE, at steady state, plasma glucose was at target, with concomitant significant (P 0.001) increases in GIR, insulin, C-peptide, ISR, and ISR/G relative to baseline. At matched plasma glucose, exenatide elicited significant (P 0.001) increases in GIR, plasma insulin, C-peptide, and ISR compared with placebo (glucose alone) at steady state and for the duration of the clamp (Table 2). During the GGI, in the NOB-EXE, plasma glucose rose progressively from baseline with each of the stepwise increases in GIR with concomitant significant (P 0.001) increases in insulin, C-peptide, ISR, and ISR/G relative to baseline and over placebo (glucose alone or NOB-PBO). The responses in insulin, C-peptide, ISR, and ISR/G were most prominent at the highest GIR of the GGI (12 mg kg 1 min 1 ), and significantly (P 0.001) greater in NOB-EXE compared with NOB-PBO (Fig. 1 and Table 4). In response to the robust insulin secretory response, glycemic excursion with exenatide was significantly blunted compared with that with glucose alone, with G max in NOB-EXE being significantly (P 0.001) lower by 54% compared with NOB-PBO (Fig. 1 and Table 4). Notably however, despite the vigorous insulin secretory response, at no point in the GGI did plasma glucose fall below 70 mg/dl in NOB-EXE. Comparison between HGC and GGI. Both methodologies detected a significant stimulation of insulin secretion in response to hyperglycemia (P 0.001) and a further significant augmentation with exenatide (P 0.001) (Fig. 2). To enable a direct head-to-head comparison between procedures, these effects were expressed using an end-point common to both procedures, the ISR/G estimated at steady state of the HGC, and at the highest GIR of the GGI. The choice of the ratio of

4 E868 Fig. 1. Time course profiles for glucose, insulin, insulin secretion rates (ISR), and ISRto-glucose ratio (ISR/G) after administration of placebo or exenatide in healthy nonobese subjects. Results are expressed as a change from baseline (means SE), using the timeweighted average across the graded glucose infusion (GGI). A highly statistically significant (P 0.001) increase from baseline in glucose, and corresponding increases in insulin, ISR, and ISR/G were observed in both groups. Highly statistically significant (P 0.001) increases in insulin, ISR, and ISR/G were observed in the exenatide group vs. the placebo group. This was accompanied by a highly statistically significant (P 0.001) blunting in glycemic excursion for the exenatide group vs. the placebo group. ISR/G was also able to account for any numerical differences in the actual level of hyperglycemia between the two procedures. ISR/G in the HGC rose a significant 1.3-fold over baseline at steady state in response to glucose alone and showed a further significant increase with exenatide (5.4-fold; Fig. 2). In the GGI, ISR/G rose a significant 2.0-fold over baseline at the highest GIR in response to glucose alone, with a further significant increase with exenatide (8.2-fold; Figs. 1 and 2). Across treatment conditions, ISR/G at steady state of the HGC was significantly correlated with ISR/G at the highest GIR for the GGI (r 0.72, P 0.01). The overall variability (expressed as the %CV) for the ISR/G end point was 6.3% for HGC and 13.9% for GGI. Operationally, the GGI was far simpler than the HGC, requiring fewer specialized resources for execution. Table 2. Summary statistics at steady state of the hyperglycemic clamp in healthy nonobese subjects Parameter NOB-PBO NOB-EXE P Value SSPG, mg/dl SSGIR, mg/kg/min TWA SSPI, miu/l 47.4 (28.9, 77.7) (202.2, 659.0) TWA SSPC, ng/dl 7.09 (5.29, 9.51) 23.3 (16.4, 33.1) TWA SSISR, ng/min 0.97 (0.80, 1.17) 4.07 (3.20, 5.18) TWA SS-ISR/G (0.0042, ) (0.0163, ) SSPG, steady-state plasma glucose; SSPI, steady-state plasma insulin; SSPC, steady-state plasma C-peptide concentration; SSGIR, steady state glucose infusion rates; TWA, time-weighted average. Comparison of -Cell Response across Nonobese Healthy, Overweight and Obese Nondiabetic, and T2DM Subjects in a GGI The second set of results describes quantitative comparisons across populations of -cell response to glucose alone measured in a GGI. Data from the placebo-treated groups from each of the three study populations were compared. Demographic characteristics of placebo-treated subjects from the three groups are shown in Table 3. Plasma glucose rose incrementally and significantly over baseline across the esca- Fig. 2. Geometric mean and 90% confidence interval for ISR/G in the placeboand exenatide-treated healthy nonobese subjects at steady state of the hyperglycemic clamp (HGC) and during the highest glucose infusion rate (GIR) of the GGI. A highly statistically significant increase (P 0.001) in ISR/G was found for the exenatide group compared with the placebo group in both the HGC and GGI.

5 Table 3. Baseline characteristics of placebo-treated subjects across metabolic spectrum E869 NOB-PBO OB-PBO T2DM-PBO Parameter Mean SD Mean SD Mean SD BMI, kg/m Age, yr Fasting plasma glucose, mg/dl Fasting insulin, IU/ml Fasting C-peptide, ng/ml ISR, ng/min ISR/G HbA 1c, % NM N/A NM N/A Duration of T2DM, yr N/A N/A N/A N/A NOB-PBO, nonobese healthy placebo-treated subjects; OB-PBO, overweight and obese nondiabetic placebo-treated subjects; T2DM-PBO, type 2 diabetes subjects treated with placebo; NM, not measured; N/A, not applicable. Fig. 3. Time course profiles for glucose, insulin, ISR, and ISR/G in placebotreated healthy nonobese, obese nondiabetic, and type 2 diabetic (T2DM) subjects. Results are expressed as change from baseline (means SE). At the highest GIR, differences were significant (P 0.001) for all between-group comparisons. lating glucose infusion in all groups, with the highest levels in T2DM-PBO, lowest in NOB-PBO, and intermediate in OB- PBO (P for all between group differences; Table 4 and Fig. 3). Concomitant robust increases over baseline in insulin, C-peptide, ISR, and ISR/G were observed in NOB-PBO and OB-PBO and were significantly (P 0.001) greater in OB- PBO than in NOB-PBO (Fig. 3). In T2DM-PBO, responses over baseline in all insulin secretory parameters were modest and were significantly (P 0.001) blunted compared with the groups without diabetes (Fig. 3). At the highest GIR, OB-PBO had the most exuberant response, followed by NOB-PBO, with T2DM-PBO having the lowest response, with significant (P 0.001) differences for all between group comparisons for insulin, C-peptide, ISR, and ISR/G (Fig. 3 and Table 4). Treatment Effects of Liraglutide on -Cell Response in T2DM in a GGI We examined the effects of a single dose of liraglutide in a GGI in a group of volunteers with T2DM, using a crossover Fig. 4. Time course profiles for glucose, insulin, ISR, and ISR/G after administration of placebo or liraglutide in T2DM subjects. Results are expressed as change from baseline (means SE), using the time-weighted average across the GGI. A highly statistically significant (P 0.001) increase from baseline in glucose observed in both treatment groups, accompanied by significant increases from baseline in insulin, ISR, and ISR/G observed in the liraglutide group and blunted changes from baseline in insulin, ISR, and ISR/G in the placebo group. At the highest GIR, highly statistically significant (P 0.001) decreases in glucose accompanied by robust (P 0.001) increases in insulin, ISR, and ISR/G in the liraglutide group vs. the placebo group.

6 E870 Fig. 5. Group comparisons of relationship of insulin secretory response to glucose for the entire duration of the GGI (slope of ISR vs. glucose across the GGI). The slope of the regression line for ISR vs. G showed a significant right shift in placebo-treated T2DM (P 0.001) vs. nondiabetic groups and a significant left shift in placebo-treated overweight and obese (P 0.001) vs. placebo-treated healthy nonobese (A). Slope of ISR vs. G showed a significant left shift in T2DM treated with liraglutide (P 0.001) vs. placebo-treated T2DM (B). Slope of ISR vs. G in T2DM treated with liraglutide was similar (P NS) to that of the placebotreated obese nondiabetic group (C). Finally, in the healthy nonobese, the slope of ISR vs. G showed a significant left shift in response to exenatide (P 0.001) vs. placebo group (D). All relationships were uniformly linear. paradigm. Baseline characteristics of the volunteers are shown in Table 3 and described under MATERIALS AND METHODS. Treatment and procedure were well tolerated, and plasma glucose prior to initiation of the GGI was similar between treatment ( mg/dl) and placebo ( mg/dl) groups. Plasma glucose rose incrementally across the escalating GIR in both groups. Insulin, C-peptide, and ISR/G responses to the glycemic excursions were significantly (P 0.001) higher by 256, 223, and 255%, respectively, in T2DM- LIRA compared with T2DM-PBO during the GGI (Fig. 4), with between-group differences especially prominent at the highest GIR (P for all parameters; Fig. 4 and Table 4). Mean plasma glucose levels and G max were significantly (P 0.001) lower in T2DM-LIRA compared with T2DM-PBO (Fig. 4 and Table 4). -Cell Glucose Sensing and Responsiveness The slopes from the linear regression of ISR vs. G and of ISR vs. G throughout the GGI were computed in all groups (Fig. 5). Taken together, these slope parameters provide an index of the ability of the -cell to first sense changes in prevailing glucose and to then adapt and respond with appropriate changes in insulin secretion. Assessment of linearity. A linear relationship between the response (ISR or ISR) and the stimulus (G or G) was deemed by calculation of R 2 (a global measure of variance explained by the glucose stimulus). In addition, plots of the model fit (residuals vs. the fitted values) showed no bias or systematic deviation across the range of glucose stimuli for any of the populations tested. Comparative examination of slope ISR vs. G [0.014 (0.009, 0.02) in NOB-PBO, (0.016, 0.026) in OB- PBO, and (0.002, 0.011) in T2DM-PBO] and slope ISR vs. G across populations showed a significant right shift in T2DM-PBO (P 0.001) compared with those without diabetes, and a significant left shift in OB-PBO (P 0.001) compared with NOB-PBO (Fig. 5A), remaining linear in all three groups. Table 4. Comparative display of key parameters at the highest GIR in GGI across populations and treatments Parameter NOB-PBO NOB-EXE OB-PBO T2DM-PBO T2DM-LIRA G max, mg/dl (213.7, 262.1) (130.2, 159.1) (247.1, 309.3)* (371.6, 433.1)* (203.6, 346.2)* Insulin levels, U/ml (38.9, 67.4) (130.7, 238.3)* (117, 203.2)* (20.5, 35.9)* (84.7, 152.2)* C-peptide levels, ng/ml (5.78, 9.11) (11.6, 18.2) (11.7, 14.9)* (3.18, 4.44)* (7.41, 10.5)* ISR, ng/min (1.83, 3.02) (3.28, 5.99)* (3.12, 5.55) (1.84, 3.09) (5.66, 7.03) ISR/G (0.007, 0.013) (0.027, 0.038) (0.013, 0.019)* (0.005, 0.007)* (0.015, 0.023)* Results are expressed as absolute values with 90% confidence interval at the highest GIR in each group. T2DM-LIRA, T2DM liraglutide; G max, maximal glycemic excursion. Statistical significance for cross-group comparisons is presented. *P vs. placebo-treated healthy nonobese; P vs. placebo-treated T2DM.

7 The treatment effect of liraglutide in T2DM was examined. The slope of ISR vs. G [0.006 (0.002, 0.011) in T2DM-PBO, and (0.008, 0.056) in T2DM-LIRA] and slope ISR vs. G showed a significant left shift in T2DM-LIRA (P 0.001) compared with T2DM-PBO (Fig. 5B). The responses displayed a linear pattern of increase. Consequent to the significant leftward shift in response to a single dose of liraglutide, slope ISR vs. G and slope ISR vs. G in T2DM-LIRA were similar (P NS) to that in OB-PBO (Fig. 5C). The treatment effect of the insulinotropic agent exenatide was assessed in NOB. The slope ISR vs. G [0.014 (0.009, 0.02) in NOB-PBO, and (0.050, 0.089) in NOB-EXE] and slope ISR vs. G showed a significant left shift in NOB-EXE (P 0.001) compared with NOB-PBO (Fig. 5D). In the healthy nonobese as well, the augmented response to pharmacological stimulation displayed a linear pattern of increase. DISCUSSION The findings obtained from this series of clinical investigations help to thoroughly characterize the GGI as a tool to measure glucose-dependent insulin secretion across a metabolic spectrum of healthy nonobese, obese nondiabetic, and T2DM and in a context of clinically relevant incretin-based pharmacotherapy using uniform procedural and analytic methods. One of the three central goals of the present set of studies was to calibrate the GGI against a HGC, which to our knowledge is the first report of such a comparison. The HGC is generally regarded as a gold standard approach to detect even small changes in insulin secretion with relatively small study populations, whether in response to glucose alone or in conjunction with an insulinotropic stimulus (10, 12). In our study, we confirm this attribute of the HGC and find that the GGI compares well with the HGC in this regard. The measurement attributes of the GGI, namely a CV of 13.9% with the ability to detect a 21% increase in GDIS in a sample size of about eight nonobese healthy subjects, seem amply sufficient to detect a clinically relevant change in -cell response. Our findings on the comparison between the HGC and GGI for assessment of the treatment effects of an incretin analog on insulin secretion support this observation. There is a noteworthy practical difference between the two procedures. A GGI is simpler to conduct than a HGC, as the latter requires expertise to achieve and maintain target hyperglycemia (9). Conversely, the operator of a GGI follows a prespecified schedule of escalations in GIR, allowing glycemia to respond dynamically (32). The technical simplicity of the GGI is a practical advantage. Another difference between the procedures is that a HGC typically examines response to a single level of hyperglycemia maintained at a steady-state plateau, whereas a GGI generates a dynamic range of incremental increases of hyperglycemia, which enables the assessment of changes in insulin secretion in response to changing glycemia (18, 32). Our findings of a strong correlation between the insulin secretory responses determined, respectively using a GGI and a HGC, indicate that testing insulin secretory response to a dynamic range for hyperglycemia vs. a single level of hyperglycemia yield similar insights for understanding insulin secretory capacity. However, the unique dynamic nature of a GGI allows the investigator to not only assess insulin secretory capacity but to also simultaneously measure the E871 impact of insulin secretion on the amplitude of glycemic rise, as well as to evaluate the interactive relationship between changes in insulin secretion and prevailing glucose treated as continuous variables. For instance, in the present set of studies, there were dramatic differences in the amplitude of glycemic rise during the GGI procedures when a GLP-1 analog was used (compared with placebo), and there were sharp differences in the amplitude of glycemic rise noted across lean, obese, and T2DM subjects. This attribute of the GGI may be especially valuable toward evaluating novel interventions, particularly pharmacological interventions, to bolster glucose-dependent insulin secretion, whereby understanding the pattern of insulin secretion as hyperglycemia ascends in a stepwise manner while simultaneously determining how the level of insulin secretion attained in turn impacts the glycemic ascent during a GGI is of translational value. Furthermore, insight into the complex relationship between insulin secretory responses to changing glucose and of the glycemic responses to changing insulin secretion across the spectrum of health and disease is likely to add to our understanding of alterations in overall -cell response behavior with disease progression. As will be discussed more fully, an intriguing observation within the present set of studies is the linearity of insulin secretory response across the dynamic range of hyperglycemia achieved during a GGI. Comparison of responses across healthy nonobese, obese nondiabetic, and T2DM subjects assessed using a GGI confirmed prior observations that insulin secretory capacity is significantly blunted in T2DM and tends to be exuberant in obese nondiabetics, presumably as a compensatory response to insulin resistance (8, 21, 22). The insulin secretory response in T2DM was diminished 3.5-fold compared with BMI-matched nondiabetic controls and was about one-half that of the healthy nonobese. In obese nondiabetics, insulin secretion was increased nearly 1.8-fold over healthy nonobese. The glucose sensing and responsiveness of the -cell showed a significant right and downward shift in T2DM compared with nondiabetic populations, and a significant left and upward shift in obese nondiabetic compared with the nonobese. While these quantitative differences between populations are consistent with prior separate reports in these groups (8, 20, 22), the current studies seem to be the first to complete these group comparisons by employing similar protocol and data analyses methodologies across populations. The results affirm the sensitivity of the GGI procedure to assess population-based differences for -cell response to hyperglycemia that are related to obesity, insulin resistance, and diabetes. Although this series of experiments did not include a formal assessment of insulin sensitivity, such measurements may further extend the value of the GGI for -cell function evaluation in the context of insulin sensitivity, particularly across populations. Another central goal of the present studies was to characterize -cell responses to an acute pharmacological intervention using two clinically validated GLP-1 analogs, exenatide and liraglutide, as measured in a GGI. Earlier studies by Kjems et al. (22) in diabetics and Brandt et al. (3) in healthy volunteers with native GLP-1 had suggested the potential usefulness of a GGI in this regard. Our findings demonstrating robust changes, significantly downward for glycemic response and significantly upward for insulin secretion, in response to an incretin analog, are highly consistent with these prior reports with native GLP-1 (3, 22). The changes we observed were

8 E872 evident in healthy individuals and in those with T2DM. Notably, the ability of the GLP-1 analog to augment insulin secretion in T2DM was less robust than in the healthy volunteers (similar to that shown by Kjems et al. with native GLP-1). However, as we have shown, following a single administration of a clinically relevant dose of a GLP-1 analog, -cell glucose sensing and responsiveness in a GGI in T2DM was restored to that observed in obese nondiabetics challenged with hyperglycemia alone. Our findings therefore confirm and extend the findings of Chang et al. on -cell function with liraglutide in a hyperglycemic clamp (10). In prior literature addressing the -cell response to hyperglycemia in vitro and in vivo, including GGI studies, some investigators have reported a plateau in the insulin secretory response (8, 19, 21, 25), whereas others have reported linear relationships in individual populations over a limited range of glycemia (7, 23, 33). In the current set of studies, we did not detect a plateau in the relationship between hyperglycemia and insulin secretory response in any of the tested populations across a fairly wide range of glycemia. Our use of the GGI to investigate the treatment effects of the GLP-1 analog exenatide in nondiabetic nonobese healthy volunteers revealed a striking augmentation of insulin secretion, by nearly 300% over hyperglycemia alone. This response is consistent with the report of Brandt et al. with native GLP-1 (3). Yet, even in the context of this robust response the relationship of ISR to glycemia (and of change in each parameter) remained distinctly linear. This linearity of response in the nondiabetic nonobese healthy volunteers denotes that the dynamic range of hyperglycemia achieved in the GGI did not maximally challenge the full capacity of the -cell functional reserve of these individuals. Arguably, more extreme hyperglycemia, bolstered with stimulation like arginine, is a better means to assess the maximal -cell insulin secretory capacity. But this is not the explicit goal of a GGI; rather, its more germane purpose is to generate a dynamic range of hyperglycemia that spans and moderately exceeds the usual physiological range of hyperglycemia and assess at each step (i.e., at each GIR) the corresponding -cell response. The linearity of the insulin secretory response to progressive hyperglycemia in nonobese healthy is, as earlier noted, consistent with a substantial reserve capacity for insulin secretion. But interestingly, and as to be discussed below, this linearity of response was also evident within the compensatory and exaggerated -cell response of obese volunteers and within the diminished -cell response of T2DM. The compensatory hyperinsulinemia associated with obese nondiabetics was robustly evident in the insulin secretory response to glucose in a GGI. We observed that this exaggerated response sustained a linear relationship to hyperglycemia. In T2DM, insulin secretory capacity was blunted during a GGI; yet here as well, the response was linear across the dynamic range of hyperglycemia. We interpret these linear relationships to indicate that neither in the context of the exaggerated response of obese nor in the diminished response of T2DM, did the stimulus generated by a broad range of clinically relevant hyperglycemia lead to a depletion in the the respective functional reserve capacity for insulin secretion. This suggests that, within this range of hyperglycemia, the activity of glucose sensing and intracellular glucose metabolism within -cells, as well as insulin granule exocytosis, remain tightly proportionate to the dynamic range of hyperglycemia. Taken together, the preservation of functional reserve capacity and proportionality of the relationship between ISR and glucose would appear to explain, in part, the rectifiable nature of the -cell defects in T2DM. Remediation of -cell response in T2DM was evident in the present studies in response to an acute administration of thea GLP-1 analog liraglutide, with GDIS augmented by 200% over placebo. Such a response is not surprising given the large precedent clinical and therapeutic experience of the past decade (2, 10, 14). Yet, it is of interest that this response also adheres to a linear relationship, augmenting insulin secretion by a governed proportion to each increased step across a dynamic range of hyperglycemia (from 126 to 401 mg/dl). It should be noted that the T2DM patients in the current series were well controlled and relatively early in the course of their disease ( 10 yr). It will be interesting to understand whether a linearity of ISR response to a dynamic range of hyperglycemia is evident in those T2DM with more advanced disease, especially in those who require insulin therapy. In summary, this is the first report demonstrating in a head-to-head setting that the GGI is comparable to the hyperglycemic clamp as a tool to measure GDIS effects. Given that a GGI is less demanding to execute than a HGC, technically it seems a more accessible tool to comprehensively measure -cell function and should be given greater consideration in many clinical investigations. We also show that the linearity of -cell glucose sensing and responsiveness is evident under physiological, pathophysiological, and pharmacological conditions across the spectrum between health and T2DM. Further work with the GGI on the effects of interventions, pharmacological and otherwise, will add to our current understanding of -cell physiology and of the capacity for improving impaired -cell function in diabetes and related disorders. ACKNOWLEDGMENTS We thank Sheila Erespe (Merck & Co., Inc.) for assistance with submission. GRANTS This work was funded by Merck & Co., Inc., Kenilworth, NJ. DISCLOSURES S.S. Shankar, R.R. Shankar, L. Mixson, D. Miller, C. Chung, C. Cilissen, C. Beals, A. Stoch, H.O. Steinberg, and D.E. Kelley are or were employees of Merck Sharp & Dohme (MSD) Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, or MSD (Europe) and may own stock or stock options in Merck. AUTHOR CONTRIBUTIONS S.S.S., R.R.S., D.M., C. Chung, C. 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