Glucagon‐like peptide 1 infusions overcome anabolic resistance to feeding in older human muscle

Abstract Background Despite its known insulin‐independent effects, glucagon‐like peptide‐1 (GLP‐1) role in muscle protein turnover has not been explored under fed‐state conditions or in the context of older age, when declines in insulin sensitivity and protein anabolism, as well as losses of muscle mass and function, occur. Methods Eight older‐aged men (71 ± 1 year, mean ± SEM) were studied in a crossover trial. Baseline measures were taken over 3 hr, prior to a 3 hr postprandial insulin (~30 mIU ml−1) and glucose (7–7.5 mM) clamp, alongside I.V. infusions of octreotide and Vamin 14 (±infusions of GLP‐1). Four muscle biopsies were taken, and muscle protein turnover was quantified via incorporation of 13C6 phenylalanine and arteriovenous balance kinetics, using mass spectrometry. Leg macro‐ and microvascular flow was assessed via ultrasound and anabolic signalling by immunoblotting. GLP‐1 and insulin were measured by ELISA. Results GLP‐1 augmented muscle protein synthesis (MPS; fasted: 0.058 ± 0.004% hr−1 vs. postprandial: 0.102 ± 0.005% hr−1, p < 0.01), in comparison with non‐GLP‐1 trials. Muscle protein breakdown (MPB) was reduced throughout clamp period, while net protein balance across the leg became positive in both groups. Total femoral leg blood flow was unchanged by the clamp; however, muscle microvascular blood flow (MBF) was significantly elevated in both groups, and to a significantly greater extent in the GLP‐1 group (MBF: 5 ± 2 vs. 1.9 ± 1 fold change +GLP‐1 and −GLP‐1, respectively, p < 0.01). Activation of the Akt‐mTOR signalling was similar across both trials. Conclusion GLP‐1 infusion markedly enhanced postprandial microvascular perfusion and further stimulated muscle protein metabolism, primarily through increased MPS, during a postprandial insulin hyperaminoacidaemic clamp.

Crucially, recent data have emerged suggesting a novel role of GLP-1 in relation to muscle protein metabolism and muscle mass regulation. For example, longer-term observational studies with inhibitors of the GLP-1-degrading enzyme, dipeptidyl peptidase-4 (DPP-4), demonstrate improved muscle mass preservation in older age (Bouchi et al., 2018). Moreover, recently a comprehensive pre-clinical study demonstrated that exendin-4-a peptide agonist of GLP-1r-suppressed the expression of molecules involved in muscle atrophy, for example atrogin 1 and MURF-1, and preserved muscle in mouse models of muscle wasting (Hong, Lee, Jeong, Choi, & Jun, 2019). However, evidence of a direct effect of GLP-1 on human muscle protein metabolism is lacking and its potential impact on 'anabolic resistance' in ageing muscle is unexplored. Therefore, the aims of this study were to determine the acute impact of GLP-1 in relation to muscle protein metabolism. We hypothesised GLP-1 would promote greater protein anabolism in older muscle in response to amino acid feeding during a postprandial insulin clamp.

| Subject characteristics
The physical and demographic characteristics of study participants are described in Table 1.

| GLP-1 and Insulin concentrations
GLP-1 levels at baseline in the two were similar; however, upon infusion of GLP-1, levels rose rapidly peaking around 40 min, but remained unchanged throughout, in the control group. Mean GLP-1 concentration over the postprandial period was 63 ± 15 pmol L −1 and 17 ± 4 pmol L −1 with and without GLP-1 infusion, respectively, and the AUC above baseline was significantly greater in the GLP-1 group ( Figure 1a)  at baseline in both groups (5.1 ± 0.5 and 5.6 ± 0.9 μIU ml −1 with and without GLP-1, respectively). During the postprandial clamp with insulin alone, levels rose to 25 ± 0.4 μIU ml −1 and to 31 ± 1.3 μIU ml −1 when GLP-1 was co-infused (p < 0.001 vs. fasting for both) (Figure 1b); AUC above baseline is shown in the inset, for −GLP-1 and +GLP-1, p = 0.24.

| Phenylalanine concentration and enrichment
Arterial phenylalanine concentrations were similar at baseline in both groups (58.5 ± 2.7 μM −GLP-1 vs. 60.8 ± 3.4 μM +GLP-1 and rose significantly over the first 90 min of the postprandial period in both groups more than doubling to 135.8 ± 6.8 μM and 130.5 ± 2.7 μM, respectively, both p < 0.0001 Figure 2a,b) and remained elevated throughout. Phenylalanine concentrations were used as a proxy to illustrate the effect of Vamin infusion on circulating AA concentrations, mimicking a feeding response.
Phenylalanine enrichment reached a steady state rapidly during the baseline period and then fell similarly in both groups to a new steady state during the postprandial clamp ( Figure 2c,d), due to the presence of unlabelled Phe in the Vamin infusion. Phenylalanine kinetics were determined over three periods as outlined below, when leg blood flow was simultaneously measured (see protocol Figure 3).

| Total leg blood flow and microvascular blood flow
Although total leg blood flow rose slightly, during the postprandial clamp in both groups, this was not significant (0.34 ± 0.02 vs.

| Gene expression of GLP1r
Our data show sufficient expression of the receptor in both skeletal muscle cells and skeletal muscle tissue. Data in Figure S2 show gene expression of GLP1r in different human tissue/cell types expressed as fold difference in relation to the housekeeping gene (RPL13A).

| D ISCUSS I ON
In the present study, we demonstrate an anabolic effect of GLP-1 in Since these pathways are involved in anabolic responses to nutrition driven by AA (Fujita et al., 2007) and insulin (Hillier, Long, Jahn, Wei, & Barrett, 2000), we investigated the activation of key sig-  (Greenhaff et al., 2008), and further work is needed to define these limitations. On long-term provision of therapeutic GLP-1r agonists, mainly to achieve weight loss and optimise glucose control, the resultant weight loss was predominantly F I G U R E 3 Schematic representation of study protocol. 8 older men studied in a crossover design in the fasted + fed state, with and without GLP-1. CEUS, contrastenhanced ultrasound; LBF, leg blood flow 0 1 2 3 4 5 6 Postprandial Insulin clamp ± GLP-1 (1.2 pmol.kg -1 .min -1 ) OctreoƟde 30 ng.kg -1 .min -1 IV Vamin 14-EF providing ≤20 g mixed AA IV 20% Glucose; 7-7.5 mM.L -1 Glucose clamp In order to seek other mechanisms relating to GLP-1's effects, we also quantified muscle perfusion. Age-related attenuation of vascular responses to feeding is well documented, and we have previously described the existence of fed-state microvascular resistance in older age and its co-existence with impaired muscle anabolic response (Phillips et al., 2014). We demonstrate in the present study a greater increase in muscle MBF in response to feed- Finally, enhanced MBF may instead play a crucial role in muscle glucose uptake (Vincent et al., 2004).
Recently, chronic provision of antiglycaemic DPP-4 inhibitors was shown to result in increases in muscle mass (Bouchi et al., 2018), although the mechanistic basis for the observed benefits was not studied. Similarly, recent work in mice has shown the potential of GLP-1 agonism in relation to skeletal muscle maintenance in catabolic disease models (Hong et al., 2019). Our data indicate that GLP-1 infusion restores the anabolic response to EAA provision in older muscle, which may help explain the increase or maintenance in muscle mass observed in these previous studies. In the context of diabetes, GLP-1 therapy is widely used in clinical practice, to improve glucose uptake and disposal, and given that muscle loss and the incidence of sarcopenia is accelerated in patients with diabetes (Park et al., 2007), GLP-1 therapies may also have a role to play in maintaining muscle mass.
Our study is limited by the nature of its short duration in demonstrating these metabolic and microvascular gains. As we have previ-

| Subjects and design
The study was approved by The University of Nottingham Ethics Committee (Reference Number: G12122013 MSGEM) and was conducted in line with the Declaration of Helsinki, 2013. Eight healthy male volunteers (65-75 years of age, see Table 1 for subject characteristics) from the local area were recruited into the study, via mailshot.
A comprehensive clinical examination and metabolic screening were conducted at the Royal Derby Medical School, Derby. Subjects with metabolic disease, lower limb musculoskeletal abnormalities, acute cerebrovascular or cardiovascular disease, active malignancy, uncontrolled hypertension, and BMI <18 or >28 kg m 2 , on medications that impact muscle protein metabolism or modulate vascular tone or have known allergy to any of the study drugs, were excluded. All volunteers were studied following overnight fasting of 10-14 hr. Each volunteer was studied on two occasions, at least 3 weeks apart. Volunteers were randomly assigned to receive either glucagon-like peptide-1 (GLP-1) infusion into the femoral artery in one leg or placebo in the contralateral leg. Volunteers were blinded to which visit they would receive GLP-1.

| Reporting and initial preparation
On the morning of the study, volunteers reported to the Clinical Physiology Laboratory at 0800. Following a DXA scan, volunteers lay supine on a bed. Three polyethylene cannulae (20G × 2 & 18G × 1) for intravenous infusions were inserted, distributed between the two forearms. This was followed by the insertion of femoral venous and arterial cannulae into the femoral artery and vein of the leg designated for study. The area below the inguinal ligament was anaesthetised before introduction of wire-guided femoral catheters using ultrasound scan guidance (Philips iU22 Ultrasound, Bothell, WA, USA).

| Labelled AA infusion
At time zero after baseline venous and arterial blood sampling, an infusion of L-[ring-13 C 6 ]-phenylalanine (Cambridge Laboratories, MA, USA) was started (prime 0.4 mg kg −1 . then infused at 0.6 mg kg −1 hr −1 ) and was continued until the end of the study (a total of 6 hr, see study protocol for timings).

| Muscle biopsies
Baseline muscle biopsies were taken at 60 min (after attainment of steady-state isotope labelling) and 180 min following initiation of infusion and immediately before start of postprandial clamp. Two further muscle biopsies were taken at 90 min and 180 min postinitiation of the clamp.

| Contrast-enhanced ultrasound
At 145 min following the start of labelled AA infusion, baseline measurement of microvascular parameters was conducted using contrast-enhanced ultrasound (CEUS-Philips iU22 Ultrasound, Bothell, WA, USA).
Sonovue TM (Bracco, Courcouronnes, France) was infused via a peripheral vein at an initial rate of 2 ml min −1 for 1 min and 1 ml min −1 for a further 2 min. During the three-min duration, three cycles of flash/replenishment videos were recorded. Further assessment of microvascular parameters was made at 120 min following the start of the postprandial clamp. At the end of the study, participants were monitored and fed before being allowed to leave. Volunteers returned after at least 3 weeks for the crossover study on the contralateral leg.

| Anthropometric indices
Body mass index (BMI) was defined as weight in kg height in m −2 .
Sarcopenic index corresponds to the appendicular skeletal muscle mass index (ASMI), which was calculated as appendicular skeletal muscle (ASM) in kg height in m −2 .

| Plasma insulin and GLP-1 concentrations
Commercial ELISA kits (Milliplex Map kit, EMS Millipore, Germany) were used to determine insulin, C peptide and GLP-1 concentrations. For GLP-1, blood was collected in P800 tubes, to stabilise

| Plasma AA concentration and Phe enrichment
For the measurement of AA concentration, internal standards were added to plasma samples, before the addition of urease solution and incubation at room temperature for 20 min. Samples were then de-proteinised with ice-cold ethanol for 20 min at 4°C, before centrifugation at 13,000 g. The supernatant containing plasma free AA was then decanted and evaporated at 90°C under N 2 . Dried AA were solubilised in 0.5 M HCl and lipids extracted in ethyl acetate, before being evaporated to dryness. AA were derivatised through the addition of equal volumes of acetonitrile (ACN) and N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) and heated to 90°C for 60 min to create tert-butyldimethylsilyl (t-BDMS) AA esters. Samples were allowed to cool before transfer to autosampler vials. AA concentrations were quantified using standard curves of known concentrations by GC-MS, as per our standard approach (Wilkinson et al., 2018). with reference to standard curves of known concentration (Smith & Rennie, 1996).

| Determination of muscle protein-bound and intracellular free phenylalanine enrichment
The muscle myofibrillar fraction was isolated as previously described (Atherton et al., 2010;Greenhaff et al., 2008), and L-[ring-13 C 6 ]-phenylalanine incorporation into myofibrillar protein was determined by gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS, Delta-plus XP, Thermo, Hemel Hampstead, UK). Separation was achieved on a 25 m · 0.25 mm · 1.0 μ-film DB 1701 capillary column (Agilent Technologies, West Lothian, United Kingdom). Gas chromatography mass spectrometry (GC-MS, Agilent-5977a, California, USA) was used to determine muscle intracellular L-[ring-13 C 6 ]-phenylalanine enrichment. The sarcoplasmic fraction containing the intramuscular free amino acid pool was precipitated, and the supernatant was purified by cationexchange chromatography, using Dowex H + resin and derivatised as their t-BDMS derivatives before measurement of phenylalanine enrichment by GC-MS (Atherton et al., 2010;Greenhaff et al., 2008;Mitchell et al., 2015).  Phenylalanine release from the leg was calculated as: LBF × C V .

| Calculation of fractional synthesis rates
Muscle protein net balance (NB) was calculated as (C A − C V ) × LBF.
The rate of appearance, that is MPB, was calculated from the dilution of tracer across the leg, as (E A /E V − 1) × LBF × C A ; the rate of disappearance of phenylalanine, that is MPS, was calculated indirectly as the sum of MPB + muscle protein net balance (NB) where NB is where C A and C V are concentration of phenylalanine in the femoral artery and vein, respectively, and E A and E V are phenylalanine enrichment in the femoral artery and vein, respectively (Bennet, Connacher, Scrimgeour, Jung, & Rennie, 1990).

| Statistical analysis
The sample size was prospectively determined based on previous local studies to detect difference in muscle metabolism. For repeated measures of MPS in the same sample, the coefficient of variation (CV) is ~3.8%.
The population CVs are ~10-12% for young and older men. For MPB, with a population CV of 15% (based on previous laboratory data) and CV of laboratory techniques also of 15% (propagated error ~21%), we were able to detect (with 80% confidence at the 5% significance level) differences in rates of MPB after feeding of ±21% (i.e. 1 SD). Given these parameters, the smallest number of subjects needed to detect (with 80% confidence, 5% significance level) a cross-sectional difference between groups, or a one-way difference on a paired basis, of 20% was 8.
Analysis was conducted using Prism 7 (GraphPad, San Diego, CA). Data are presented as mean ± SEM. Normality of distribution was tested using

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest in relation to this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.