Coppin, Lucy

Jackson, Nicola

Mari, Andrea

McCreight, Laura J.

Pearson, Ewan

Umpleby, A. Margot

University of Dundee Online Publications

                                                                    University of DundeeMetformin increases fasting glucose clearance and endogenous glucose production innon-diabetic individualsMcCreight, Laura; Mari, Andrea; Coppin, Lucy ; Jackson, Nicola ; Umpleby, A. Margot;Pearson, EwanPublished in:DiabetologiaDOI:10.1007/s00125-019-05042-1Publication date:2019Document VersionPublisher's PDF, also known as Version of recordLink to publication in Discovery Research PortalCitation for published version (APA):McCreight, L., Mari, A., Coppin, L., Jackson, N., Umpleby, A. M., & Pearson, E. (2019). Metformin increasesfasting glucose clearance and endogenous glucose production in non-diabetic individuals. Diabetologia, 1-4.https://doi.org/10.1007/s00125-019-05042-1General rightsCopyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or othercopyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated withthese rights. • Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal.Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 07. Dec. 2019RESEARCH LETTERMetformin increases fasting glucose clearance and endogenousglucose production in non-diabetic individualsLaura J. McCreight1 & Andrea Mari2 & Lucy Coppin3 & Nicola Jackson3 & A. Margot Umpleby3 & Ewan R. Pearson1Received: 14 August 2019 /Accepted: 1 October 2019# The Author(s) 2019Keywords Endogenous glucose production . Glucose clearance .Metformin .Mixed-meal test . Stable isotopesAbbreviationsAMPK 5′ AMP-activated protein kinaseEGP Endogenous glucose productionMMT Mixed-meal testTo the Editor: It is currently accepted that metformin inhibitscomplex I of the respiratory chain, altering the ratio of AMP orADP to ATP and subsequently inhibiting gluconeogenesis [1,2]. The original insulin clamp studies of the 1990s investigatingthe effect of metformin on glucose metabolism in obese andlean individuals with type 2 diabetes support this mechanism ofaction [3], demonstrating a reduction in endogenous glucoseproduction (EGP) following metformin treatment. However,an increasing number of alternative pathways of metforminaction have been proposed, including 5′AMP-activated proteinkinase (AMPK)-dependent and -independent effects, as well asgastrointestinal mechanisms of action [4, 5].It is, therefore, with great interest that we read the articlerecently published in Diabetologia entitled ‘Metforminincreases endogenous glucose production in non-diabeticindividuals and individuals with recent-onset type 2 diabetes’[6]. In the article, using studies of whole-body glucose metab-olism, including non-steady state glucose tracer kinetics andhyperinsulinaemic–euglycaemic clamping, Gormsen et alreport that metformin treatment is associated with an increasein the glucose rate of disappearance (Rd), glucagon levels andEGP both in healthy control individuals and in individualswith recent-onset type 2 diabetes with good glycaemiccontrol. These changes were absent in a placebo-treatedgroup.In this letter we present data from our recent study of non-diabetic individuals, which aimed to investigate the effect ofmetformin on glucose kinetics. Our findings corroborate thefindings of Gormsen et al, adding to the pool of evidenceindicating that the mechanism of action of metformin maybe extra-hepatic.Our study was conducted in the Clinical Research Centre atNinewells Hospital, Dundee, between September 2016 andAugust 2017. It was co-sponsored by the University ofDundee and NHS Tayside, and ethical approval was grantedby the East of Scotland Research Ethics Committee. The studywas conducted in accordance with the Good Clinical Practiceguidelines and the Declaration of Helsinki. The study wasregistered on the public database clinicaltrials.gov (identifierNCT02733679). Formal written informed consent wasobtained from each individual prior to inclusion. Here wereport the data from the non-diabetic controls.In this non-randomised, non-blinded, open-label crossoverstudy we treated non-diabetic individuals with two commonlyused diabetes drugs, namely, metformin and pioglitazone. Thetime in study was 17 weeks, consisting of two 8 week treat-ment periods separated by 1 week washout. During treatmentperiod one, participants were treated with metformin, titratedto a maximum of 1000 mg twice daily. During treatment peri-od two, participants were treated with pioglitazone, titrated to30 mg once daily. Each participant had three study visits:baseline, post-metformin and post-pioglitazone. Each visitincluded a dual-tracer mixed-meal test (MMT), to assessglucose kinetics in the fasted and non-steady state.In brief, participants received a standardised meal theevening before the MMT. They were fasted from midnight* Ewan R. Pearsone.z.pearson@dundee.ac.uk1 Division of Population Health and Genomics, University of Dundee,Ninewells Hospital, Dundee DD19SY, UK2 Institute of Neuroscience, National Research Council, Padova, Italy3 Faculty of Health and Medical Sciences, University of Surrey,Guildford, UKDiabetologiahttps://doi.org/10.1007/s00125-019-05042-1and attended the Clinical Research Centre at 08:00 hours. Aftertaking a baseline blood sample from each participant, a primed(6 mg/kg infused for 1 min) continuous (0.06 mg kg−1 min−1for 120 min) infusion of [6,6-2H2]glucose (Euriso-Top, Saint-Aubin, France) was administered over an initial 2 h period,from −120 to 0 min, to reach steady state. Once in steady state,a liquid mixed meal labelled with [U-13C]glucose (1.7 g addedto 71.9 g glucose drink, 220 ml total volume; CambridgeIsotope Laboratories, Andover, MA, USA), along with liquidparacetamol (1 g in 20 ml), was ingested. Over the following6 h, a variable infusion of [6,6-2H2]glucose was administered,with the rate mimicking the expected EGP post meal. Bloodsamples were taken at 21 time points over the 8 h period.Samples were analysed for glucose at each time point, usinggas chromatography–mass spectrometry to differentiate endog-enous from labelled glucose (Agilent 7890A GasChromatographer and Agilent 5975 Mass Spectrometry detec-tor, Agilent Technologies, Wokingham, UK). Insulin (ELISA80-INSHU-E01.1, Alpco, Salem, MA, USA), glucagon(radioimmunoassay RIA; GL-32 K, Millipore, UK) andparacetamol level (enzymatic bichromatic endpoint techniqueon a Dimension VISTA 1500, Siemens; used as a measure ofgastric emptying) were also measured. Glucose kinetics weremodelled using the circulatory model [7], to assess EGP,glucose clearance and the rate of appearance of oral glucose.Statistical analyses were performed using R studio (rstudio.com; R version 3.1.0, R Foundation for StatisticalComputing, Vienna, Austria). Variables are reported as fastingor meal mean (whereby the average increment was calculatedby dividing the AUC of the variable in question by the durationof the meal test). Data were tested for normality using Shapiro–Wilk test. Normally distributed data are presented as mean ±SEM, and compared within group using the paired t test. Non-normally distributed data are presented as median with inter-quartile range (IQR) and compared using theMann–WhitneyUtest. Statistical significance was defined as p < 0.05.In total, 18 non-diabetic individuals were recruited. Ofthese, 15 completed the study, six of whom (40%) werefemale. The mean age was 22.4 years, BMI was 24.0 kg/m2,and HbA1c was 33 mmol/mol (5.2%). One individual051015202530Baseline Metformin Pioglitazone01234567Baseline Pioglitazone***01234567Baseline Metformin Pioglitazone051015202530Fasting EGP(µmol min–1 kg–1 )Fasting glucose clearance(ml min–1 kg–1 )Meal mean EGP(µmol min–1 kg–1 )Meal mean glucose clearance(ml min–1 kg–1 )Baseline Metformin Pioglitazone***Metformina bc dFig. 1 Fasting EGP (a); meal mean EGP (b); fasting glucose clearance (c) and meal mean glucose clearance (d) at all visits. Data are median (IQR) of allparticipants (n=15) in each cohortDiabetologiawithdrew from the study due to metformin side effects, andtwo participants withdrew due to vomiting during the MMT.Metformin did not affect gastric emptying, nor did it impactupon the rate of appearance of oral glucose. Fasting glucose andinsulin concentrations were unchanged from baseline, followingmetformin treatment. However, there was an increase in fastingglucose clearance compared with baseline (baseline 2.7 [IQR2.3–2.8] vs post metformin 3.0 [IQR 2.7–3.4] ml min−1 kg−1,p< 0.001). This was associated with an increase in the meanglucagon during the MMT (baseline 54.5 [IQR 35.8–71.0] vspost metformin 58.4 [IQR 42.3–78.4] pg/ml, p = 0.007).Fasting glucagon was elevated from baseline, though did notreach significance (baseline 54.8 [IQR 42.0–70.5] vs postmetformin 65.2 [IQR 36.9–83.4] pg/ml, p = 0.28).During fasting, participants are in steady state, where EGP isequal to Rd. Therefore, as there is an increase in glucose clear-ance, metformin treatment also resulted in an increase in EGPunder fasting conditions (baseline 10.9 [IQR 10.1–12.4] vs postmetformin 13.8 [11.7–15.0] μmol min−1 kg−1, p < 0.001) (Fig.1). Mean EGP during the MMTwas elevated but did not reachsignificance (baseline 5.2 [IQR 4.8–6.5] vs post metformin 6.1[IQR 4.4–7.0] μmol min−1 kg−1, p = 0.28). Pioglitazone had noimpact on glucose clearance, EGP or glucagon.This data from dual-tracer MMTs has provided some inter-esting insight into the mechanism of action of metformin.Metformin treatment was associated with increased fastingglucose clearance, which has been previously reported inclamp data [3, 8–10]. The increase in clearance could repre-sent increased glucose uptake by the intestine, which hasrecently been postulated as a ‘glucose sink’ [11, 12].Our non-diabetic group showed a metformin-related coun-ter-regulatory increase in their mean glucagon level, andperhaps this occurs to prevent hypoglycaemia in the contextof increased glucose clearance in these normoglycaemic indi-viduals. In keeping with this, they show an increase in theirfasting EGP, which may be due to glucagon-induced glyco-genolysis or gluconeogenesis. This finding is consistent withthe data presented by Gormsen et al [6].The metformin-associated increase in EGP demonstratesthat the mechanism of action of metformin is not entirelyexplained by a reduction in hepatic gluconeogenesis.However, these metformin response pathways are not neces-sarily mutually exclusive. In the context of markedhyperglycaemia, such as in individuals with poorly controlleddiabetes, the metformin-associated increase in glucose clear-ance may not be sufficient to induce hypoglycaemia or stimu-late counter-regulatory mechanisms, such as glucagon. In thissetting, when baseline hepatic glucose output is high, thehepatic effects of metformin to reduce gluconeogenesis maydominate. A previous clamp study on the effect of metformineffect on glucose metabolism recruited individuals with poorlycontrolled diabetes (baseline HbA1c 84 mmol/mol [9.8%]) [3].These study participants were hyperglycaemic and hadelevated EGP. It is interesting that metformin augmentednon-insulin mediated glucose uptake in both our study and thatby Gormsen et al, but this would have a small impact and, inthe context of hyperglycaemia, would be unlikely to trigger acounter-regulatory response. However, in the healthy individ-ual with normoglycaemia, increased glucose clearance mayput the individual at risk hypoglycaemia, thereby stimulatingglucagon secretion. The inhibitory effect of metformin ongluconeogenesis would be outweighed by the stimulatoryeffects of glucagon on glycogenolysis and gluconeogenesis.In summary, the effect of metformin on glucose metabo-lism appears to be dependent on the glycaemic state of theindividual. Using an alternative study design to that used byGormsen et al, our data lends support to the suggestion thatmetformin increases glucose clearance, which results in anincrease in EGP in the healthy individual, via counter-regulatory hormones, which is in contrast to the reduction inEGP seen in the individual with poorly controlled diabetes.Acknowledgements The authors would like to thankC.Glen (PA to E. R.Pearson, University of Dundee, Dundee, UK) for logistical managementof the participants’ study visits. They would also like to thank L. Cabrelliand H. Loftus (research nurses, CRC, University of Dundee) forconducting study visits and general assistance in running the study, andG. Kiddie (lab manager) and L. King (lab technician) (both CRC,University of Dundee) for initial processing and organising sample stor-age and transfers.Data availability The datasets generated during and / or analysed duringthe current study are available from the corresponding author on reason-able request.Funding This work was funded by the Wellcome Trust New InvestigatorAward (102820/Z/13/Z), awarded to ERP.Duality of interest The authors declare there is no duality of interestassociated with this manuscript.Contribution statement LJM designed and led the study, performedstatistical analysis, and drafted and revised the article. LC and NJperformed mass spec analysis and reviewed the manuscript. AMperformed the glucose kinetics modelling and reviewed the manuscript.AMU contributed to study design, mass spec analysis and reviewed themanuscript. ERP was chief investigator of the study, contributed to studyconception and design, contributed to and reviewed the manuscript and isthe guarantor of this work. All authors reviewed and approved the finalversion for publication.Open Access This article is distributed under the terms of the CreativeCommons At t r ibut ion 4 .0 In te rna t ional License (h t tp : / /creativecommons.org/licenses/by/4.0/), which permits unrestricted use,distribution, and reproduction in any medium, provided you give appro-priate credit to the original author(s) and the source, provide a link to theCreative Commons license, and indicate if changes were made.References1. Hunter RW, Hughey CC, Lantier L et al (2018) Metformin reducesliver glucose production by inhibition of fructose-1-6-Diabetologiabisphosphatase. Nat Med 24(9):1395–1406. https://doi.org/10.1038/s41591-018-0159-72. Miller RA, Birnbaum MJ (2010) An energetic tale of AMPK-independent effects of metformin. J Clin Invest 120(7):2267–2270. https://doi.org/10.1172/JCI436613. DeFonzo RA, Barzilai NIR, Simonson DC (1991) Mechanism ofmetformin action in obese and lean noninsulin-dependent diabeticsubjects. J Clin Endocrinol Metab 73(6):1294–1301. https://doi.org/10.1210/jcem-73-6-12944. McCreight LJ, Bailey CJ, Pearson ER (2016) Metformin and thegastrointestinal tract. Diabetologia 59(3):426–435. https://doi.org/10.1007/s00125-015-3844-95. Rena G, Hardie DG, Pearson ER (2017) The mechanisms of actionof metformin. Diabetologia 60(9):1577–1585. https://doi.org/10.1007/s00125-017-4342-z6. Gormsen LC, Søndergaard E, Christensen NL, Brøsen K, Jessen N,Nielsen S (2019) Metformin increases endogenous glucose produc-tion in non-diabetic individuals and individuals with recent-onsettype 2 diabetes. Diabetologia 62(7):1251–1256. https://doi.org/10.1007/s00125-019-4872-77. Mari A, Stojanovska L, Proietto J, Thorburn AW (2003) A circula-tory model for calculating non-steady-state glucose fluxes.Validation and comparison with compartmental models. ComputMethods Prog Biomed 71(3):269–281. https://doi.org/10.1016/s0169-2607(02)00097-48. McIntyre HD, Ma A, Bird DM, Paterson CA, Ravenscroft PJ,Cameron DP (1991) Metformin increases insulin sensitivity andbasal glucose clearance in type 2 (non-insulin dependent) diabetesmellitus. Aust NZ J Med 21(5):714–719. https://doi.org/10.1111/j.1445-5994.1991.tb01375.x9. Hother-Nielsen O, Schmitz O, Andersen PH, Beck-Nielsen H,Pedersen O (1989) Metformin improves peripheral but not hepaticinsulin action in obese patients with type II diabetes. ActaEndocrinol 120(3):257–265. https://doi.org/10.1530/acta.0.120025710. Chiswick CA, Reynolds RM, Denison FC et al Does metforminreduce excess birthweight in offspring of obese pregnant women?A randomised controlled trial of efficacy, exploration of mecha-nisms and evaluation of other pregnancy complications. Efficacyand Mechanism Evaluation, no. 3.7. NIHR Journals Library,Southampton11. Zarrinpar A, Chaix A, Xu ZZ et al (2018) Antibiotic-inducedmicrobiome depletion alters metabolic homeostasis by affectinggut signaling and colonic metabolism. Nat Commun 9(1):2872.https://doi.org/10.1038/s41467-018-05336-912. Koffert JP, Mikkola K, Virtanen KA et al (2017) Metformin treat-ment significantly enhances intestinal glucose uptake in patientswith type 2 diabetes: results from a randomized clinical trial.Diabetes Res Clin Pract 131:208–216. https://doi.org/10.1016/j.diabres.2017.07.015Publisher’s note Springer Nature remains neutral with regard to jurisdic-tional claims in published maps and institutional affiliations.Diabetologia

                                                                    University of DundeeMetformin increases fasting glucose clearance and endogenous glucose production innon-diabetic individualsMcCreight, Laura J.; Mari, Andrea; Coppin, Lucy ; Jackson, Nicola ; Umpleby, A. Margot;Pearson, EwanPublished in:DiabetologiaDOI:10.1007/s00125-019-05042-1Publication date:2020Licence:CC BYDocument VersionPublisher's PDF, also known as Version of recordLink to publication in Discovery Research PortalCitation for published version (APA):McCreight, L. J., Mari, A., Coppin, L., Jackson, N., Umpleby, A. M., & Pearson, E. (2020). Metformin increasesfasting glucose clearance and endogenous glucose production in non-diabetic individuals. Diabetologia, 63, 444-447. https://doi.org/10.1007/s00125-019-05042-1General rightsCopyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or othercopyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated withthese rights. • Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal.Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 19. Apr. 2023RESEARCH LETTERMetformin increases fasting glucose clearance and endogenousglucose production in non-diabetic individualsLaura J. McCreight1 & Andrea Mari2 & Lucy Coppin3 & Nicola Jackson3 & A. Margot Umpleby3 & Ewan R. Pearson1Received: 14 August 2019 /Accepted: 1 October 2019# The Author(s) 2019Keywords Endogenous glucose production . Glucose clearance .Metformin .Mixed-meal test . Stable isotopesAbbreviationsAMPK 5′ AMP-activated protein kinaseEGP Endogenous glucose productionMMT Mixed-meal testTo the Editor: It is currently accepted that metformin inhibitscomplex I of the respiratory chain, altering the ratio of AMP orADP to ATP and subsequently inhibiting gluconeogenesis [1,2]. The original insulin clamp studies of the 1990s investigatingthe effect of metformin on glucose metabolism in obese andlean individuals with type 2 diabetes support this mechanism ofaction [3], demonstrating a reduction in endogenous glucoseproduction (EGP) following metformin treatment. However,an increasing number of alternative pathways of metforminaction have been proposed, including 5′AMP-activated proteinkinase (AMPK)-dependent and -independent effects, as well asgastrointestinal mechanisms of action [4, 5].It is, therefore, with great interest that we read the articlerecently published in Diabetologia entitled ‘Metforminincreases endogenous glucose production in non-diabeticindividuals and individuals with recent-onset type 2 diabetes’[6]. In the article, using studies of whole-body glucose metab-olism, including non-steady state glucose tracer kinetics andhyperinsulinaemic–euglycaemic clamping, Gormsen et alreport that metformin treatment is associated with an increasein the glucose rate of disappearance (Rd), glucagon levels andEGP both in healthy control individuals and in individualswith recent-onset type 2 diabetes with good glycaemiccontrol. These changes were absent in a placebo-treatedgroup.In this letter we present data from our recent study of non-diabetic individuals, which aimed to investigate the effect ofmetformin on glucose kinetics. Our findings corroborate thefindings of Gormsen et al, adding to the pool of evidenceindicating that the mechanism of action of metformin maybe extra-hepatic.Our study was conducted in the Clinical Research Centre atNinewells Hospital, Dundee, between September 2016 andAugust 2017. It was co-sponsored by the University ofDundee and NHS Tayside, and ethical approval was grantedby the East of Scotland Research Ethics Committee. The studywas conducted in accordance with the Good Clinical Practiceguidelines and the Declaration of Helsinki. The study wasregistered on the public database clinicaltrials.gov (identifierNCT02733679). Formal written informed consent wasobtained from each individual prior to inclusion. Here wereport the data from the non-diabetic controls.In this non-randomised, non-blinded, open-label crossoverstudy we treated non-diabetic individuals with two commonlyused diabetes drugs, namely, metformin and pioglitazone. Thetime in study was 17 weeks, consisting of two 8 week treat-ment periods separated by 1 week washout. During treatmentperiod one, participants were treated with metformin, titratedto a maximum of 1000 mg twice daily. During treatment peri-od two, participants were treated with pioglitazone, titrated to30 mg once daily. Each participant had three study visits:baseline, post-metformin and post-pioglitazone. Each visitincluded a dual-tracer mixed-meal test (MMT), to assessglucose kinetics in the fasted and non-steady state.In brief, participants received a standardised meal theevening before the MMT. They were fasted from midnight* Ewan R. Pearsone.z.pearson@dundee.ac.uk1 Division of Population Health and Genomics, University of Dundee,Ninewells Hospital, Dundee DD19SY, UK2 Institute of Neuroscience, National Research Council, Padova, Italy3 Faculty of Health and Medical Sciences, University of Surrey,Guildford, UKDiabetologiahttps://doi.org/10.1007/s00125-019-05042-1and attended the Clinical Research Centre at 08:00 hours. Aftertaking a baseline blood sample from each participant, a primed(6 mg/kg infused for 1 min) continuous (0.06 mg kg−1 min−1for 120 min) infusion of [6,6-2H2]glucose (Euriso-Top, Saint-Aubin, France) was administered over an initial 2 h period,from −120 to 0 min, to reach steady state. Once in steady state,a liquid mixed meal labelled with [U-13C]glucose (1.7 g addedto 71.9 g glucose drink, 220 ml total volume; CambridgeIsotope Laboratories, Andover, MA, USA), along with liquidparacetamol (1 g in 20 ml), was ingested. Over the following6 h, a variable infusion of [6,6-2H2]glucose was administered,with the rate mimicking the expected EGP post meal. Bloodsamples were taken at 21 time points over the 8 h period.Samples were analysed for glucose at each time point, usinggas chromatography–mass spectrometry to differentiate endog-enous from labelled glucose (Agilent 7890A GasChromatographer and Agilent 5975 Mass Spectrometry detec-tor, Agilent Technologies, Wokingham, UK). Insulin (ELISA80-INSHU-E01.1, Alpco, Salem, MA, USA), glucagon(radioimmunoassay RIA; GL-32 K, Millipore, UK) andparacetamol level (enzymatic bichromatic endpoint techniqueon a Dimension VISTA 1500, Siemens; used as a measure ofgastric emptying) were also measured. Glucose kinetics weremodelled using the circulatory model [7], to assess EGP,glucose clearance and the rate of appearance of oral glucose.Statistical analyses were performed using R studio (rstudio.com; R version 3.1.0, R Foundation for StatisticalComputing, Vienna, Austria). Variables are reported as fastingor meal mean (whereby the average increment was calculatedby dividing the AUC of the variable in question by the durationof the meal test). Data were tested for normality using Shapiro–Wilk test. Normally distributed data are presented as mean ±SEM, and compared within group using the paired t test. Non-normally distributed data are presented as median with inter-quartile range (IQR) and compared using theMann–WhitneyUtest. Statistical significance was defined as p < 0.05.In total, 18 non-diabetic individuals were recruited. Ofthese, 15 completed the study, six of whom (40%) werefemale. The mean age was 22.4 years, BMI was 24.0 kg/m2,and HbA1c was 33 mmol/mol (5.2%). One individual051015202530Baseline Metformin Pioglitazone01234567Baseline Pioglitazone***01234567Baseline Metformin Pioglitazone051015202530Fasting EGP(µmol min–1 kg–1 )Fasting glucose clearance(ml min–1 kg–1 )Meal mean EGP(µmol min–1 kg–1 )Meal mean glucose clearance(ml min–1 kg–1 )Baseline Metformin Pioglitazone***Metformina bc dFig. 1 Fasting EGP (a); meal mean EGP (b); fasting glucose clearance (c) and meal mean glucose clearance (d) at all visits. Data are median (IQR) of allparticipants (n=15) in each cohortDiabetologiawithdrew from the study due to metformin side effects, andtwo participants withdrew due to vomiting during the MMT.Metformin did not affect gastric emptying, nor did it impactupon the rate of appearance of oral glucose. Fasting glucose andinsulin concentrations were unchanged from baseline, followingmetformin treatment. However, there was an increase in fastingglucose clearance compared with baseline (baseline 2.7 [IQR2.3–2.8] vs post metformin 3.0 [IQR 2.7–3.4] ml min−1 kg−1,p< 0.001). This was associated with an increase in the meanglucagon during the MMT (baseline 54.5 [IQR 35.8–71.0] vspost metformin 58.4 [IQR 42.3–78.4] pg/ml, p = 0.007).Fasting glucagon was elevated from baseline, though did notreach significance (baseline 54.8 [IQR 42.0–70.5] vs postmetformin 65.2 [IQR 36.9–83.4] pg/ml, p = 0.28).During fasting, participants are in steady state, where EGP isequal to Rd. Therefore, as there is an increase in glucose clear-ance, metformin treatment also resulted in an increase in EGPunder fasting conditions (baseline 10.9 [IQR 10.1–12.4] vs postmetformin 13.8 [11.7–15.0] μmol min−1 kg−1, p < 0.001) (Fig.1). Mean EGP during the MMTwas elevated but did not reachsignificance (baseline 5.2 [IQR 4.8–6.5] vs post metformin 6.1[IQR 4.4–7.0] μmol min−1 kg−1, p = 0.28). Pioglitazone had noimpact on glucose clearance, EGP or glucagon.This data from dual-tracer MMTs has provided some inter-esting insight into the mechanism of action of metformin.Metformin treatment was associated with increased fastingglucose clearance, which has been previously reported inclamp data [3, 8–10]. The increase in clearance could repre-sent increased glucose uptake by the intestine, which hasrecently been postulated as a ‘glucose sink’ [11, 12].Our non-diabetic group showed a metformin-related coun-ter-regulatory increase in their mean glucagon level, andperhaps this occurs to prevent hypoglycaemia in the contextof increased glucose clearance in these normoglycaemic indi-viduals. In keeping with this, they show an increase in theirfasting EGP, which may be due to glucagon-induced glyco-genolysis or gluconeogenesis. This finding is consistent withthe data presented by Gormsen et al [6].The metformin-associated increase in EGP demonstratesthat the mechanism of action of metformin is not entirelyexplained by a reduction in hepatic gluconeogenesis.However, these metformin response pathways are not neces-sarily mutually exclusive. In the context of markedhyperglycaemia, such as in individuals with poorly controlleddiabetes, the metformin-associated increase in glucose clear-ance may not be sufficient to induce hypoglycaemia or stimu-late counter-regulatory mechanisms, such as glucagon. In thissetting, when baseline hepatic glucose output is high, thehepatic effects of metformin to reduce gluconeogenesis maydominate. A previous clamp study on the effect of metformineffect on glucose metabolism recruited individuals with poorlycontrolled diabetes (baseline HbA1c 84 mmol/mol [9.8%]) [3].These study participants were hyperglycaemic and hadelevated EGP. It is interesting that metformin augmentednon-insulin mediated glucose uptake in both our study and thatby Gormsen et al, but this would have a small impact and, inthe context of hyperglycaemia, would be unlikely to trigger acounter-regulatory response. However, in the healthy individ-ual with normoglycaemia, increased glucose clearance mayput the individual at risk hypoglycaemia, thereby stimulatingglucagon secretion. The inhibitory effect of metformin ongluconeogenesis would be outweighed by the stimulatoryeffects of glucagon on glycogenolysis and gluconeogenesis.In summary, the effect of metformin on glucose metabo-lism appears to be dependent on the glycaemic state of theindividual. Using an alternative study design to that used byGormsen et al, our data lends support to the suggestion thatmetformin increases glucose clearance, which results in anincrease in EGP in the healthy individual, via counter-regulatory hormones, which is in contrast to the reduction inEGP seen in the individual with poorly controlled diabetes.Acknowledgements The authors would like to thankC.Glen (PA to E. R.Pearson, University of Dundee, Dundee, UK) for logistical managementof the participants’ study visits. They would also like to thank L. Cabrelliand H. Loftus (research nurses, CRC, University of Dundee) forconducting study visits and general assistance in running the study, andG. Kiddie (lab manager) and L. King (lab technician) (both CRC,University of Dundee) for initial processing and organising sample stor-age and transfers.Data availability The datasets generated during and / or analysed duringthe current study are available from the corresponding author on reason-able request.Funding This work was funded by the Wellcome Trust New InvestigatorAward (102820/Z/13/Z), awarded to ERP.Duality of interest The authors declare there is no duality of interestassociated with this manuscript.Contribution statement LJM designed and led the study, performedstatistical analysis, and drafted and revised the article. LC and NJperformed mass spec analysis and reviewed the manuscript. AMperformed the glucose kinetics modelling and reviewed the manuscript.AMU contributed to study design, mass spec analysis and reviewed themanuscript. ERP was chief investigator of the study, contributed to studyconception and design, contributed to and reviewed the manuscript and isthe guarantor of this work. All authors reviewed and approved the finalversion for publication.Open Access This article is distributed under the terms of the CreativeCommons At t r ibut ion 4 .0 In te rna t ional License (h t tp : / /creativecommons.org/licenses/by/4.0/), which permits unrestricted use,distribution, and reproduction in any medium, provided you give appro-priate credit to the original author(s) and the source, provide a link to theCreative Commons license, and indicate if changes were made.References1. Hunter RW, Hughey CC, Lantier L et al (2018) Metformin reducesliver glucose production by inhibition of fructose-1-6-Diabetologiabisphosphatase. Nat Med 24(9):1395–1406. https://doi.org/10.1038/s41591-018-0159-72. Miller RA, Birnbaum MJ (2010) An energetic tale of AMPK-independent effects of metformin. J Clin Invest 120(7):2267–2270. https://doi.org/10.1172/JCI436613. DeFonzo RA, Barzilai NIR, Simonson DC (1991) Mechanism ofmetformin action in obese and lean noninsulin-dependent diabeticsubjects. 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McIntyre HD, Ma A, Bird DM, Paterson CA, Ravenscroft PJ,Cameron DP (1991) Metformin increases insulin sensitivity andbasal glucose clearance in type 2 (non-insulin dependent) diabetesmellitus. Aust NZ J Med 21(5):714–719. https://doi.org/10.1111/j.1445-5994.1991.tb01375.x9. Hother-Nielsen O, Schmitz O, Andersen PH, Beck-Nielsen H,Pedersen O (1989) Metformin improves peripheral but not hepaticinsulin action in obese patients with type II diabetes. ActaEndocrinol 120(3):257–265. https://doi.org/10.1530/acta.0.120025710. Chiswick CA, Reynolds RM, Denison FC et al Does metforminreduce excess birthweight in offspring of obese pregnant women?A randomised controlled trial of efficacy, exploration of mecha-nisms and evaluation of other pregnancy complications. Efficacyand Mechanism Evaluation, no. 3.7. NIHR Journals Library,Southampton11. Zarrinpar A, Chaix A, Xu ZZ et al (2018) Antibiotic-inducedmicrobiome depletion alters metabolic homeostasis by affectinggut signaling and colonic metabolism. Nat Commun 9(1):2872.https://doi.org/10.1038/s41467-018-05336-912. Koffert JP, Mikkola K, Virtanen KA et al (2017) Metformin treat-ment significantly enhances intestinal glucose uptake in patientswith type 2 diabetes: results from a randomized clinical trial.Diabetes Res Clin Pract 131:208–216. https://doi.org/10.1016/j.diabres.2017.07.015Publisher’s note Springer Nature remains neutral with regard to jurisdic-tional claims in published maps and institutional affiliations.Diabetologia

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Metformin increases fasting glucose clearance and endogenous glucose production in non-diabetic individuals

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