Title : Exercise training plus metformin , but not exercise training alone , decreases insulin 1 production and increases insulin clearance in adults with prediabetes

45 46 Adding metformin to exercise does not augment the effect of training alone to boost whole-body insulin 47 sensitivity and lower circulating insulin concentrations. While lower insulin concentrations (lower 48 supply) following lifestyle and/or pharmacological interventions are primarily attributed to reductions in 49 insulin secretion that match increases in peripheral insulin sensitivity (lower demand), it is unclear if 50 exercise and/or metformin exert direct effects on insulin production, extraction or clearance. Thirty-six 51 middle-aged, obese, sedentary adults with prediabetes were randomized to either: placebo (P), metformin 52 (M), exercise+placebo (E+P), or exercise+metformin (E+M) for 12 weeks. Fasting plasma proinsulin (an 53 indicator of insulin production), C-peptide, insulin and glucose were collected before and after the 54 intervention. Peripheral insulin sensitivity (euglycemic-clamp), hepatic insulin extraction, insulin 55 clearance, body weight and cardiorespiratory fitness were also measured. Fasting proinsulin was 56 unchanged following P (19.4 ± 10.1 vs. 22.6 ± 15.0 pmol/L), E+P (15.1 ± 7.4 vs 15.5 ± 7.4 pmol/L) or M 57 (24.8 ± 18.9 vs. 16.7 ± 20.3 pmol/L) but was significantly reduced after E+M (18.6 ± 11.9 vs. 13.9 ± 6.7 58 pmol/L, p=0.04). Insulin clearance was significantly greater following M (384.6 ± 19.4 vs. 477.4 ± 49.9, 59 p=0.03) and E+M (400.1 ± 32.0 vs 482.9 ± 33.9, p=0.02) but was unchanged in P or E+P. In this study, 60 metformin combined with exercise training reduced circulating proinsulin, and both groups taking 61 metformin increased insulin clearance. This suggests that adding metformin to exercise may augment or 62 attenuate training effects depending on the outcome or organ system being assessed. 63 64 New and Noteworthy 65 Exercise is increasingly viewed as medication, creating a need to understand its interactions with other 66 common medications. Research suggests metformin, a widely prescribed diabetes medication, may 67 diminish the benefits of exercise when used in combination. In this study however, metformin combined 68 with exercise training, but not exercise alone, lowered proinsulin concentrations and increased insulin 69 clearance in adults with prediabetes. This may directly influence personalized prescriptions of lifestyle 70 and/or pharmacology in order to reduce diabetes risk. 71 by 10.0.33.6 on A uust 5, 2017 http://jaysiology.org/ D ow nladed fom Introduction 72 A key aspect of type 2 diabetes (T2D) prevention is the ability to appropriately match the 73 production and secretion of insulin with the demand of whole-body insulin resistance. Up to 70% of 74 individuals with prediabetes (fasting and/or post-challenge glucose concentrations above normal but 75 below the range of frank T2D) are characterized by an inappropriate matching of insulin secretion and 76 sensitivity (7), and without lifestyle or pharmacological intervention the transition from prediabetes to 77 T2D is highly likely (25). Results from the United States Diabetes Prevention Program suggest that both 78 lifestyle intervention (comprised of weight loss and increased physical activity) and the anti79 hyperglycemia medication metformin delay the transition from prediabetes to T2D (13). Despite a 80 plausible expectation of additivity, recent evidence suggests that combining metformin and exercise 81 training confers no added benefit to whole-body insulin sensitivity and markers of cardiovascular risk 82 when compared to exercise training alone (9, 19, 20). Lifestyle interventions such as physical activity 83 typically increase whole-body insulin sensitivity, which leads to a compensatory reduction in circulating 84 insulin concentrations (6). While most of this compensatory response is a result of reduced insulin 85 secretion (13), insulin concentrations may also be influenced by changes in insulin production (i.e. the 86 conversion of proinsulin to insulin and C-peptide) within the beta cells of the pancreas and/or adjustments 87 to insulin extraction and clearance by the liver. 88 Proinsulin is the precursor prohormone to insulin and C-peptide primarily contained within the 89 beta cells of the pancreas, and serves as a biomarker of insulin production. Elevated circulating proinsulin 90 concentrations represent a mismatch between glycemia and the synthesis and release of insulin. It is 91 therefore not surprising that total proinsulin concentrations, as well as the ratio between proinsulin and 92 both insulin (PI/I) and C-peptide (PI/C), are elevated in adults with prediabetes (15, 30). Additionally, 93 hyperproinsulinemia is associated with both the development and severity of T2D (17, 26), and may serve 94 as a link between impaired glycemic control and cardiovascular disease (4, 31). Circulating proinsulin 95 concentrations are reduced following intervention with lifestyle or metformin (13), but the effects of 96 combining the two interventions are unknown. The removal, extraction and clearance of insulin after it is 97 by 10.0.33.6 on A uust 5, 2017 http://jaysiology.org/ D ow nladed fom produced and released from the beta cell is comprised of two distinct components; First pass hepatic 98 insulin extraction (HIE) and insulin clearance (IC). HIE involves the degradation of insulin by the liver 99 after secretion from beta cells but prior to reaching the general circulation (27). Obese individuals and 100 individuals with hyperinsulinemia often display reduced HIE (12, 24), which may partly contribute to 101 their elevated circulating insulin concentrations. HIE may be higher following interventions that reduce 102 hyperinsulinemia or improve glucose tolerance (14), however it is rarely measured in lifestyle and 103 pharmacological interventions. As a result little is known about how HIE changes with respect to 104 alterations to insulin secretion and sensitivity. HIE may act as a temporary means by which circulating 105 insulin concentrations can be adjusted to match daily fluctuations to insulin sensitivity without the need 106 for upor down-regulation of insulin production within the beta cell, and HIE is often reduced following 107 short-term (i.e. <2 week) interventions that induce transient insulin resistance (e.g. overfeeding) and 108 increased following short-term interventions that increase peripheral insulin sensitivity, such as exercise 109 (14, 24). Insulin clearance (IC), or the systemic breakdown of insulin following release into the general 110 circulation, occurs primarily in the liver (~80%) and kidney (~20%) and is also reduced in obesity (1, 29) 111 and prediabetes (3, 21). IC may respond to pharmacologically augmented weight loss (11) but the effects 112 of lifestyle interventions such as exercise training on IC are unclear. 113 Insulin production and clearance may lie outside the closed-loop relationship between whole114 body insulin sensitivity and compensatory insulin secretion. As a result, changes to these tissue-specific 115 outcomes following lifestyle and/or pharmacological interventions may not be effectively captured with 116 tools (e.g. euglycemic clamp) and metrics (e.g. HOMA-IR) used to evaluate systemic changes to insulin 117 sensitivity and secretion. There is a pressing public health need to maximize the efficacy and precision of 118 T2D prevention, which requires evaluating the independent and interactive effects of lifestyle and 119 pharmacological interventions. Given that exercise training and metformin may regulate glycemic 120 control via different physiological mechanisms of action, their independent and combined effects on 121 insulin production and clearance could impact their utility as agents to prevent or delay diabetes. Thus 122 the purpose of this study was to evaluate the effects of a 12-week exercise training and/or metformin 123 by 10.0.33.6 on A uust 5, 2017 http://jaysiology.org/ D ow nladed fom intervention on insulin production, clearance and extraction. Given our previous work suggesting that 124 combined metformin and exercise training is not more beneficial than exercise alone with respect to 125 cardiometabolic health (19, 20), we hypothesized that metformin would not confer any added benefit, and 126 may even blunt, the beneficial effects of exercise training on IC, HIE and proinsulin processing. 127 128 Methods 129 Overview: The protocol has been previously described in detail elsewhere (19, 20). Briefly, overweight to 130 obese sedentary women and men (Table 1) with impaired glucose tolerance, as determined by a 75 g oral 131 glucose tolerance test, were randomly assigned to one of four groups: Placebo (P, n=8), Metformin (M, 132 n=9), exercise training plus placebo (E+P, n=9), and exercise training plus metformin (E+M, n=10). 133 Exclusion criteria were smoking, weight instability (> 5 kg change over previous 6 months), regular 134 physical activity (> 60 min/wk), or contraindications to metformin. Peak aerobic capacity (VO2peak, 135 cycle ergometer), maximal strength (1 repetition max for key muscle groups), and body composition (dual 136 X-ray absorptiometry, DEXA, Lunar Technologies, Chicago IL) were tested before and after the 137 intervention. All participants were verbally briefed about the study and signed informed consent 138 documents approved by the University of Massachusetts Amherst Institutional Review Board. 139 140 Intervention Protocol: All participants were instructed to maintain baseline diet and physical activity 141 levels throughout the 12-week intervention, and no change in diet (3-day diet records) or habitual 142 ambulation (pedometer) was observed (19). Participants were randomly assigned to receive metformin 143 (1000 mg twice per day separated by 8-12 hours) or an identical placebo and further subdivided into 144 exercise training (3 days/week, 225 total minutes of supervised aerobic and resistance exercise) or non145 training groups. Aerobic exercise consisted of 135 minutes/week on a cycle ergometer at a heart rate 146 corresponding to 65% VO2peak. Resistance training consisted of two 45-minute strength training sessions 147 on non-consecutive days, which focused on upperand lower-body major muscle groups (e.g. bench 148 press, leg extension) at a resistance of 60-70% 1-repition maximum. 149 by 10.0.33.6 on A uust 5, 2017 http://jaysiology.org/ D ow nladed fom 150 Blood collection and hyperinsulinemic-euglycemic clamp: Following 24 hours of dietary and physical 151 activity control (meals provided to ensure caloric and macronutrient balance) and a 10-12 hour overnight 152 fast, blood samples were taken from an indwelling catheter placed in an antecubital vein. Blood samples 153 were collected and plasma was separated in tubes containing EDTA (proinsulin, insulin and C-peptide) 154 and NaF (glucose) and stored at -80° for subsequent analysis. Following the fasting blood draw a 120155 minute hyperinsulinemic-euglycemic clamp (5mmol, 80 mU/m/min) with stable isotope tracers was used 156 to determine peripheral (skeletal muscle) and hepatic insulin sensitivity. Details of the isotope analysis 157 techniques used to calculate rates of glucose disappearance (Rd) and suppression of endogenous glucose 158 appearance (Ra, a metric of hepatic insulin sensitivity) can be found in Malin et al. 2012 (19). 159 160 Biochemical Analysis: Fasting blood glucose was determined using the glucose oxidase method (GM7 161 analyzer, Analox Instruments, Lunenberg MA). Fasting plasma proinsulin, insulin and C-peptide 162 concentrations were determined using a commercial radioimmunoassay (Millipore, Billerica MA). The 163 cross-reactivity of the human proinsulin RIA with insulin and C-Peptide is <0.1%, and the cross164 reactivity of the human C-Peptide RIA for total proinsulin is <4%. The intra-assay coefficient of variation 165 was 4.7%, and the interassay coefficient of variation was <10%. 166 167 Proinsulin processing, hepatic insulin extraction and insulin clearance: Total fasting proinsulin, 168 proinsulin to insulin (PI/I) and proinsulin to C-peptide (PI/C) ratios were calculated to depict proinsulin 169 secretion. Hepatic insulin extraction (HIE) was assessed using the volume-adjusted insulin to C-Peptide 170 ratio as defined by Cobelli and colleagues (5), in which the proportion of insulin reaching the circulation 171 (1-([Insulin]/[C-Peptide)) is divided by preand post-intervention body volume (to account for the 172 confounding variable of body mass changes over the intervention period) before conversion into a 173 percentage. Insulin clearance was determined during the last 30 minutes of the clamp by dividing the 174 insulin infusion rate by the circulating steady-state plasma insulin concentration (SSPI), as previously 175 by 10.0.33.6 on A uust 5, 2017 http://jaysiology.org/ D ow nladed fom reported by Marini and colleagues (21, 22). Whole-body insulin sensitivity was defined as the glucose 176 disposal rate (Rd) during the last 30 minutes of the clamp divided by the SSPI. 177 178 Statistics: Data were analyzed using R statistical software (Vienna AU 2010, http://www.R-project.org). 179 Baseline and group differences were evaluated using a one-way ANOVA. Pre to post differences within 180 groups were determined using paired t-tests. Pearson product moment correlation coefficients were used 181 to determine associations between changes in proinsulin, HIE and IC as well as changes in insulin action 182 and markers of cardiometabolic health. Statistical significance was accepted as p<0.05. 183 184 Results 185 Baseline characteristics and effects of training: Baseline body weight, fitness, insulin sensitivity, and 186 physical activity levels were similar among groups (Table 1). As reported previously (19, 20), VO2peak 187 increased in both E+P (+18%) and E+M (+10%), and weight loss was greater after M (-4%) and E+M (188 7%) compared with P (0%) and E+P (-0.2%). Insulin sensitivity increased following all treatments (Table 189 2), and the rise in sensitivity was 25-30% greater in E+P compared with E+M (19). While there were no 190 changes to fasting glucose, insulin or C-peptide in the P and M groups, there were significant reductions 191 in fasting plasma insulin and C-peptide following E+P and significant decreases in fasting plasma glucose 192 and C-peptide in the E+M group (Table 2). 193 194 Proinsulin and Proinsulin ratios: Baseline proinsulin concentrations did not differ among groups. 195 Compared to baseline, fasting plasma proinsulin was not different in P, M, or E+P, however proinsulin 196 concentrations were significantly lower following E+M (Figure 1). There were no significant differences 197 in the PI/I ratio or PI/C ratio across the interventions (Table 2). There was also no significant correlation 198 between the change in fasting proinsulin and change in insulin sensitivity (r=-0.127, p=0.46), fasting 199 plasma glucose (r=0.199, p=0.25), or body fat percentage (r=0.323, p=0.64). 200 201 by 10.0.33.6 on A uust 5, 2017 http://jaysiology.org/ D ow nladed fom Hepatic extraction and insulin clearance: There were no significant changes to first pass HIE in the 202 control group or any of the intervention groups (Table 2). There was also no significant correlation 203 between the change in HIE and the change in fasting proinsulin (r=0.053, p=0.76), insulin sensitivity (r=204 0.064, p=0.96), body fat percentage(r=0.139, p=0.88) or body weight (r=-0.208, p=0.43). Steady state 205 plasma insulin was significantly lower in M and E+M, but there was no difference in P or E+P (Figure 206 2A). Similarly, insulin clearance was also significantly increased in M and E+M, and unchanged in P and 207 E+P (Figure 2B). Post-intervention insulin clearance was also significantly greater than insulin clearance 208 in the placebo group, both prior to and following completion of the intervention period (Figure 2B). There 209 was a significant association between the change in insulin clearance and change in whole-body insulin 210 sensitivity (r=0.344, p=0.014), however there was no association between insulin clearance and fitness 211 (r=0.291, p=0.15), body fat (r=-0.143, p=0.42) or hepatic insulin sensitivity (r=0.081, p=0.78). 212 213 Discussion 214 In this study, fasting proinsulin was significantly reduced (-24%) only after exercise training was 215 combined with metformin. There was a 20% decrease with metformin alone that was comparable to the 216 combined intervention, but this was not statistically significant. Additionally, rates of steady state insulin 217 clearance during a hyperinsulinemic-euglycemic clamp were also significantly greater following 12 218 weeks of metformin, with or without exercise training, but not with placebo or exercise training alone. 219 These results surprised us because we expected both proinsulin concentrations and insulin clearance to 220 follow a similar pattern to that of insulin sensitivity, which was most strongly enhanced by exercise 221 training alone (19, 20). Consistent with findings from our previous studies, these changes to proinsulin 222 and insulin clearance were not influenced by age or sex differences. The decoupling of changes to 223 proinsulin and insulin clearance from changes to insulin sensitivity and fasting insulin suggests that 224 metformin and exercise differentially impact the relationship between insulin demand (tissue sensitivity) 225 and supply (production and secretion). 226 by 10.0.33.6 on A uust 5, 2017 http://jaysiology.org/ D ow nladed fom There are several explanations for these results. In the current study, only the two groups that 227 received metformin lost weight. Weight loss drives many of the beneficial outcomes of lifestyle or 228 pharmacologic interventions on cardiometabolic health/disease risk, and this may be true for insulin 229 clearance as well. However the driving force behind these positive changes is generally considered to be 230 loss of fat, particularly loss of abdominal fat (19). In our study it was only the two exercise groups, not 231 the metformin-only group, who lost total and central adiposity, and a causal relationship between 232 weight/fat loss and both lower proinsulin and greater insulin clearance is not supported. Similarly, 233 although greater whole-body insulin sensitivity was strongly associated with higher cardiorespiratory 234 fitness (CRF), there was no change to fasting proinsulin or insulin clearance in the exercise-only group, 235 which showed the largest rise in CRF. In contrast, metformin alone caused a 20% reduction in proinsulin 236 and a 20% increase in insulin clearance despite no change in CRF. These results suggest that changes in 237 CRF are not necessary to alter fasting proinsulin or insulin clearance. 238 It is also possible that in men and women with prediabetes, elevated fasting proinsulin levels and 239 increased insulin clearance more closely reflect fasting hyperglycemia than hepatic or peripheral insulin 240 resistance. If so, fasting proinsulin may only decline subsequent to, or concomitantly with, lowering of 241 fasting glucose concentrations. We observed no association between change in proinsulin and changes in 242 fasting glucose. However, it is conceivable that any relationship between the two was obscured by the 243 relatively modest fasting hyperglycemia at baseline, restricting the magnitude of any declines in fasting 244 glycemia and, therefore, proinsulin. The only group that exhibited a statistically significant decline in 245 proinsulin was the E+M group, which also had a statistically significant reduction in fasting glucose 246 concentration. Additionally, while there were significant reductions in proinsulin (E+M) and increases 247 insulin clearance (M, E+M) over the intervention period, there were no significant differences in 248 proinsulin or insulin clearance between groups at baseline or after each intervention. It is unclear why 249 these within-group changes did not manifest as significant differences between groups following the 250 intervention period, however the high degree of variability within groups, especially the metformin only 251 group, may have played a role. 252 by 10.0.33.6 on A uust 5, 2017 http://jaysiology.org/ D ow nladed fom In addition to suppressing hepatic gluconeogenesis (18), metformin may also affect signaling 253 pathways within the beta cell (16), and it is possible that metformin, but not exercise, has a direct impact 254 on insulin production in humans but exercise does not. While only exercise training plus metformin 255 significantly lowered proinsulin concentrations, the pattern of changes in the metformin groups suggests 256 that metformin may act directly on the beta cell to lower insulin production. This hypothesis is clearly 257 preliminary, but is supported by recent work in isolated pancreatic beta cells, in which metformin altered 258 intracellular insulin processing via an AMP kinase-related mechanism (10, 23). It is also possible that 259 metformin directly influences hepatic and renal insulin clearance via upregulation of key insulin 260 degrading enzymes and functions (2, 8), but that exercise training has a minimal effect on this degradation 261 process. Directly testing how the combination of exercise training and metformin impacts insulin 262 synthesis within the beta cells of the pancreas, as well as insulin degradation within hepatocytes, will 263 require animal models and cell culture work in follow-up studies. 264 In contrast to the effects of metformin, discord between changes in circulating insulin, proinsulin, 265 insulin sensitivity and insulin clearance in the exercise groups suggests that exercise training may modify 266 glycemic control primarily by enhancing peripheral insulin sensitivity and consequently reducing insulin 267 secretion. In addition to a lack of effect of exercise training alone on fasting proinsulin concentrations, 268 we observed no effect of exercise training alone on either measure of insulin clearance (HIE or IC). 269 However, the calculation of hepatic insulin extraction using fasting C-peptide and insulin kinetics relies 270 on several assumptions (5), and without a preand post-intervention glucose challenge it is hard to argue 271 that hepatic extraction has no role in the adaptations to glycemic control following exercise training. The 272 lack of a preand post-intervention intravenous or oral glucose challenge also limits the scope of insulin 273 secretion to fasting C-peptide and insulin alone, and therefore the potential relationship between changes 274 to insulin secretion and insulin production/clearance following exercise training and/or metformin is 275 incomplete. A complete understanding of how exercise training or metformin alters insulin supply and 276 demand will require cleverly designed studies to tease apart several interrelated processes. 277 by 10.0.33.6 on A uust 5, 2017 http://jaysiology.org/ D ow nladed fom Understanding the effects of exercise or metformin on measures of glycemic control requires 278 considering the larger context. The hyperbolic law of insulin kinetics suggests that the relationship 279 between insulin demand (sensitivity or resistance) and supply (secretion and clearance) is coupled, such 280 that decreasing sensitivity leads to higher circulating insulin concentrations, and vice versa (28). The 281 current study suggests that metformin, but not exercise alone, could change insulin production and 282 clearance without the necessity for upstream changes to insulin sensitivity. If true, there are potentially 283 important clinical ramifications. For example, one of the oft-cited benefits to improving insulin sensitivity 284 is reducing hyperinsulinemia and “resting” the pancreas to preserve beta cell function. If lower circulating 285 insulin is a result of changes in post-secretion insulin clearance instead of reductions in first and/or second 286 phase insulin secretion, there may be little or no reduction in beta cell “stress.” Additionally, if there is 287 recognized value in reducing insulin secretion by the islets regardless of the degree of insulin reaching the 288 general circulation, there may be practical reasons to choose between metformin or exercise for patients 289 who are still able to compensate for insulin resistance with hyperinsulinemia. The divergent impact of 290 exercise and/or metformin on tissue-specific (e.g. proinsulin and insulin clearance) compared to whole291 body (e.g. insulin action) metrics of glycemic control suggests that the utility of selecting one treatment 292 versus the other, or combining both treatments, is outcome-specific. Scaling up to the critical public 293 health issue of preventing diabetes and cardiovascular disease in humans, the independent and combined 294 actions of physical activity and/or metformin on the transition from prediabetes to T2D is difficult to 295 predict from studies of insulin sensitivity or biomarkers. To understand the comparative efficacy of 296 physical activity and/or metformin on T2D prevention, studies will need to be conducted in the target 297 population with the development of frank T2D as the primary outcome. 298 299 Acknowledgements: 300 The authors would like to thank Dr. Stuart Chipkin for his contributions towards study design and 301 assistance with the hyperinsulinemic-euglycemic clamp procedures 302 Author Disclosure: 303 by 10.0.33.6 on A uust 5, 2017 http://jaysiology.org/ D ow nladed fom The authors report no conflict of interest related to the research presented within this article.304Funding: NIH 5 R56 DK081038 and ACSM Doctoral Student Foundation305306307308309310311312313314315316317318319320321322323324325326327328329by10.0.33.6onAuust5,2017http://jaysiology.org/Downladedfom