combined glp-1, oxyntomodulin and peptide yy improves body … · 2019-11-13 · 1 combined glp-1,...

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Combined GLP-1, oxyntomodulin and peptide YY improves body weight and glycaemia in obesity and prediabetes/type 2 diabetes: a randomized single-blinded placebo controlled study

Running title: GOP combination in obesity and type 2 diabetes

Preeshila Behary PhD1*, George Tharakan PhD1*, Kleopatra Alexiadou PhD1, Nicholas Johnson MSc2, Nicolai J. Wewer Albrechtsen PhD3,4, Julia Kenkre MB BChir1, Joyceline Cuenco PhD1, David Hope MBBS1, Oluwaseun Anyiam MBBS1, Sirazum Choudhury MBBS1, Haya Alessimii MSc1, Ankur Poddar MBBS1, James Minnion PhD1, Chedie Doyle BSN1, Gary Frost PhD1, Carel Le Roux PhD1,5, Sanjay Purkayastha MD6, Krishna Moorthy MD6, Waljit Dhillo PhD1, Jens J. Holst MD7, Ahmed R. Ahmed PhD6, Toby Prevost PhD2, Stephen R. Bloom MD1, Tricia M. Tan PhD1†

*These authors contributed equally1Section of Investigative Medicine, Imperial College London, London W12 0HS, UK.2Imperial Clinical Trials Unit, Imperial College London, London W12 7RH, UK.3Department of Clinical Biochemistry, Rigshospitalet, 2100 Copenhagen Ø, Denmark.4NNF Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.5School of Medicine, University College Dublin, Dublin 4, Ireland.6Department of Surgery and Cancer, Imperial College Healthcare NHS Trust, London SW7 2AZ, UK.7Panum Institute, Department of Biomedical Sciences and the NNF Center for Basic Metabolic Research, University of Copenhagen, 2200 Copenhagen N, Denmark.

†Corresponding author:Prof Tricia TanDivision of Diabetes, Endocrinology and MetabolismSection of Investigative Medicine6th Floor Commonwealth BuildingImperial College LondonDu Cane Road, London W12 0HSEmail: [email protected]; Tel: 020 75942665

Word Count: 3995; Main Text Tables and Figures: 4

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CONFIDENTIAL-For Peer Review Only

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Abstract

Objective

Roux-en-Y gastric bypass (RYGB) augments post-prandial secretion of glucagon-like peptide-

1 (GLP-1), oxyntomodulin (OXM) and peptide YY (PYY). Subcutaneous infusion of these

hormones (‘GOP’) mimicking post-prandial levels reduces energy intake. Our objective was

to study the effects of GOP on glycaemia and body weight when given for 4 weeks to patients

with diabetes and obesity.

Research Design and Methods

In this single-blinded mechanistic study, prediabetic/diabetic obese patients were randomized

to GOP (n=15) or Saline (n=11) infusion for 4 weeks. We also studied 21 patients who had

RYGB, and 22 patients on very low calorie diet (VLCD) as unblinded comparators. Outcomes

measured were: (a) body weight; (b) fructosamine levels; (c) glucose and insulin during mixed

meal test (MMT); (d) energy expenditure (EE); (e) energy intake (EI); (f) mean glucose and

measures of glucose variability during continuous glucose monitoring.

Results

GOP infusion was well tolerated over the 4 week period. There was a greater weight loss

(p=0·025) with GOP (mean change -4·4 [95% CI -5·3, -3·5] kg) vs Saline (-2·5 [-4·1, -0·9]

kg). GOP led to a greater improvement (p=0·0026) in fructosamine (-44·1 [62·7, -25·5]

µmol/L) vs Saline (-11·7 [-18·9, -4·5] µmol/L). Despite a smaller weight loss compared to

RYGB and VLCD, GOP led to superior glucose tolerance after a mixed meal stimulus and

reduced glycaemic variability compared to RYGB and VLCD.

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Conclusion

GOP infusion improves glycaemia and reduces body weight. It achieves superior glucose

tolerance and reduced glucose variability compared to RYGB and VLCD. GOP is a viable

alternative for the treatment of diabetes with favourable effects on body weight.

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Bariatric surgery, in particular Roux-en-Y Gastric Bypass (RYGB), remains the most

efficacious treatment for obesity and type 2 diabetes (T2DM). Glycaemic improvement after

RYGB is superior to intensive medical management and is sustained, with diabetes remission

rates of 26-29% at 5 years (1). Mechanistic studies have shown an early improvement in hepatic

insulin sensitivity and later in peripheral insulin sensitivity as well as beta-cell function (2).

Proposed mechanisms include: early post-surgical calorie restriction; post-prandial secretion

of the satiety gut hormones glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM) and

peptide YY (PYY); reduced secretion of the orexigenic hormone Ghrelin; bypass of the

proximal small bowel and reduced secretion of an ‘anti-incretin’; changes in bile acid

metabolism; changes in gut microbiota composition, reprogramming of intestinal glucose

metabolism and increase in energy expenditure, among others (3). However, the exact

contributions from each mechanism are not clear.

GLP-1, OXM and PYY are released from the L cells of the small intestine and colon, and their

post-prandial secretion is augmented several-fold after RYGB (4; 5). GLP-1 is insulinotropic

and reduces food intake. Its analogues are an established treatment for diabetes (6) as well as

obesity (7). OXM is a dual agonist of the GLP-1 and glucagon receptors (8) which reduces

food intake (9), increases energy expenditure (10), leading to weight loss (11). PYY acts to

reduce appetite and food intake post-prandially (12; 13). Combinations of GLP-1 and PYY

(14; 15), OXM and PYY (16) and GLP-1 and glucagon (17) have synergistic effects on food

intake. Replicating the post-prandial gut hormone levels after RYGB is possible using a

continuous subcutaneous infusion of combination of GLP-1, OXM and PYY (‘GOP’). This is

safe, acceptable and led to a reduction in total ad libitum energy intake compared to placebo,

over a 10·5 hour infusion (5). A continuous subcutaneous infusion obviates the development

of sharp peaks and leads to a gradual rise towards steady gut hormone plasma levels (5) which

enhances tolerability. If needed to improve tolerability, the infusion can be stopped or the rate

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reduced, with hormone levels responding within 1 hour because of their short half-lives. This

is not possible with injections of analogues possessing extended pharmacokinetics. Lastly,

continuous subcutaneous infusions are commonplace in diabetes treatment, demonstrating its

practicality in daily life. We therefore hypothesized that a GOP infusion, given for 4 weeks in

‘free living’ conditions, would reduce body weight and improve glycaemia in comparison to a

placebo 0·9% saline (hereafter referred to as Saline) infusion. To compare the metabolic effects

of GOP with those of RYGB we also studied a group of patients who underwent surgery. To

compare the effects of GOP with simple dietary restriction, we also studied a group of patients

who followed an 800 kcal/day very low-calorie diet (VLCD).

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Research Design and Methods

Study Design and Participants

This mechanistic study took place at the NIHR Imperial Clinical Research Unit Facility at

Hammersmith Hospital, London from Jul 2016 to Oct 2018. It was a single-blinded randomized

controlled study comparing two infusion groups (GOP or Saline) in patients with obesity, and

either prediabetes or T2DM. Two further similar non-blinded groups of patients undergoing

RYGB and patients undergoing a VLCD diet were recruited. Potential volunteers for the GOP,

Saline and VLCD groups were recruited from clinics at the Imperial Weight Centre (IWC) or

from newspaper advertising whereas patients already listed for surgery at the IWC were

recruited to form the RYGB group.

Inclusion and Exclusion Criteria

Key eligibility criteria were male or female participants aged between 18-70 years, meeting the

UK National Health Service criteria for bariatric surgery, with a diagnosis of prediabetes

(impaired fasting glucose, impaired glucose tolerance or HbA1c of 6·0-6·4% [42-47

mmol/mol]) or T2DM according to WHO criteria. If diabetic, patients had a stable HbA1c of

≤9·0% (75 mmol/mol) either on diet or a single oral hypoglycemic agent. Key exclusion criteria

were any co-morbidities or medications which could compromise the validity and safety

aspects of the study, a current history of smoking, pregnancy and a history of eating disorders

or restrained eating habits as assessed by SCOFF and DEB-Q questionnaires (18-20).

Interventions

Infusion groups (GOP or Saline)

GLP-1(7-36)amide, PYY(3-36)amide and OXM were manufactured to GMP standards

(AmbioPharm). They were mixed under sterile conditions and freeze-dried in single vials.

Visually identical vials containing freeze-dried 0·9% saline were also manufactured. Vials

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were reconstituted with sterile water for injections and added to 0·9% saline. Reconstituted

peptides were verified to be stable for over 12 hours at 37°C by high-performance liquid

chromatography (Figure S1). A Cane Crono Infusion pump (Applied Medical Technology) and

an infusion set (Medtronic) was used to deliver the subcutaneous infusion of GOP at a dose of

4/4/0.4 pmol/kg/min respectively (5). Volunteers were allocated to receive either GOP or

Saline infusion for 28 days, by simple randomization, performed by an independent

investigator. Volunteers’ usual treatment for diabetes was suspended for the duration of the

study. Only volunteers remained blinded to the nature of the infusion from allocation until the

end of the 28-day infusion period. They were instructed to run the infusion an hour prior to

breakfast and disconnect after their last meal of the day, for 12 hours per day. On study days,

the infusions were commenced at least 2 hours prior to any study procedures. All volunteers

also received dietetic advice on healthy eating and weight loss from a qualified dietician.

RYGB group

Volunteers undergoing RYGB attended the research unit for a baseline visit prior to surgery

and were reviewed at 2, 4 and 12 weeks post-surgery. RYGB surgery was performed

laparoscopically according to standardized techniques by three designated surgeons at IWC.

Very Low Calorie Diet (VLCD) group

Volunteers attended the research unit for a baseline visit, before starting a complete meal

replacement VLCD of 800 kcal/day for 4 weeks (Cambridge Weight Plan). They were

reviewed by a dietician pre-diet and then on a weekly basis till completion.

Method Details

Anthropometry and body composition

Body weight and body composition were measured using a Tanita T8148.

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Mixed meal test (MMT)

A standardised MMT (Ensure Compact®, 14 g protein, 12·9 g of fat, 39·6 g of carbohydrates,

330 kcal, 137·5 ml, Abbott), was administered prior to and at the end of each intervention.

Blood for glucose, insulin and glucagon levels were sampled at T=-30, 0, 15, 30, 60, 120, 180

and 240 minutes from time of meal ingestion, via an indwelling cannula placed in the ante-

cubital fossa. Estimates of hepatic insulin sensitivity (iHOMA2 %S) were derived from fasting

glucose and insulin levels (21).

Continuous Glucose Monitoring (CGM)

Subjects were fitted with a blinded CGM (Dexcom G4 Platinum) before their baseline visit and

on week 3 of the intervention. Data was collected for 7 days under free living conditions and

were analysed using the easyGV calculator (http://www.phc.ox.ac.uk/research/technology-

outputs/easygv) for measures of glycaemic variability (GV).

Indirect Calorimetry and 3-D Accelerometry for Energy Expenditure (EE)

Indirect calorimetry using a ventilated hood system (GEM Nutrition) was used to measure

Resting Energy Expenditure (REE) and Diet-induced Thermogenesis (DIT) (5). After resting

for 30 minutes in a recliner chair, REE was measured in the fasted state for 15-20 minutes. The

EE measurement was repeated at 30 minutes after the ingestion of the MMT to estimate DIT.

Activity-related EE (AEE) was estimated using an Actiheart device (CamNTech).

Ad Libitum and 24h Energy Intake (EI) Measurement

Ad libitum EI studies (5) were carried out at baseline and at 4 weeks following the infusions

but at 12 weeks after RYGB (as this group could not tolerate solid meals at 4 weeks and had

resumed normal eating by 12 weeks). To estimate ‘free living’ 24h EI, volunteers were asked

to fill in 72h food diaries and mean intakes over 24h calculated using Dietplan7 (Forestfield

Software).

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http://www.phc.ox.ac.uk/research/technology-outputs/easygv

http://www.phc.ox.ac.uk/research/technology-outputs/easygv

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Blood Sampling and Assays

Blood for fructosamine was collected in clot activator tubes and analysed using the colorimetric

method (within-batch CV <1%, between-batch CV <2·5% at between 260 and 500 μmol/L;

Sandwell and West Birmingham Hospitals NHS Trust). Blood for gut hormones was collected

in lithium heparin tubes containing Aprotinin (Nordic Pharma) and the DPP-IV inhibitor

Diprotin A (Enzo Life Sciences). Samples were placed on ice and centrifuged at 4°C within 10

minutes of collection. They were stored at -80°C until analysis. Active GLP-1 levels were

measured by the Milliplex magnetic bead-based multi-analyte, metabolic panel, four-plex

immunoassay (Millipore). Total PYY was measured by an in-house radio-immunoassay. OXM

was measured using a specific and sensitive mass-spectrometry validated immunoassay (Holst

Laboratory, University of Copenhagen). The intra-assay and inter-assay coefficient of variation

for active GLP-1, PYY, and OXM assays was <10% and the lowest limit of detection was 0·8

pmol/L for GLP-1, 8·7 pmol/L for PYY and 5 pmol/L for OXM. Glucagon was measured using

an ELISA (Mercodia AB), with a detection limit of 1·5 pmol/L. Blood for glucose was

collected in fluoride oxalate tubes and for insulin and lipids in plain tubes. Glucose, insulin and

lipid levels were measured by NW London Pathology (Abbott Architect; CVs <5%, <10%,

<5% respectively).

Outcomes and Statistical analysis

The primary outcome of the study was the change in body weight at 4 weeks. Secondary

outcomes were: change in glycaemia, as assessed by fructosamine measurement, change in ad

libitum and 24-hour total EI, fasting and post-prandial glucose and insulin levels in response

to a mixed meal test, GV as assessed by Continuous Glucose Monitoring (CGM); energy

expenditure. Twenty subjects per group were calculated to give an 85% power to detect a

minimal clinically significant difference in weight loss of 2·5 kg between Saline and GOP at 4

weeks (SD 2·6 kg based on pilot data). With regards to fructosamine, a sample size of

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approximately 13 volunteers per group would provide 95% power (alpha 5%, S.D. 28 µmol/L,

two-tailed t-test) to detect a change of ~32 µmol/L, equivalent to a minimal clinically

significant HbA1c change of 0·5%. Analysis was carried out based on a per-protocol approach.

The primary comparison between GOP and Saline, and the secondary comparisons of GOP vs

RYGB and GOP vs VLCD were made using an unpaired Student t-test for the primary and

secondary outcomes and a p value of <0·05 was considered significant. Glycaemic parameters,

metabolic profiles in response to a mixed meal test (MMT), energy balance parameters and

lipid parameters were assessed using the same methodology for the primary outcome analysis.

To look for potential baseline effects for fructosamine, analysis of covariance (ANCOVA) was

used for the corresponding tests of GOP vs RYGB and GOP vs VLCD incorporating baseline

fructosamine as a covariate. Additional sensitivity tests investigated outliers highlighted during

normality testing to estimate outlier influence. A sensitivity analysis showed that the

conclusions were robust with and without imputation. Post-prandial insulin and glucose levels

from t=0 to 240 minutes were analysed using area-under-the-curve (AUC0-240) derived using

the trapezoidal method. Where values were missing at t=0, the preceding t=-30 value was

carried forward. Software packages used for analysis were the R environment (v 3·5, R

Foundation) and GraphPad Prism (v.8·0·1, GraphPad Software).

Study Approvals

The described study is part of a series of experimental medicine studies on the mechanisms of

bariatric surgery (ClinicalTrials.gov NCT01945840). This was not a clinical trial of an

investigational medicinal product, confirmed after a protocol review with the UK Medicines

and Healthcare Regulatory Agency. Ethical approval was obtained from the UK National

Health Service Health Research Authority West London National Research Ethics Committee

(reference 13/LO/1510). Substantial amendments to study methods were approved in July 2014

(to increase HbA1c cut-off for inclusion), Dec 2014 (refinements to study visit procedures),

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June 2015 (increased upper limit for age to 70), Oct 2015 (added CGM to study procedures),

Dec 2015 (added food preferences study, not reported here), May 2016 (removed upper limit

for BMI for inclusion, previously 50 kg/m2), Dec 2017 (to include future arms to the study, not

reported here). All participants provided written informed consent and the study was conducted

according to the principles of the Declaration of Helsinki.

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Results

Baseline characteristics

The disposition of volunteers in the study are shown in Figure S2. Data were available for a

total of 21 who underwent a RYGB, 22 who had a VLCD, 15 who were infused GOP and 11

who received Saline. Nine were excluded from the analysis set (6 from GOP and 3 from Saline)

due to technical issues with the infusion pump, inability to attend study visits and unrelated

conditions occurring during the study. Baseline characteristics are listed in Table 1. Regarding

the primary comparison of GOP vs Saline, there were no significant differences in baseline

characteristics. RYGB and VLCD volunteers were similar in weight to GOP but had lower

baseline fructosamine levels. Most volunteers were diabetic (68-87%) and baseline treatments

were for the most part evenly split between diet-only and metformin monotherapy.

Gut hormone levels during GOP are maintained at post-prandial levels observed

4 weeks post RYGB

The fasting and post-prandial levels of GLP-1, OXM and PYY following an MMT are shown

in Figure S3. Target levels of GLP-1, OXM and PYY equivalent to post-prandial peak levels

post RYGB were achieved and maintained using a GOP infusion at doses of 4/4/0·4

pmol/kg/min respectively.

GOP leads to significantly more weight loss than Saline

GOP was significantly more effective than Saline in reducing weight at -4·4 kg (percentage

change from baseline -4·0%), versus Saline at -2·5 kg (-2·0%) – see Table 2. The changes in

weight for RYGB were -10·3 kg (-8·8%) and for VLCD, -8·3 kg (-7·6%). Both RYGB and

VLCD lost significantly more weight than GOP. Reductions in both fat mass and muscle mass

proportionate with body weight reductions were recorded with all interventions (Table 2).

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Glycaemia improves after GOP, RYGB and VLCD interventions

Fructosamine was used as the biomarker for average glycaemia because of its more rapid

response to a glucose-lowering intervention in comparison to glycated haemoglobin. The mean

absolute change in fructosamine after 4 weeks GOP (Table 3) was -44·1 µmol/L, compared to

Saline at -11·7 µmol/L, difference in treatment effect 32·4 µmol/L (p = 0·0026, unpaired t-

test). A subsequent sensitivity analysis to explore any potential baseline effect on the results

using ANCOVA indicated a similar statistically significant difference in treatment effect

between GOP and Saline (p=0·0001). As expected, RYGB led to a significant reduction in

fructosamine (-34·0 µmol/L) as did VLCD (-28·5 µmol/L). There was no significant difference

in treatment effects when comparing either GOP and RYGB, or GOP and VLCD.

Fasting glucose was reduced significantly both by GOP and Saline infusion but the treatment

effect with GOP was significantly larger than Saline (Table 3). Fasting glucose fell also after

RYGB and VLCD. GOP had a significantly better treatment effect on fasting glucose compared

to RYGB. The treatment effects of GOP vs VLCD on fasting glucose were not significantly

different (Table 3).

To assess the impact of the interventions on ‘free living’ glycaemia, we measured mean glucose

and mean amplitude glucose change (MAG: an index of GV which measures summed absolute

differences between sequential readings divided by the time between the first and last glucose

measurement) from CGM records taken at baseline and during the 3rd week of each

intervention (Figure S4). GOP significantly reduced mean glucose by -3.6 mmol/L whereas the

Saline group saw no significant change in mean glucose (Table 3). RYGB also reduced mean

glucose by -2·5 mmol/L; comparison of GOP vs Saline showed that the treatment effect was

significantly better with GOP. VLCD patients did not undergo CGMS.

In terms of GV, there was a significant reduction in MAG with GOP whereas the Saline group

saw no significant change in MAG (Table 3). The difference in treatment effect on MAG

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between GOP and Saline was significant. There was no significant reduction of MAG with

RYGB . Comparing GOP with RYGB, the difference in treatment effects on this parameter

was significant.

The glucose and insulin dynamics during Mixed Meal Test differ between

interventions

Given that average glycaemia was improved by both GOP and RYGB, but GV was improved

by GOP but not RYGB, we analyzed glucose and insulin dynamics during a mixed meal test

(MMT) before and after interventions. During the post-intervention MMT in RYGB, there

was a marked post-prandial glucose peak at 30 minutes reflecting rapid transit and absorption

in the jejunum (Figure 1A), accompanied by a large insulin peak at 60 min (Figure 1B), leading

to glucose disposal and a subsequent fall in glucose. In contrast, the glycaemic response with

GOP was nearly flat (Figure 1E), accompanied by a relatively small insulinotropic response

(Figure 1F). VLCD led to an overall reduction of glucose levels by ~2 mmol/L compared to

baseline (Figure 1C), but there was no change in the insulin secretion profile (Figure 1D).

The post-prandial glucose excursion during the MMT as measured by the area under the curve

(Glu AUC0-240) was compared between baseline and after 4 weeks of each intervention. There

was a profound reduction of Glu AUC0-240 after GOP, whereas there was no significant change

after Saline. GOP led to a reduction in the AUC for insulin (Ins AUC0-240) and again there was

no significant change in Ins AUC0-240 after Saline (Table S2). RYGB led to a significant overall

reduction in Glu AUC0-240, but the treatment effect was similar between GOP and RYGB.

However, RYGB caused an increase in Ins AUC0-240 in comparison to the fall with GOP (Table

S2). Comparing GOP and VLCD, although there was a significant reduction in Glu AUC0-240

with VLCD the treatment effect was significantly better with GOP than VLCD. VLCD also

led to a reduction in Ins AUC0-240 (Table S2).

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We also examined the dynamics of glucagon secretion in response to the MMT in the GOP and

RYGB groups. Fasting glucagon levels were not altered pre and post interventions. GOP

volunteers, at baseline, exhibited a post-prandial elevation in glucagon secretion. When given

GOP, this post-prandial elevation was suppressed. In comparison, RYGB exhibited a peak in

glucagon secretion both pre and post intervention (Figure 1I).

GOP and RYGB reduce Energy Intake and Resting Energy Expenditure

Ad libitum EI was reduced significantly after 4 weeks of GOP by a mean of -292·7 kcal (Table

S1) vs Saline at -168·5 kcal, with no statistically significant difference in treatment effects

between the GOP and Saline arms. The RYGB group was assessed for ad libitum EI at week

12: as expected, they ate less compared to baseline (treatment effect -276·8 kcal). Ad libitum

EI was not assessed in the VLCD group as they were on a calorie restriction a priori. The 24-

hour EI estimated from food diaries are consistent with the above findings. GOP led to a

significant reduction in 24-hour EI at -803·7 kcal/24 hours, as did Saline at -437·3 kcal/24

hours, although there was no statistically significant difference between GOP and Saline.

RYGB led to a numerically larger reduction in 24-hour EI by -957·7 kcal/24 hours but this was

not significantly larger than GOP. The VLCD group ate 822·7 kcal/24 hours on average, i.e.

they were compliant with the specified calorie restriction. REE was significantly reduced in

the GOP group and in the RYGB group but not in any other treatment group (Table S1). When

corrected for body weight or fat-free mass, no significant treatment effects were observed in

all groups. No significant treatment effects were observed for diet-induced thermogenesis

(DIT) apart from a small reduction in Saline. AEE was significantly reduced after RYGB but

not in the other groups.

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Safety and Tolerability

Overall, GOP infusion was well tolerated with no significant change in nausea scores during

the infusion. Some of the participants in the infusion groups experienced a temporary, mild

erythema around the infusion sites resolving with improvements in set insertion technique. We

did not observe any hypoglycemic episodes in any of the GOP volunteers during the infusion

but 4 volunteers in the RYGB group had a documented glucose level of <4·0 mmol/L (Table

S3).

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Discussion

GOP infusion at home was feasible and well tolerated over a 4 week period. It led to a

substantial mean weight loss of -4.4 kg. The GLP-1/glucagon dual agonist MEDI0382 (22) and

the GLP-1 agonist Semaglutide (23) led to mean weight losses of -3.8 kg and -1 kg respectively

at roughly similar timepoints, although these studies were double-blinded with larger sample

sizes. Our previous study showed a 32% reduction in ad libitum EI with an acute GOP infusion

(5), and this study shows that the effect persists over 4 weeks with no tachyphylaxis. However,

the treatment effects vis-à-vis EI in this study were not statistically different between the GOP

and Saline groups, likely due to small sample sizes. The differential effects on body weight

from GOP (-4·0%) compared to RYGB (-8·8%) and VLCD (-7·6%) parallel the reductions in

24h EI of 30·6%, 49·2% and 53·6% in the respective groups. Our earlier study showed no

change in REE when GOP is given acutely (5). There was a decrease in REE after GOP and

RYGB, like Schmidt et al. (24), but this disappeared after adjustment for body weight or fat-

free mass. A small reduction in AEE after RYGB was seen which is likely because patients

were still recovering from surgery. The effects of the interventions on body weight are therefore

driven by the changes in EI.

GOP also led to improvements in fructosamine comparable to RYGB and VLCD, both of

which can lead to diabetes remission (1; 25). GOP achieved these improvements with a smaller

weight loss compared to RYGB and VLCD. The average measures of glycaemia mask a

marked difference in glucose/insulin dynamics between interventions. GOP led to a significant

reduction in GV compared to RYGB. During the MMT, after RYGB there was a rapid spike

in glucose levels followed by a peak in insulin secretion from the combination of

hyperglycaemia and insulinotropy from GLP-1 and possibly glucagon (5). In certain patients,

this phenomenon may lead to an ‘overswing’ hypoglycaemia. Post-bariatric hypoglycaemia is

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an increasingly recognized clinical problem and this is linked to increased GV after RYGB

(26). In contrast, GOP leads to nearly static glucose and a minimal increase in insulin. This

may be due to a delay in gastric emptying induced by GLP-1 and OXM (not directly tested

here) though this is usually subject to a rapid tachyphylaxis (27). It is also possible that GOP

could directly improve insulin sensitivity, although this was not directly tested in this study.

Another explanation might be the flattened post-prandial glucagon response with GOP

(mediated by GLP-1 and OXM), thus leading to suppression of hepatic glucose output (28).

With regards to VLCD, although there was some improvement in glucose tolerance, GOP was

superior in this regard.

The limitations of our study are as follows. We infuse only for 12 out of 24 hours. However,

this pattern of elevation of gut hormones during the day is likely to be seen in RYGB patients

who tend to eat frequent small meals during the day, thus leading to consistent gut hormone

elevations, and whose gut hormone levels fall back to baseline during the night. Although GOP

replicates post-surgical post-prandial gut hormone levels it does not replicate anatomical

changes. GOP does not replicate any changes in a notional ‘anti-incretin’ from the bypassed

gut (29), nor does it replicate any changes in ghrelin. The study is limited by a relatively small

sample size and short duration. It was not powered to show differences in the secondary

endpoints nor in safety and tolerability characteristics. Lastly, the study is not designed to

examine the contribution of the individual hormones to the overall effect.

We conclude that the post-prandial elevations in GLP-1, OXM and PYY after RYGB may be

responsible for the glycaemic improvements and some of the weight loss benefits from surgery.

There may be other contributions from surgical anatomical changes which lead to larger weight

loss. GOP achieves superior glucose tolerance to VLCD, reduces glucose variability and lowers

the risk of provoking hypoglycaemia compared to RYGB. Together, this suggests that ‘triple

agonism’ with GLP-1, OXM and PYY is a viable alternative to RYGB for the treatment of

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diabetes, with favourable effects on body weight, in patients who may not be able to have

bariatric surgery.

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Acknowledgments

The research study was funded by the UK Medical Research Council (MRC) Experimental

Challenge Grant (MR/K02115X/1). The Section of Investigative Medicine is funded by grants

from the MRC and Biotechnology and Biological Sciences Research Council and is supported

by the NIHR Imperial Biomedical Research Centre Funding Scheme. The research study was

also supported by the Imperial NIHR Clinical Research Facility at Imperial College Healthcare

NHS Trust. TT and SRB are funded by the MRC and by the NIHR. WSD is funded by an

NIHR Research Professorship. GF, SC, JK are funded by the NIHR. CD and JC are funded by

the NIHR Imperial Biomedical Research Centre Funding Scheme. JJH is supported by the

Novo Nordisk Foundation. We would like to thank Anthony Leeds and Cambridge Weight

Plan for their support. We would also like to thank the staff at the Imperial Weight Centre

(Samantha Scholtz, Harvey Chahal, Alex Miras, Karen O’Donnell, Debbie O’Rourke) for their

support of this research study. TT and SRB contributed to study design, data collection,

statistical analysis, data interpretation, and drafting of the manuscript. PB, GT, KA, JK, JC,

DH, OA, SC, HA, AP, JM, CD contributed to the running of the study, data collection and data

interpretation. PB and GT also contributed to the drafting of the manuscript. AA, SP and KM

were the surgeons of the study. TP and NJ performed the statistical analysis. WD, CLR and

GF contributed to data interpretation. JJH and NJWA contributed to the analysis of samples

and data interpretation. All authors contributed to critical review of the manuscript. TT is the

guarantor of this work and had full access to all the data in the study; she takes responsibility

for the integrity of the data and the accuracy of the data analysis. The views expressed are those

of the authors and not necessarily those of the abovementioned funders, the NHS, the NIHR,

or the Department of Health.

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Conflicts of Interest

JJH is a member of advisory boards for Novo Nordisk. No potential conflicts of interest

relevant to this article were reported by the other authors.

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References

1. Schauer PR, Bhatt DL, Kirwan JP, Wolski K, Aminian A, Brethauer SA, Navaneethan SD, Singh RP, Pothier CE, Nissen SE, Kashyap SR, Investigators S: Bariatric Surgery versus Intensive Medical Therapy for Diabetes - 5-Year Outcomes. N Engl J Med 2017;376:641-6512. Bojsen-Moller KN, Dirksen C, Jorgensen NB, Jacobsen SH, Serup AK, Albers PH, Hansen DL, Worm D, Naver L, Kristiansen VB, Wojtaszewski JF, Kiens B, Holst JJ, Richter EA, Madsbad S: Early enhancements of hepatic and later of peripheral insulin sensitivity combined with increased postprandial insulin secretion contribute to improved glycemic control after Roux-en-Y gastric bypass. Diabetes 2014;63:1725-17373. Miras AD, le Roux CW: Mechanisms underlying weight loss after bariatric surgery. Nat Rev Gastroenterol Hepatol 2013;10:575-5844. le Roux CW, Aylwin SJ, Batterham RL, Borg CM, Coyle F, Prasad V, Shurey S, Ghatei MA, Patel AG, Bloom SR: Gut hormone profiles following bariatric surgery favor an anorectic state, facilitate weight loss, and improve metabolic parameters. Ann Surg 2006;243:108-1145. Tan T, Behary P, Tharakan G, Minnion J, Al-Najim W, Albrechtsen NJW, Holst JJ, Bloom SR: The Effect of a Subcutaneous Infusion of GLP-1, OXM, and PYY on Energy Intake and Expenditure in Obese Volunteers. J Clin Endocrinol Metab 2017;102:2364-23726. Drucker DJ: The Ascending GLP-1 Road From Clinical Safety to Reduction of Cardiovascular Complications. Diabetes 2018;67:1710-17197. Pi-Sunyer X, Astrup A, Fujioka K, Greenway F, Halpern A, Krempf M, Lau DC, le Roux CW, Violante Ortiz R, Jensen CB, Wilding JP, SCALE Obesity and Prediabetes NN8022-1839 Study Group: A Randomized, Controlled Trial of 3.0 mg of Liraglutide in Weight Management. N Engl J Med 2015;373:11-228. Pocai A: Action and therapeutic potential of oxyntomodulin. Mol Metab 2014;3:241-2519. Cohen MA, Ellis SM, Le Roux CW, Batterham RL, Park A, Patterson M, Frost GS, Ghatei MA, Bloom SR: Oxyntomodulin suppresses appetite and reduces food intake in humans. J Clin Endocrinol Metab 2003;88:4696-470110. Wynne K, Park AJ, Small CJ, Meeran K, Ghatei MA, Frost GS, Bloom SR: Oxyntomodulin increases energy expenditure in addition to decreasing energy intake in overweight and obese humans: a randomised controlled trial. Int J Obes (Lond) 2006;30:1729-173611. Wynne K, Park AJ, Small CJ, Patterson M, Ellis SM, Murphy KG, Wren AM, Frost GS, Meeran K, Ghatei MA, Bloom SR: Subcutaneous oxyntomodulin reduces body weight in overweight and obese subjects: a double-blind, randomized, controlled trial. Diabetes 2005;54:2390-239512. Batterham RL, Cohen MA, Ellis SM, Le Roux CW, Withers DJ, Frost GS, Ghatei MA, Bloom SR: Inhibition of food intake in obese subjects by peptide YY3-36. N Engl J Med 2003;349:941-94813. Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM, Brynes AE, Low MJ, Ghatei MA, Cone RD, Bloom SR: Gut hormone PYY(3-36) physiologically inhibits food intake. Nature 2002;418:650-65414. Neary NM, Small CJ, Druce MR, Park AJ, Ellis SM, Semjonous NM, Dakin CL, Filipsson K, Wang F, Kent AS, Frost GS, Ghatei MA, Bloom SR: Peptide YY3-36 and glucagon-like peptide-17-36 inhibit food intake additively. Endocrinology 2005;146:5120-5127

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15. Schmidt JB, Gregersen NT, Pedersen SD, Arentoft JL, Ritz C, Schwartz TW, Holst JJ, Astrup A, Sjodin A: Effects of PYY3-36 and GLP-1 on energy intake, energy expenditure, and appetite in overweight men. Am J Physiol Endocrinol Metab 2014;306:E1248-125616. Field BC, Wren AM, Peters V, Baynes KC, Martin NM, Patterson M, Alsaraf S, Amber V, Wynne K, Ghatei MA, Bloom SR: PYY3-36 and oxyntomodulin can be additive in their effect on food intake in overweight and obese humans. Diabetes 2010;59:1635-163917. Cegla J, Troke RC, Jones B, Tharakan G, Kenkre J, McCullough KA, Lim CT, Parvizi N, Hussein M, Chambers ES, Minnion J, Cuenco J, Ghatei MA, Meeran K, Tan TM, Bloom SR: Coinfusion of low-dose GLP-1 and glucagon in man results in a reduction in food intake. Diabetes 2014;63:3711-372018. Morgan JF, Reid F, Lacey JH: The SCOFF questionnaire: assessment of a new screening tool for eating disorders. BMJ 1999;319:1467-146819. van Strien T, Frijters JER, Bergers GPA, Defares PB: The Dutch Eating Behavior Questionnaire (DEBQ) for assessment of restrained, emotional, and external eating behavior. Int J Eat Disord 1986;5:295-31520. Wardle J: Eating style: a validation study of the Dutch Eating Behaviour Questionnaire in normal subjects and women with eating disorders. J Psychosom Res 1987;31:161-16921. Hill NR, Levy JC, Matthews DR: Expansion of the homeostasis model assessment of beta-cell function and insulin resistance to enable clinical trial outcome modeling through the interactive adjustment of physiology and treatment effects: iHOMA2. Diabetes Care 2013;36:2324-233022. Ambery P, Parker VE, Stumvoll M, Posch MG, Heise T, Plum-Moerschel L, Tsai LF, Robertson D, Jain M, Petrone M, Rondinone C, Hirshberg B, Jermutus L: MEDI0382, a GLP-1 and glucagon receptor dual agonist, in obese or overweight patients with type 2 diabetes: a randomised, controlled, double-blind, ascending dose and phase 2a study. Lancet 2018;391:2607-261823. Blundell J, Finlayson G, Axelsen M, Flint A, Gibbons C, Kvist T, Hjerpsted JB: Effects of once-weekly semaglutide on appetite, energy intake, control of eating, food preference and body weight in subjects with obesity. Diabetes Obes Metab 2017;19:1242-125124. Schmidt JB, Pedersen SD, Gregersen NT, Vestergaard L, Nielsen MS, Ritz C, Madsbad S, Worm D, Hansen DL, Clausen TR, Rehfeld JF, Astrup A, Holst JJ, Sjodin A: Effects of RYGB on energy expenditure, appetite and glycaemic control: a randomized controlled clinical trial. Int J Obes (Lond) 2016;40:281-29025. Lean ME, Leslie WS, Barnes AC, Brosnahan N, Thom G, McCombie L, Peters C, Zhyzhneuskaya S, Al-Mrabeh A, Hollingsworth KG, Rodrigues AM, Rehackova L, Adamson AJ, Sniehotta FF, Mathers JC, Ross HM, McIlvenna Y, Stefanetti R, Trenell M, Welsh P, Kean S, Ford I, McConnachie A, Sattar N, Taylor R: Primary care-led weight management for remission of type 2 diabetes (DiRECT): an open-label, cluster-randomised trial. Lancet 2018;391:541-55126. Tharakan G, Behary P, Wewer Albrechtsen NJ, Chahal H, Kenkre J, Miras AD, Ahmed AR, Holst JJ, Bloom SR, Tan T: Roles of increased glycaemic variability, GLP-1 and glucagon in hypoglycaemia after Roux-en-Y gastric bypass. Eur J Endocrinol 2017;177:455-46427. Nauck MA, Kemmeries G, Holst JJ, Meier JJ: Rapid tachyphylaxis of the glucagon-like peptide 1-induced deceleration of gastric emptying in humans. Diabetes 2011;60:1561-1565

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28. Hare KJ, Knop FK, Asmar M, Madsbad S, Deacon CF, Holst JJ, Vilsboll T: Preserved inhibitory potency of GLP-1 on glucagon secretion in type 2 diabetes mellitus. J Clin Endocrinol Metab 2009;94:4679-468729. Salinari S, Mingrone G, Bertuzzi A, Previti E, Capristo E, Rubino F: Downregulation of Insulin Sensitivity After Oral Glucose Administration: Evidence for the Anti-Incretin Effect. Diabetes 2017;66:2756-2763

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Tables

Table 1: Baseline characteristics of studied patients. Data displayed as Mean (SD) except where indicated. M, male; F, female; BMI, body mass index; T2DM, type 2 diabetes mellitus; Prediabetic, includes impaired glucose tolerance, impaired fasting glucose or HbA1c of 6·0-6·4% (42-48 mmol/mol); DPP-IV, dipeptidyl peptidase-IV. aGOP vs SALINE, bGOP vs RYGB, cGOP vs VLCD (unpaired two-tailed t-test for continuous data or Fisher two-tailed exact probability test for categorical data).

GOP (n=15) SALINE (n=11) p-valuea RYGB (n=21) p-valueb VLCD (n=22) p-valuec

Mean Age (years) 55·9 (8·5) 53·5 (8·5) 0·50 48·2 (13·2) 0·06 47·0 (10·2) 0·01Gender (F:M) 6:9 5:6 1·00 17:4 0·02 12:10 0·51Weight (kg) 112·6 (26·7) 119·2 (25·1) 0·53 117·8 (25·3) 0·56 109·0 (20·5) 0·65BMI (kg/m2) 38·4 (6·9) 39·2 (5·4) 0·74 43·2 (6·2) 0·03 39·1 (4·3) 0·70DiagnosisT2DM (n/%) 13/87% 8/73% 18/86% 15/68%Prediabetic (n/%) 2/13% 3/27%

0·613/14%

1·007/32%

0·26

TreatmentDiet only (n/%) 8/53% 6/55% 7/33% 9/41%Metformin only (n/%) 7/47% 5/45% 13/62% 13/59%DPP-IV inhibitor only (n/%) 0 0

1·001/5%

0·310

0·51

Fructosamine (µmol/L) 304·6 (66·5) 278·1 (42·3) 0·26 250·3 (35·9) 0·003 252·4 (37·2) 0·004Fasting glucose (mmol/L) 9·9 (4·7) 7·7 (1·5) 0·15 8·4 (2·0) 0·19 7·9 (2·7) 0·11Fasting Insulin (mIU/L) 16·3 (10·9) 14·4 (7·3) 0·63 19·7 (6·7) 0·25 18·0 (11·6) 0·66iHOMA2 %S 49·9 (21·3) 57·1 (21·7) 0·41 40·2 (17·0) 0·14 47·8 (18·3) 0·74Total Cholesterol (mmol/L) 4·8 (0·7) 5·1 (1·3) 0·49 4·3 (1·1) 0·09 5·0 (1·1) 0·65LDL cholesterol (mmol/L) 2·9 (0·7) 3·1 (1·2) 0·63 2·5 (0·9) 0·14 3·1 (0·9) 0·48HDL cholesterol (mmol/L) 1·1 (0·3) 1·3 (0·3) 0·12 1·1 (0·2) 0·56 1·0 (0·2) 0·21Triglycerides (mmol/L) 1·7 (0·9) 1·5 (0·4) 0·44 1·5 (0·5) 0·50 2·1 (2·3) 0·57

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Table 2: GOP leads to significantly more weight loss than Saline. Data displayed as Means [95% CI] for comparisons against baseline and for comparison of treatment effects between groups. BL, baseline; W4, week 4; ΔW4, treatment effect between baseline and week 4; ΔW4 vs GOP, difference in treatment effect compared to GOP arm. * p<0·05, ** p<0·01 for primary comparison (unpaired t-test) of treatment effect between GOP and Saline arms.

Primary Comparison (GOP vs Saline) Secondary Comparisons (GOP vs RYGB, GOP vs VLCD)

GOP SALINE RYGB VLCD

BL 112·6 119·3 117·8 109·0

W4 108·2 116·8 107·6 100·8

∆W4 -4·4 [-5·4, -3·5] -2·5 [-4·1, -0·9] -10·3 [-11·8, -8·8] -8·3 [-9·5, -7·1]Weight (kg)

∆W4 vs GOP 1·9 [0·3, 3·5] * -5·9 [-7·5, -4·2] -3·9 [-5·3, -2·4]

∆W4 -4·0 [-4·8, -3·3] -2·0 [-3·3, -0·8] -8·8 [-10·0, -7·7] -7·6 [-8·3, -6·8]Percentage Weight Change ∆W4 vs GOP 2·0 [0·7, 3·3] ** -4·8 [-6·2, -3·5] -3·6 [-4·6, -2·5]

BL 43·4 48·3 55·5 44·4

W4 40·9 47·0 49·4 39·3

∆W4 -2·4 [-3·2, -1·7] -1·3 [-2·9, 0·3] -6·1 [-7·1, -5·1] -5·1 [-6·3, -3·9]Fat Mass (kg)

∆W4 vs GOP 1·1 [-0·4, 2·7] -3·6 [-4·9, -2·3] -2·7 [-4·2, -1·1]

BL 63·3 67·3 59·3 60·9W4 61·6 66·4 55·7 57·8

∆W4 -1·7 [-2·8, -0·7] -0·9 [-1·7, -0·2] -3·6 [ -5·0, -2·3] -3·0 [-3·8, -2·3]Muscle Mass (kg)

∆W4 vs GOP 0·8 [-0·5, 2·1] -1·9 [-3·6, -0·3] -1·3 [-2·5, -0·1]

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Table 3: Glycaemia improves after GOP, RYGB and VLCD interventions. Data displayed as Means [95% CI] for comparisons against baseline and for comparison of treatment effects between groups. BL, baseline; W4, week 4; ΔW4, treatment effect between baseline and week 4; ΔW4 vs GOP, difference in treatment effect compared to GOP arm. N/A not applicable. aAdditional results based on sensitivity analysis (unpaired t-test) of fitted values derived from ANCOVA model, adjusting for baseline fructosamine levels and any potential interaction between baseline and treatment effect. * p<0·05, ** p<0·01, *** p<0·001 for primary comparison of treatment effect (unpaired t-test) between GOP and Saline arms.



BL 304·6 278·1 250·3 252·4

W4 260·5 266·4 216·4 223·9

∆W4 -44·1 [-62·7, -25·5] -11·7 [-18·9, -4·5] -34·0 [-45·6, -22·4] -28·5 [-40·4, -16·7]Fructosamine

(μmol/L)

∆W4 vs GOP 32·4 [12·9, 51·9] ** 10·2 [-9·8, 30·2] 15·6 [-6·6, 35·8]

∆W4 -44·1 [-57(2, -31·1] -11·7 [-15·6, -7·9] N/A N/AFructosaminea (μmol/L) ∆W4 vs GOP 33·2 [19·0, 45·8] *** N/A N/A

BL 9·9 7·7 8·3 7·9

W4 5·8 6·7 5·8 5·9

∆W4 -4·1 [-6·3, -1·9] -1·0 [-1·7, -0·3] -2·5 [-3·3, -1·7] -2·0 [-3·0, -1·0]Fasting Glucose

(mmol/L)

∆W4 vs GOP 3·1 [0·8, 5·3] * 1·6 [0·7, 3·9] 2·0 [-0·3, 4·5]

BL 16·3 14·4 19·7 18·0

W4 18·2 12·0 12·3 10·1

∆W4 1·9 [-0·8, 4·6] -2·4 [-5·2, 0·4] -7·4 [-10·4, -4·3] -7·9 [-11·2, -4·6]Fasting Insulin

(mmol/L)

∆W4 vs GOP -4·8 [-8·0, -0·5] * -9·2 [-13·4, -5·1] -9·8 [-14·3, -5·3]

BL 10·4 7·4 8·5 N/A

W4 6·9 7·1 6·0 N/AMean Glucose from CGM (mmol/L)

∆W4 -3·6 [-5·2, -1·9] -0·4 [-1·4, 0·7] -2·5 [-3·6, -1·4] N/A

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∆W4 vs GOP 3·2 [1·1, 5·3] ** 1·1 [-0·9, 3·0] N/A

BL 2·3 1·7 1·7 N/A

W4 1·55 1·6 1·6 N/A

∆W4 -0·75 [-1·08, -0·41] -0·1 [-0·5, 0·2] -0·1 [-0·5, 0·3] N/AMAG

∆W4 vs GOP 0·6 [0·2, 1·1] * 0·6 [0·1, 1·1] N/A

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Figure Legends

Figure 1: The Glucose (A, C, E, G) and Insulin (B, D, F, H) dynamics during a Mixed Meal Tolerance test differ between interventions. (A,B) Pre and post RYGB intervention; (C,D) Pre and post VLCD intervention; (E, F) Pre and post GOP intervention; (G, H) Pre and post Saline intervention. (I) Glucagon responses to MMT pre and post GOP infusion compared to the RYGB group. Means and 95% CI plotted.

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Online-Only Supplementary Information

Combined GLP-1, oxyntomodulin and peptide YY improves body weight and glycaemia in obesity and prediabetes/type 2 diabetes: a randomized single-blinded placebo controlled study

Preeshila Behary, George Tharakan, Kleopatra Alexiadou, Nicholas Johnson, Nicolai J. Wewer Albrechtsen, Julia Kenkre. Joyceline Cuenco, David Hope, Oluwaseun Anyiam, Sirazum Choudhury, Haya Alessimii, Ankur Poddar, James Minnion, Chedie Doyle, Gary Frost, Carel Le Roux, Sanjay Purkayastha, Krishna Moorthy, Waljit Dhillo, Jens J. Holst, Ahmed R. Ahmed, Toby Prevost, Stephen R. Bloom, Tricia M. Tan

Supplemental Results

List of Supplemental TablesTable S1: GOP Reduces Energy Intake and Resting Energy Expenditure.Table S2: GOP improves glucose tolerance and reduces the insulin excursion during the MMT. Table S3: Safety Data. Table S4: All interventions reduce fasting total cholesterol.

List of Supplemental FiguresFigure S1: GOP peptide stability as measured by HPLCFigure S2: CONSORT Diagram showing patient flow in the studyFigure S3: Gut hormone levels after GOP infusion are maintained at post-prandial levels observed 4 weeks post RYGB.Figure S4: Representative examples of ambulatory glucose profiles from CGM for two volunteers at baseline and during GOP infusion.

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Supplemental ResultsGOP infusion is well tolerated over 4 weeksRested blood pressure and pulse were recorded in the fasted state, in all volunteers during the study visits. Nausea was subjectively assessed using a Visual Scale Analogue scale, both in the fasted (t=0) and fed state (t=30 and 240 minutes from the ingestion of Ensure) at the baseline visit and after 4 weeks of each intervention (23). Treatment-emergent adverse effects were recorded in the case record files for each volunteer. Significant improvements from baseline in fasting blood pressure were noted within the VLCD (systolic and diastolic) and GOP (systolic) groups at the end of 4 weeks. No significant improvements in fasting BP were observed in the RYGB and Saline groups (Table S3). Comparing the two primary analysis groups indicated no significant mean [95% CI] difference in effect between the two groups for both systolic (4·8 [-1·3, 10·9]) mmHg and diastolic (-1·2 [-6·9, 4·5] mmHg) fasting BP. A significantly reduced pulse rate was observed in the VLCD group but no other groups (Table S3). Nausea, as assessed by visual analogue scores in the fasted state and after feeding were unchanged following the GOP infusion. In contrast, after surgery, the volunteers experienced a significant increase in post-prandial nausea (Table S3). Some of the participants in the infusion groups experienced a temporary, mild erythema around the infusion sites; however, these resolved with additional training in infusion set insertion technique. We did not observe any hypoglycemic episodes in any of the GOP volunteers during the infusion on any study days. Four volunteers in the RYGB group had a documented glucose level of <4·0 mmol/L.

All interventions reduce fasting total cholesterolFasting lipid profiles were measured for each intervention group before and after the interventions. As noted in Table S4, all interventions led to a statistically significant reduction in total cholesterol from baseline. This was mainly driven by significant reductions in LDL cholesterol (seen with the GOP, Saline, VLCD interventions, but not with the RYGB intervention). The Saline and RYGB intervention groups also had statistically significant reductions in HDL cholesterol. The GOP intervention led to a statistically significant reduction in triglyceride levels which was not seen in other interventions. No statistically significant differences in treatment effects between GOP and the other interventions were observed.

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Table S1: GOP reduces Energy Intake and Resting Energy Expenditure. Data displayed as Means [95% CI] for comparisons against baseline and for comparison of treatment effects between groups. BL, baseline; W4, week 4; ΔW4, treatment effect between baseline and week 4; ΔW4 vs GOP, difference in treatment effect compared to GOP arm. N/A not applicable. aAssessed at week 12 for the RYGB group. REE, resting energy expenditure. DIT, diet-induced thermogenesis. AEE, activity-related energy expenditure.



BL 785·1 1002·6 655·1 N/A

W4 492·4 834·1 378·3a N/A∆W4 -292·7 [-389·5, -196·0] -168·5 [-300·5, -36·5] -276·8 [-398·4, -155·3]a N/A

Ad Libitum Energy Intake (kcal)

∆W4 vs GOP 124·2 [-26·6, 275·0] 15·9 [-140·0, 171·8]a N/A

BL 2073·2 2133·9 1882·6 2192·1

W4 1269·5 1696·6 924·9# 822·7

∆W4 -803·7 [-1239·6, -367·9] -437·3 [-823·4, -51·2] -957·7 [-1239·5, -675·9]a -1369·4 [-2059·3, -679·5]24-hour EI (kcal)

∆W4 vs GOP 366·4 [-212·3, 945·2] -154·0 [-643·6, 335·6]a -565·7 [-1374·3, 243·0]

BL 1984·3 1936·2 1994·7 1829·7REE (kcal/24 hours) W4 1780·2 1891·4 1806·0 1705·6

∆W4 -204·1 [-356·2, -52·0] -44·8 [-229·4, 139·9] -188·8 [-363·8, -13·7] -124·2 [-325·8, 77·5]∆W4 vs GOP 159·4 [-65·0, 383·7] 15·4 [-221·4, 252·1] 80·0 [-184·1, 344·1]

BL 120·6 229·5 123·8 239·4W4 118·6 63·7 220·9 192·3

∆W4 -2·0 [-330·7, 326·7] -165·8 [-307·4, -24·3] 97·1 [-86·2, 280·4] -47·1 [-187·9, 93·8]DIT (kcal/24 hours)

∆W4 vs GOP -163·8 [-510·2, 182·5] 99·1 [-252·7, 450·8] -45·1 [-354·5, 264·3]

BL 626·7 653·8 735·5 711·2W4 688·2 530·7 520·1 728·5

∆W4 61·5 [-98·5, 221·5] -123·1 [-278·6, 32·4] -215·4 [-426·9, -3·9] 17·3 [-155·6, 190·2]AEE (kcal/24 hours)

∆W4 vs GOP -184·6 [-414·9, 61·5] -276·9 [-532·8, 21·0] -44·2 [-279·1, 190·7]

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Table S2: GOP improves glucose tolerance and reduces the insulin excursion during the MMT. Data displayed as Means [95% CI] for comparisons against baseline and for comparison of treatment effects between groups. BL, baseline; W4, week 4; ΔW4, treatment effect between baseline and week 4; ΔW4 vs GOP, difference in treatment effect compared to GOP arm. N/A not applicable. ** p<0·01 for primary comparison (unpaired t-test) of treatment effect between GOP and Saline arms.



BL 2584·1 1970·5 2168·3 2145·9

W4 1471·6 1799·9 1528·2 1614·9

∆W4 -1112·6 [-1624·8, -600·3] -170·5 [-362·1, 21·0] -640·1 [-898·7, -381·5] -531·0 [-818·4, -243·6]Glucose AUC0-240

(mmol/L·min)

∆W4 vs GOP 942·5 [407·6, 1476·6] ** 472·5 [-37·6, 982·6] 582.0 [57·5, 1105·6]

BL 9669·2 8811·1 10766·1 9831·5

W4 6254·5 9724·8 12849·9 8261·0

∆W4 -3414·8 [-5983·7, -845·8] 913·6 [-463·7, 2291·0] 2083·8 [-933·1 ,5100·7] -1570·5 [-2906·6, - 234·3]Insulin AUC0-240

(mU/L·min)

∆W4 vs GOP 4402·6 [1520·5, 7136·2] ** 5498·6 [1503·3, 9493·8] -1844·3 [-704·3, 4392·9]

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Table S3: Safety Data. Means [95% CI] displayed. * p <0·05, ** p<0·01 for comparison between pre and following 4 weeks of each intervention, Student Paired t-test.

GOP (n=15) SALINE (n=11) RYGB (n=21) VLCD (n=22) Pre 4 weeks Pre 4 weeks Pre 4 weeks Pre 4 weeks

Fasting Systolic BP (mmHg)

132·0 [126·2, 137·8]

123·6 [117·8,129·3]**

128·1 [118·8, 137·5]

124·5 [113·7,135·2]

128·6 [122·0,135·2]

122·9 [116·4, 129·4]

132·3 [126·1, 138·6]

124·7[120·3, 129·2]*

Fasting Diastolic BP (mmHg)

73·6 [68·5, 78·8]

71·5 [67·3, 75.7]

76·3 [70·0, 82·5]

72·9 [65·9, 80·0]

73·0 [68·8, 77·1]

72·2 [68·0, 76·4]

78·5 [74·2, 82·9]

72·5 [69·0, 76·0]**

Fasting Pulse rate (min-1)

70·6 [65·2, 76·1]

69·5 [64·5, 74·6]

68·5 [60·9, 76·1]

69·1 [61·1, 77·1]

78·3 [72·7, 83·9]

74·0 [69·2, 78·7]

74·8 [71·0, 78·5]

70·2 [66·2, 74·2]**

Fasted Nausea VAS (mm)

3·1 [-1·6, 7·9]

9·5 [-2·05, 21·0]

2·9 [-1·6, 7·4]

2·0 [-2·0, 6·0]*

3·3 [0·3, 6·3]

4·2 [0·5, 7·9]

4·3 [1·2, 7·4]

4·0 [-0·7, 8·6]

Nausea AUC0-240 (mm·min)

1604 [159, 3049]

2193 [-61, 4447]

758 [-305, 1821]

310·9 [-304, 926]

990 [61·5, 1919]

5222 [2475, 7970]**

1276 [450·2, 2101]

842·7 [397, 1288]

Treatment-emergent adverse events, n (%)

Mild transient erythema 8 (53%) 3 (27%) N/A N/AHypoglycemia (< 4.0

mmol/L)0 (0%) 0 (0%) 4 (19%) 0 (0%)

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Table S4: All interventions reduce fasting total cholesterol. Data displayed as Means [95% CI] for comparisons against baseline and for comparison of treatment effects between groups. BL, baseline; W4, week 4; ΔW4, treatment effect between baseline and week 4; ΔW4 vs GOP, difference in treatment effect compared to GOP arm.



BL 4·8 5·1 4·3 5·0W4 4·2 4·5 3·7 3·8

∆W4 -0·7 [-1·0, -0·3] -0·6 [-1·0, -0·3] -0·6 [-1·1, -0·2] -1·2 [-1·6, -0·8]Total Cholesterol

(mmol/L)

∆W4 vs GOP 0·0 [-0·4, 0·5] 0·1 [-0·5, 0·6] -0·5 [-1·1, 0·0]

BL 1·1 1·3 1·1 1·0W4 1·0 1·2 0·9 0·9

∆W4 -0·1 [-0·2, 0·0] -0·2 [-0·2, -0·1] -0·2 [-0·3, -0·1] -0·1 [-0·2, 0·0]HDL cholesterol

(mmol/L)

∆W4 vs GOP -0·1 [-0·2, 0·1] -0·1 [-0·3, 0·4] 0·0 [ -0·1, 0·1]

BL 2·9 3·1 2·5 3·1W4 2·6 2·7 2·2 2·4

∆W4 -0·4 [-0·6, -0·1] -0·4 [-0·7, -0·1] -0·3 [-0·7, 0·1] -0·7 [-1·0, -0·5]LDL cholesterol

(mmol/L)

∆W4 vs GOP -0·0 [-0·4, 0·3] 0·0 [-0·3, 0·4] -0·4 [-0·7, 0·0]

BL 1·7 1·5 1·5 2·1W4 1·2 1·3 1·3 1·1

∆W4 -0·5 [-0·8, -0·1] -0·2 [-0·5, 0·2] -0·2 [-0·6, 0·1] -1·0 [-2·0, 0·1]Triglycerides (mmol/L)

∆W4 vs GOP 0·3 [-0·2, 0·8] 0·2 [-0·2, 0·7] -0·5 [-1·6, 0·5]

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Figure S1: Peptide stability in the GOP mixture when loaded into infusion syringes at room temperature as measured by HPLC. Means plotted with 95% CI.

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Figure S2: CONSORT Diagram showing patient flow in the study

Infusion GroupsScreened 80

Randomised 35

GOP 21

Discontinued 6Not adherent to Protocol 5

Technical difficulties w/ pump 1

Completed Follow Up 15

Saline 14

Discontinued 3Not adherent to Protocol 1Other condition requiring

investigation 2


Did not meet criteria 37Excluded b/f Randomisation 8

Other incidental diagnosis 1Withdrew consent 7

RYGB GroupScreened 40

Eligible 27

[Not randomised]

Discontinued 6Sleeve Gastrectomy 4 Open Laparotomy 1 Surgery postponed 1


Did not meet criteria 13

VLCD GroupScreened 40

Eligible 28

[Not randomised]

Discontinued 6Unable to commit to Protocol 5

Acute Flare of Rheumatoid Arthritis 1


Did not meet criteria 12

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Figure S3: Gut hormone levels after GOP infusion are maintained at post-prandial levels observed 4 weeks post RYGB (a) Active GLP-1; (b) OXM; (c) total PYY in response to an MMT test at week 4 of intervention. For the GOP group, the patients have been receiving the infusion for 120 minutes prior to the MMT test start time. Means plotted with 95% CI. Shaded areas denote the target zone for each hormone (95%CI for the peak values for each hormone).

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Figure S4: Representative examples of ambulatory glucose profiles from CGM for two volunteers at baseline and during GOP infusion. Y-axis represents the interstitial glucose values (mmol/L), X-axis represents clock time for each day. Blue lines/shading represents baseline recording (before infusion). Green lines/shading represents GOP infusion recording with thick green line on X-axis indicating time on infusion. Solid lines represent median glucose levels. Shading represents interquartile range of glucose readings. Dotted lines represent 10th and 90th percentiles of glucose readings. Yellow shading represents desired range of glucose between 3.8 to 7.2 mmol/L.

GOP infusion

46

810

12

Glu

cose

(mm

ol/L

)

2400 0600 1200 1800 2300Clock time (hours)

Base pc10/90 Base Median

Base pc25/Base pc75 GOP pc10/90

GOP Median GOP pc25/GOP pc75

GOP infusion

510

1520

25

Glu

cose

(mm

ol/L

)

2400 0600 1200 1800 2300Clock time (hours)

Base pc10/90 Base Median

Base pc25/Base pc75 GOP pc10/90

GOP Median GOP pc25/GOP pc75

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Diabetes Care

combined glp-1, oxyntomodulin and peptide yy improves body … · 2019-11-13 · 1 combined glp-1,...

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