Mechanism of action of pre-meal consumption of whey protein on glycemic control in young adults

    Mechanism Of Action Of Pre-Meal Consumption Of Whey Protein On Glycemic Control In Young Adults Tina Akhavan, Bohdan L. Luhovyy, Shirin Panahi, Ruslan Kubant, Peter H. Brown, G. Harvey Anderson PII: DOI: Reference:

S0955-2863(13)00183-6 doi: 10.1016/j.jnutbio.2013.08.012 JNB 7093

To appear in:

The Journal of Nutritional Biochemistry

Received date: Revised date: Accepted date:

12 February 2013 27 August 2013 30 August 2013

Please cite this article as: Akhavan Tina, Luhovyy Bohdan L., Panahi Shirin, Kubant Ruslan, Brown Peter H., Anderson G. Harvey, Mechanism Of Action Of Pre-Meal Consumption Of Whey Protein On Glycemic Control In Young Adults, The Journal of Nutritional Biochemistry (2013), doi: 10.1016/j.jnutbio.2013.08.012

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MECHANISM OF ACTION OF PRE-MEAL CONSUMPTION OF WHEY PROTEIN

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ON GLYCEMIC CONTROL IN YOUNG ADULTS1,2

Tina Akhavana, Bohdan L. Luhovyya, Shirin Panahia, Ruslan Kubanta, Peter H. Brownb, G.

Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto,

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Harvey Andersona*

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Ontario, Canada (TA, BLL, SP, RK, GHA) Kraft Foods Global LLC, US (PHB)

*Corresponding author. Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, 150 College Street, M5S 3E2, Toronto, ON, Canada. Tel: (416) 978-1832, Fax: (416) 978-5882, E-mail: [email protected] 1

We would like to thank Kraft Foods Global LLC for providing the whey protein and McCain

Foods Ltd. (Toronto, ON) for providing pizza for the experimental meals. This project has been supported by a Collaborative Research and Development Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) and Kraft Canada Inc.

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Author disclosure: T Akhavan, B.L. Luhovyy, S Panahi, R Kubant, P.H. Brown, and G.H

Anderson have no conflicts of interest to report. PHB is an employee of Kraft Foods Global LLC

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(Chicago, IL).

RUNNING TITLE: Whey protein consumption and glycemia

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WORD COUNT: 5354; NUMBER OF FIGURES: 5; NUMBER OF TABLES: 2

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ABBREVIATION USED: WP (whey protein), GIP (gastric inhibitory polypeptide), GLP-1 (glucagon-like peptide-1), CCK (cholecystokinin), PYY (peptide tyrosine-tyrosine), CHO

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(carbohydrate), BMI (body mass index), VAS (visual analogue scales), ANOVA (analysis of variance), Insulin SECretion (ISEC), DPP-IV (dipeptidyl peptidase IV), BBB (blood-brain

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barrier), CNS (central nervous system)

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ABSTRACT Whey protein (WP), when consumed in small amounts prior to a meal, improves postmeal glycemic control more than can be explained by insulin-dependent mechanisms alone. The

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objective of the study was to identify the mechanism of action of WP beyond insulin on the

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reduction of post-meal glycemia. In a randomized crossover study, healthy young men received

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preloads (300 mL) of WP (10 and 20 g), glucose (10 and 20 g) or water (control). Paracetamol

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(1.5 g) was added to the preloads to measure gastric emptying. Plasma concentrations of

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paracetamol, glucose, and ß-cell and gastrointestinal hormones were measured before preloads

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(baseline) and at intervals before (0-30 min) and after (50-230 min) a preset pizza meal (12

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kcal/kg). Whey protein slowed pre-meal gastric emptying rate compared to the control and 10 g

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glucose (P < 0.0001), and induced lower pre-meal insulin and C-peptide than the glucose

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preloads (P < 0.0001). Glucose, but not WP, increased pre-meal plasma glucose concentrations

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(P < 0.0001). Both WP and glucose reduced post-meal glycemia (P = 0.0006) and resulted in

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similar CCK, amylin, ghrelin and GIP responses (P < 0.05). However, compared with glucose,

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WP resulted in higher post-meal GLP-1 and PYY and lower insulin concentrations, without

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altering insulin secretion and extraction rates. For the total duration of this study (0-230 min),

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WP resulted in lower mean plasma glucose, insulin and C-peptide, but higher GLP-1 and PYY

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concentrations than the glucose preloads. In conclusion, pre-meal consumption of WP lowers

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post-meal glycemia by both insulin-dependent and insulin-independent mechanisms.

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INTRODUCTION When consumed with carbohydrate (CHO), proteins in general [1, 2] and milk proteins specifically [3, 4] reduce glycemic response compared with CHO alone. This effect has been

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attributed to the is insulinotropic effect of milk proteins [5, 6], and more specifically to whey

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protein (WP) [7]. The addition of WP [3, 8] or whey peptides [4] to a glucose drink or CHO

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meal reduces glycemic response, which has been attributed to its rapid digestion [9], and release

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of amino acids and bioactive peptides during digestion stimulate release of insulin [10], and

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many gastrointestinal hormones [7].

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Proteins are known to be insulinotrophic but whether WP is more so, as has been

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suggested, and it is the only cause of the lowered glycemia after its consumption is unclear. A

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breakfast and lunch, each containing 28 g of WP and 50 g CHO served to adults with T2D,

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resulted in higher blood insulin concentrations after both breakfast and lunch and lower glucose

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after the lunch than when the meals contained lean ham [3]. Additionally, 50 g WP in a meal

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lowered glycemia more than a similar amount of protein from turkey or egg albumin, and

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resulted in higher insulin concentrations than the meals with turkey, egg albumin and tuna over

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240 min [11].

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However, the reduced glycemia after protein consumption either with CHO [2] or alone

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[9, 12] may be due to increased insulin release but also to release of gut hormones that delay

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stomach emptying and to the release of incretins that increase efficacy of insulin [7]. Small

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amounts (as low as 9-10 g) consumed 30 min prior to a meal [13] reduce post-meal

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concentrations of both glucose and insulin, indicating that insulin cannot be the only cause of the

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reductions. Only one study has related decreased post-meal glycemia to slower gastric emptying.

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Pre-meal consumption of 55 g WP increased glucagon-like peptide-1 (GLP-1) and delayed

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gastric emptying more than when consumed with a small CHO meal [14]. Therefore, the hypothesis of this study was that WP consumed alone prior to a meal

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improves post-meal glycemic control by both insulin-dependent and -independent mechanisms.

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The objective was to describe and compare the effect of WP and glucose consumed 30 min

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before a fixed meal in healthy men on pre- and post-meal and plasma concentrations of glucose,

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insulin, gastrointestinal hormones regulating stomach emptying and incretins involved in

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potentiating insulin efficacy and on pre-meal gastric emptying rate.

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METHODS AND MATERIALS

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Participants

In a randomized crossover design, 10 men (18-29 y, 18.5-29.4 kg/m2) received preloads

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(300 mL) of WP (10 and 20 g), glucose (10 and 20 g) or water (control). Paracetamol (1.5 g) was

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added to the preloads to measure gastric emptying. Plasma concentrations of paracetamol,

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glucose, ß-cell hormones and gastrointestinal hormones were measured before preloads

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(baseline) as well as at intervals before (0-30 min) and after (50-230 min) a preset pizza meal (12

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kcal/kg).

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Participants were recruited through advertisements posted on the University of Toronto

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campus. At initial contact by phone or e-mail, eligibility requirements were described to the

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potential subjects and they were asked for their age, body weight, height, if they smoke or were

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taking any medications. Breakfast skippers, smokers, dieters and individuals with diabetes or

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other metabolic diseases were ineligible to participate in the study. Individuals who fulfilled

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eligibility requirements were asked to come to the Department for a second screening to

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ACCEPTED MANUSCRIPT complete questionnaires regarding food habits, food preference and dietary restraint [15] and to

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read and sign the consent form. Their height and weight were measured to calculate their body

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mass index (BMI). Qualified subjects were invited to participate in the study. Subjects were

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financially compensated for completing the study. The procedures of the study were approved by

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the Human Subject Review Committee, Ethics Review Office at the University of Toronto.

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Based on previous clinical studies on gut hormones with the sample size required for

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blood glucose response [4, 16, 17], 10 male subjects, aged 18-29 years with a BMI between

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18.5-24.9 kg/m2, were recruited and completed the sessions.

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Protocol

Experimental sessions took place at the Department of Nutritional Sciences at University

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of Toronto and subjects participated in the study twice per week. Similar to our previous study

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[13], a breakfast (300 kcal) of a single serving of a ready-to-eat breakfast cereal (Honey Nut

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Cheerios; General Mills, Mississauga, Canada), a 250-mL box of 2% milk (Sealtest Skim Milk,

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Markham, Canada) and a 250-mL box of orange juice (Tropicana Products Inc, Bradenton, FL)

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was provided to subjects to be consumed at their preferred time in the morning (0600–0900)

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after a 10-h overnight fast. Subjects were asked not to consume anything between the breakfast

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and the study session 4 h later (1000 to 1300). Participants were permitted to consume water

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until 1 h before the session. Each subject arrived at the same chosen time for each session. They

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were instructed to refrain from alcohol consumption and any unusual exercise and activity the

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night before the study sessions.

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Upon arrival, subjects filled out the Sleep Habits and Stress Factors Questionnaire and

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Food Intake and Activity Questionnaire forms. Visual Analogue Scales (VAS) questionnaires

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were then completed to measure Physical Comfort and Fatigue/Energy [16, 18]. If they reported

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significant deviations from their usual patterns, they were rescheduled. Following completion of the VAS questionnaires, an indwelling intravenous catheter was

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inserted in the antecubital vein by a registered nurse and a baseline blood sample obtained.

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Immediately after, participants drank one of the five preloads within 5 min, followed by

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completion of a palatability VAS (at 5 min). Blood samples were collected at 10, 20 and 30 min

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(pre-meal) and 50, 60, 70, 80, 110, 140, 170, 200 and 230 min (post-meal).

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Preloads

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Pre-meal drinks included iso-volumetric amounts (300 mL) of 10 and 20 g intact WP (NZMP Whey Protein Concentration 392, Fonterra Co- operative Group Limited, New Zealand),

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and 10 and 20 g glucose control (D-Glucose Monohydrate , Grain Process Enterprises LTD.

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Scarborough, ON) and a water control. Paracetamol (1.5 g, Panadol, GlaxoSmithKline) was also

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dissolved in each of the five preloads. Preloads were served chilled with an additional 100 mL

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of water (in a separate glass) to be consumed upon completion of the drinks to reduce aftertaste.

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Lemon flavor (0.5 mL, Flavorganics, Newark, NJ), lemon juice (2 tsp, Equality; The Great

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Atlantic and Pacific Company of Canada Ltd, Toronto, Canada) and sucralose (0.15 g, McNeil

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Specialty Products Company, New Brunswick, NJ) were added to the drinks to equalize

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palatability and sweetness and to blind the subjects to the preloads.

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Blood Parameters Blood was collected in 8.5 mL BD™ P800 tubes (BD Diagnostics, Franklin Lakes, NJ) containing spray-dried K2EDTA anticoagulant and proprietary additives to prevent their

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ACCEPTED MANUSCRIPT immediate proteolytic activity. The tubes were centrifuged at 1300 RCF for 20 min at 4°C.

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Collected plasma samples were aliquoted in Eppendorf tubes and stored at -70 C for analyses.

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Plasma concentrations of glucose, paracetamol, insulin, C-peptide, amylin, ghrelin, GLP-1, GIP,

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CCK and PYY were measured.

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Plasma glucose was measured using the enzymatic hexokinase method (Roche

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Diagnostic, Laval, QC, Canada). Insulin and C-peptide were assessed with

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electrochemiluminescence assays ―ECLIA‖ (Roche Diagnostic, Laval, QC, Canada). These

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analyses were performed by the Pathology and Laboratory Medicine Division at Mount Sinai

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Hospital (Toronto, ON, Canada). However, the remaining biomarkers were measured at the

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Department of Nutritional Sciences, University of Toronto.

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Free paracetamol was determined with a commercially-available paracetamol

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(acetaminophen) enzymatic assay (Cambridge Life Sciences, Ely, Cambridge, UK). Human

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active GLP-1 (# EGLP-35K), total ghrelin (# EZGRT-89K), total GIP (# EZHGIP-54K), total

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PYY (# EZHPYYT66K) and total amylin (# EZHAT-51K) were measured with ELISA kits

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(Millipore, Billerica, MA). Human CCK was measured by enzyme immunoassay (# EIA-CCK-1,

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RayBiotech Inc, Norcross, GA).

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Plasma concentrations of glucose, insulin and C-peptide were measured at all sampling

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times, however, due to the high cost of the kits and measurements, plasma concentrations of

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hormones were measured at only baseline, 20 and 30 min (pre-meal) and 60, 80, 140 and 230

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min (post-meal).

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Meal

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ACCEPTED MANUSCRIPT Subjects were served a preset pizza meal (12 kcal/ kg body weight of subjects) with a

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bottled spring water (500 mL Crystal Springs) at 30 min because the primary objective of the

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study was to determine the mechanisms of pre-meal consumption of WP on post-meal glycemic

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control. Subjects were given 20 min to consume the pizza meal. Pizza (McCain Foods Ltd.

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Florenceville, NB) was served at all sessions after cooking from frozen according to

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manufacturer‘s directions. Detailed information of the nutrient content of the pizza and method

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of cooking was reported previously [16, 18]. They completed a VAS questionnaire on the

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palatability of the pizza after finishing the pizza meal. Because there were no differences in

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palatability or appetite among the preloads, the results are not reported here.

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Data Analysis and Calculation

A two-way repeated measures analysis of variance (ANOVA), using SAS Proc Mixed

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model followed by Tukey‘s post hoc test, was conducted on pre-meal (0-30 min), post-meal (50-

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230 min) and total (0-230 min) mean plasma concentrations of the dependent measures to test for

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time and preload effects. Means of the pre-meal, post-meal and total concentrations were used as

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an estimate of response in treatment measures rather than area under the curve because post-meal

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concentrations were above baseline and varied in response to preload amount and composition.

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When an interaction was found, one-way ANOVA was performed to identify the effect of

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preload at each time of sampling. One-way ANOVA was also used to make summary mean

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comparisons. Because there were no differences at baseline glucose, hormone and paracetamol

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concentrations, only the absolute concentrations are reported. Significance was set at P < 0.05.

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Data were presented as mean ± standard error of the mean (SEM).

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The pre-hepatic insulin secretion (Figure 2) was calculated from deconvolution of plasma concentrations of C-peptide by using the ISEC computer program [19].

RESULTS

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Participants

Ten subjects completed the study. After completing the study and measurements of the

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entire samples, two subjects had higher mean insulin (704 ± 341 vs. 197 ± 5 pmol/L) and C-

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peptide (2293 ± 797 vs. 1455 ± 26 pmol/L) concentrations compared to other subjects both at

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baseline and throughout the sessions, most possibly due to either consumption of food before

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starting the sessions, or physiological differences such as hyperinsulinemia [8]. They were

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informed and asked to check with their physicians. They were excluded from the analyses,

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therefore the results are based on eight healthy males with a mean age of 22.9 ± 1.2 y, body

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weight of 66.4 ± 2.9 kg, height of 1.7 ± 0.0 m, and BMI of 21.8 ± 0.6 kg/m2.

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Plasma glucose, insulin, C-peptide and amylin

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Pre-meal, post-meal and total mean concentrations of plasma glucose, insulin, C-peptide

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and amylin, followed by p-values for the preload, time and time by preload interaction effects are

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shown in Table 1.

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Pre-meal Mean Plasma Concentrations

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Over the pre-meal period (0-30 min), mean plasma concentrations of glucose, insulin, C-

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peptide and amylin were higher (P < 0.0001) after glucose preloads compared with WP and

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control (Table 1). Both doses of WP increased insulin and C-peptide, however only the 20 g WP

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increased amylin concentrations above control (P < 0.0001).

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Pre-meal concentrations of glucose, insulin, C-peptide (P < 0.0001) and amylin (P = 0.002) were affected by a time by preload interaction (Table 1). One-way ANOVA at each time

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showed that preloads of glucose, but not WP, increased plasma glucose concentrations in a dose-

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dependent manner (P < 0.0001) (Figure 1 A). Higher concentrations of insulin and C-peptide

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after both doses of glucose were observed at 10 min (P < 0.05), 20 min (P < 0.0001) and 30 min

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(P < 0.0001) (Figure 1 B). Only 20 g WP increased insulin concentrations (P < 0.0001), but C-

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peptide concentrations were higher after both doses of WP (P < 0.0001) at 20 and 30 min

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compared to the control, but less than equivalent doses of glucose (Figure 1 C). Amylin

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concentrations were higher after 20 g WP and both glucose preloads than after 10 g WP or

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control (Table 1). At 20 min, glucose preloads (P = 0.0008) and at 30 min, glucose preloads and

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20 g WP (P < 0.0001) increased amylin concentrations compared to control (Figure 1 D).

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Post-meal Mean Plasma Concentrations

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Over the post-meal period (50-230 min), mean plasma glucose concentrations were lower (P = 0.0006) than control after WP and glucose preloads, which were not different from each

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other (Table 1). Mean insulin concentrations were lower (P = 0.0003) after 20 g WP than both

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glucose preloads, and lower after 10 g WP than 20 g glucose. There was no difference between

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control and WP preloads. Mean C-peptide concentrations were higher (P < 0.0001) after glucose

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than after WP, and after 20 g glucose than control. Post-meal amylin concentrations were not

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affected by preload, but were affected similarly by time (P = 0.006) (Table 1).

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Post-meal plasma concentrations of glucose (P < 0.005) and C-peptide (P = 0.03), but not

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insulin, were affected by a time by preload interaction (Table 1). One-way ANOVA at each time

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showed that plasma glucose concentrations were lower after both WP doses and 10 g glucose

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preloads at 60 min (P = 0.003), and after 20 g WP at 70 min (P < 0.05) compared to control

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ACCEPTED MANUSCRIPT (Figure 1 A). Plasma glucose concentrations at 200 min were lower after 10 g compared to 20 g

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glucose (P < 0.04). Plasma insulin concentrations, after an initial post-meal rise from 50-70 min

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decreased to 230 min (Figure 1 B). While there was no difference between WP and control at

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any of the post-meal times, C-peptide concentrations were higher after 20 g glucose compared

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with control and WP at 50 min (P < 0.0001), 60 min (P < 0.002) and 70 min (P = 0.0002), with

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WP at 80 min (P = 0.0009), with control and 20 g WP at 110 min (P = 0.002), and with 10 g

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glucose and 10 g WP at170 min (P = 0.01) (Figure 1 C). The 10 g glucose preload led to a

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greater C-peptide concentration compared with control at 50 min (P < 0.0001) and compared

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with 20 g WP at 70 min (P = 0.0002) (Figure 1 C).

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Total Mean Plasma Concentrations

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Over the entire pre- and post-meal period (0-230 min), mean plasma glucose

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concentrations were lower after WP, but higher after glucose, compared to control (P < 0.0001)

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(Table 1). Mean plasma concentrations of insulin and C-peptide after WP were lower than after

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glucose and not different from control (P < 0.0001). Mean plasma amylin concentrations were

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higher (P = 0.0002) after both glucose and the 20 g WP preloads compared with control (Table

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1).

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PLASMA GLP-1, GIP, PYY, CCK AND GHRELIN CONCENTRATIONS Pre-meal, post-meal and total mean concentrations of plasma concentrations of

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gastrointestinal hormones, followed by p-values for the preload, time and time by preload

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interaction effects are shown in Table 2.

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Pre-meal Mean Plasma Concentrations

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During the pre-meal period (0-30 min), both doses of WP led to higher mean plasma GLP-1 concentrations than the equivalent dose of glucose or the control (P < 0.0001) (Table 2).

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Mean plasma GIP concentrations were higher (P = 0.0002) after the treatments than control, but

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did not differ from each other. Mean plasma PYY concentrations (P = 0.01) were higher than

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control only after 20 g WP. Pre-meal CCK concentrations were not affected by preloads. Mean

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plasma concentrations of ghrelin were not different from the preloads compared to control,

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however, 20 g glucose suppressed (P < 0.05) mean ghrelin concentrations compared with 10 g

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WP (Table 2).

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Pre-meal plasma concentrations of GLP-1 (P = 0.01) and GIP were affected by a time by

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preload interaction (Table 2). Plasma concentrations of GLP-1 were higher after 20 g WP and 20

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g glucose at 20 min (P = 0.0004), and after 20 g WP at 30 min (P = 0.0001) than control (Figure

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3 A). Plasma GIP concentrations were higher after all preloads at 20 min (P < 0.0001), and after

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all preloads except 10 g glucose at 30 min (P = 0.004) than the control (Figure 3 B).

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Pre-meal concentrations of PYY and ghrelin were not affected by a time by preload

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interaction. Pre-meal mean CCK concentrations were not affected by time or preload, but there

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was an interaction (P < 0.04) (Table 2), because the response to the preloads, while not different

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among them, changed over time.

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Post-meal Concentrations

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Over the post-meal period (60-230 min), mean plasma concentrations of GLP-1 and PYY

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were higher (P < 0.0001) after WP than glucose (Table 2). Compared to control, 20 g WP

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resulted in higher concentrations of PYY (P < 0.0001) and lower GIP (P = 0.03). All preloads

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raised mean plasma CCK concentrations above control (P < 0.0001). Mean plasma ghrelin

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concentrations were lower (P < 0.002) only after 20 g glucose compared with 10 g WP (Table 2).

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ACCEPTED MANUSCRIPT Post-meal plasma concentrations of GLP-1 (P < 0.0001) and PYY (P = 0.0009) were

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affected only by time, rising to 80 min and then decreasing to 230 min. There was no time by

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preload interaction effects on these hormones (Table 2).

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Total Mean Plasma Concentrations

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Over the entire pre- and post-meal period (0-230 min), mean concentrations of GLP-1

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and PYY were higher (P < 0.0001) after both doses of WP compared with glucose and control

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(Table 2). However, mean GIP was not affected by preloads. Mean concentrations of CCK were

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higher (P < 0.0001) after both glucose and WP than the control. Mean ghrelin concentrations

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were lower (P = 0.0001) after 20 g glucose than 10 g WP (Table 2).

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GASTRIC EMPTYING RATE (PLASMA PARACETAMOL CONCENTRATIONS)

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Pre-meal (0-30 min) concentrations of paracetamol were affected by preload (P < 0.0001)

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and time (P < 0.0001), but there was no time by preload interaction. Mean plasma concentrations

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of paracetamol were lower after both doses of WP compared to control and 10 g glucose (Figure

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4).

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Post-meal (50-230 min) concentrations of paracetamol decreased with time (P < 0.0001), but were not affected by preloads. Over the entire pre- and post-meal period (0-230 min), mean plasma paracetamol concentrations were lower after WP compared to 10 g glucose (P < 0.004).

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DISCUSSION

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The hypothesis that the post-meal plasma glucose concentrations after pre-meal

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consumption of WP are determined in part by mechanisms beyond insulin is supported by this

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ACCEPTED MANUSCRIPT study. Both WP and glucose preloads similarly reduce post-meal glycemia. However, their

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mechanism of action differed. For the total duration of the study, WP resulted in lower mean

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plasma glucose, insulin and C-peptide, but higher GLP-1 and PYY concentrations than glucose

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preloads.

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Several lines of evidence show that lower post-meal glycemia after WP, unlike glucose, occurs in part by insulin-independent mechanisms. First, these results are consistent with

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previous studies indicating that consumption of WP prior to a meal [13] and or fed with CHO

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[14] improves post-meal glycemia without an increase in post-meal insulin concentrations.

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However the results are novel not only because they show that relatively small amounts are

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efficacious when consumed before a meal, but also show that the lower blood insulin

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concentrations did not occur through alterations in the balance of the kinetics of insulin secretion

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and extraction. The pre-meal consumption of WP led to lower pre- and post-meal plasma

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concentrations of glucose, but also of both insulin and C-peptide (Table 1). Because blood

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insulin concentrations are a product of both secretion and liver extraction C-peptide was

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measured. C-peptide is secreted in equimolar concentrations with insulin from pancreatic ß cells

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but unlike insulin is not extracted by the liver. Therefore, the lower plasma concentrations of C-

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peptide are an indication of lower insulin secretion [20]. When expressed relative to insulin, it is

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also an indicator of hepatic insulin extraction by the liver. These ratios were similar for WP and

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glucose, indicating that insulin extractions rates were unchanged (Figure 5) [8][20].

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Furthermore, the post-meal ratios of C-peptide/insulin were similar after WP and glucose

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preloads and the control, but glucose concentrations were lowered after both WP and glucose;

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demonstrating that insulin-independent mechanisms contributed to post-meal regulation of blood

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glucose concentrations.

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Second, pre-meal consumption of WP, compared with glucose preloads, led to higher plasma concentrations of GLP-1 and PYY (Table 2). GLP-1 [21] and PYY [22] inhibit gut

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motility and slow gastric emptying and both rapidly cross the blood-brain barrier (BBB) to

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directly transmit signals that inhibit gastric emptying. Their increased blood concentration may

299

be explained by both increased synthesis in enteroendocrine L-cells but also by an inhibitory

300

action of WP on dipeptidyl peptidase IV (DPP-IV) which rapidly breaks down circulating

301

peptides[23-25] [7]. Additionally, GLP-1 receptors in the stomach regulate gastric acid secretion

302

and slow motility through ascending vagal afferent signals to the CNS [26, 27]. Taken together,

303

these actions are consistent with the 27% lower pre-meal gastric empting rate after WP compared

304

to glucose and the water control (Figure 4).

RI P

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MA

Third, the increase in amylin concentrations after WP, similar to glucose (Table 1), has

ED

305

T

296

not been shown previously, but may be an additional factor in the improved glucose control

307

without additional insulin. Amylin is secreted simultaneously with insulin by the ß-cell with the

308

amylin to glucose ratio of approximately 1:100 [28]. Amylin affects gastric emptying [29-32]

309

and also regulates glucose homeostasis [33, 34] by acting centrally to suppress nutrient-

310

stimulated glucagon secretion from the α-cells [35], which in turn suppresses the release of

311

endogenous hepatic glucose [29-32].

312

AC

CE

PT

306

Fourth, GLP-1 [36, 37] and PYY [38, 39] in the portal vein stimulate hepatic vagal

313

afferents and pancreatic vagal afferents that activate and enhance glucose disposal, thereby,

314

augmenting portal-mediated glucose clearance.

315

The plasma concentrations of ghrelin decreased with time by 30% after the preloads and

316

meal, with the lowest at 140 min (34%). Others have shown that WP suppresses ghrelin release

317

[40]. However, neither WP nor glucose suppressed pre-meal ghrelin concentrations compared

16

ACCEPTED MANUSCRIPT 318

with the control, possibly due to the small total energy content of the WP or glucose preloads

319

(40-80 kcal) [41]. There are some limitations in the study. First, the sample size of eight is relatively small,

T

320

but was based on previous studies of sample sizes required to detect treatment effects on short-

322

term physiological responses in healthy individuals [13]. However, it is unlikely this affected the

323

conclusion. Based on a calculation of sample size using the variability found in this study, post-

324

meal PYY concentrations after 10 g WP and control would require a sample size of 32 subjects

325

to identify a difference, if it existed. Secondly, the paracetamol absorption test is an indirect

326

method used to measure of liquid gastric emptying rate in humans [12, 42, 43] and provides a

327

reliable, reasonably accurate and inexpensive estimate [44], but it does not have the precision of

328

scintigraphy, which is technically challenging and requires expensive equipment and special

329

licensing for use of radioactive substances [45]. However, while the quantitative rates of

330

stomach emptying may not be as accurate, the primary purpose of the measure was to compare

331

the response to treatments, and differences were found. Third, total ghrelin accounting for both

332

active (acyl ghrelin) and inactive forms of ghrelin (des-acyl ghrelin) was measured in this study.

333

Active ghrelin is now recognized as the preferred measure as it relates more clearly to

334

functionality [46]. Fourth, because this study assessed the acute and short-term effect of WP on

335

glycemia and hormonal responses in healthy young men, the effectiveness of WP consumption

336

on long-term glycemic control is unclear. However, the results of this study adds to the

337

accumulative evidence that whey protein has potential in the dietary management of obesity and

338

T2D [7] and provides encouragement for both short and longer term studies in such individuals.

339 340

AC

CE

PT

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321

In conclusion, WP consumption prior to a meal results in post-meal glucose control by both insulin-dependent and –independent mechanisms.

17

ACCEPTED MANUSCRIPT 341

344 345

We thank Drs. Paul Pencharz, Thomas Wolever, and Ravi Retnakaran for their

T

343

ACKNOWLEDGMENTS

intellectual contribution and constructive guidance during the research.

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342

The authors’ responsibilities were as follows—TA, PHB and GHA: conceived and designed the study; GHA: applied for and received the grant from NSERC; TA and BLL:

347

prepared the ethics application, recruited the volunteers, conducted the clinical session TA, RK,

348

SP and BLL: performed the biochemical analyses; TA: obtained and collated the data and

349

performed the statistical analyses; all authors contributed to the writing of this manuscript.

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18

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478

24

ACCEPTED MANUSCRIPT Table 1 Mean plasma concentrations of glucose, insulin, C-peptide, and amylin after the

Control

10 g Glucose

20 g Glucose

(mmol/L)

meal2 Post-

Preload

Time

Interaction

5.3 ± 0.1c

<0.0001

<0.0001

<0.0001

5.7 ± 0.1b

5.6 ± 0.1b

0.0006

<0.0001

<0.005

5.5 ± 0.1c

5.5 ± 0.1c

<0.0001

<0.0001

<0.0001

82.8 ± 9.1c

106.0 ± 13.6c

<0.0001

<0.0001

<0.0001

266.6 ± 13.4a

218.1 ± 11.3bc

208.5 ± 9.4c

0.0003

<0.0001

NS

5.0 ± 0.1c

6.5 ± 0.2b

6.8 ± 0.3a

6.0 ± 0.1a

5.7 ± 0.1b

6.0 ± 0.1b

5.7 ± 0.1b

6.0 ± 0.1a

6.0 ± 0.1a

36.4 ± 3.8d

153.1 ± 16.3b

207.9 ± 24.3a

229.8 ± 11.5bc

240.5 ± 11.8ab

170.3 ± 11.9c

213.6 ± 10.4b

248.6 ± 12.1a

176.5 ± 10.3c

177.0 ± 9.0c

<0.0001

<0.0001

<0.0001

530.7 ± 24.2d

1081.8 ± 73.8b

1280.3 ± 112.3a

772.4 ± 50.1c

847.8 ± 48.9c

<0.0001

<0.0001

<0.0001

1604.7 ± 47.7bc

1725.8 ± 45.7b

2024.9 ± 63.4a

1589.8 ± 44.4c

1558.2 ± 40.7c

<0.0001

<0.0001

0.03

1274.2 ± 59.4c

1527.6 ± 48.6b

1795.8 ± 65.0a

1338.3 ± 50.6c

1339.6 ± 45.3c

<0.0001

<0.0001

<0.0001

18.5 ± 2.6b

27.3 ± 3.5a

28.7 ± 3.6a

21.3 ± 2.2b

26.7 ± 3.5a

<0.0001

0.0004

0.002

35.7 ± 2.8

38.2 ± 3.1

39.5 ± 3.5

36.4 ± 2.6

35.8 ± 3.3

NS

0.006

NS

28.3 ± 2.3c

33.5 ± 2.4a

34.9 ± 2.6a

29.9 ± 2.0bc

31.9 ± 2.5ab

0.0002

<0.0001

<0.005

3

Pre-

(pmol/L)

meal Post-

ED

Insulin

(pmol/L)

meal Postmeal Total Pre-

CE

Pre-

AC

C-peptide

PT

meal Total

5.1 ± 0.1c

MA

meal

Total4

P (Two-way ANOVA)

SC

Pre-

(pM)

20 g WP

NU

Glucose

Amylin

10 g WP

RI P

Biomarkers

T

preloads1

meal Postmeal Total

1

All values are ± SEM. n = 8. Data were analyzed for pre-meal, post-meal and total for preload ,

time, and preload x time interaction by 2-factor ANOVA (Proc Mixed) and significance was assessed using Tukey's post hoc, P < 0.05 for all, NS (not significant) 25

ACCEPTED MANUSCRIPT 2

Pre-meal values are means of all plasma concentrations before the test meal and calculated from

0-30 min Post-meal values are means of all plasma concentrations after the test meal and calculated from

T

3

4

RI P

50-230 min for plasma glucose, insulin and C-peptide and from 60-230 for plasma amylin Total values are means of all plasma concentrations before and after the test meal and

AC

CE

PT

ED

MA

NU

SC

calculated from 0-230 min

26

ACCEPTED MANUSCRIPT Table 2 Mean plasma concentrations of gastrointestinal hormones after the preloads1

10 g WP

20 g WP

Preload

Time

Interaction

4.9 ± 0.4c

5.3 ± 0.4c

5.8 ± 0.4bc

5.9 ± 0.3b

7.2 ± 0.4a

<0.0001

0.01

6.4 ± 0.4b

5.9 ± 0.3b

6.3 ± 0.4b

8.0 ± 0.4a

RI P

<0.0001

8.5 ± 0.5a

<0.0001

5.8 ± 0.3c

5.6 ± 0.2c

6.1 ± 0.3c

7.1 ± 0.3b

7.9 ± 0.3a

<0.0001

<0.0001

NS

82.7 ± 9.7b

157.6 ± 14.5a

139.3 ± 14.8a

152.9 ± 15.1a

166.5 ± 13.5a

0.0002

0.002

<0.04

393.9 ± 21.1a

372.5 ± 17.7ab

367.4 ± 20.3ab

364.5 ± 16.1ab

319.6 ± 18.6b

0.03

<0.0001

NS

260.5 ± 24.3

280.4 ± 18.6

269.6 ± 20.1

273.8 ± 18.0

254.0 ± 15.8

NS

<0.0001

0.0021

199.6 ± 11.2b

202.1 ± 8.1ab

204.9 ± 9.0ab

223.0 ± 7.8ab

230.3 ± 11.3a

0.01

NS

NS

231.0 ± 10.3bc

223.2 ± 6.5c

220.4 ± 6.5c

249.6 ± 8.6ab

270.4 ± 12.7a

<0.0001

0.0009

NS

217.6 ± 7.8b

214.2 ± 5.2b

213.8 ± 5.4b

238.2 ± 6.2a

253.2 ± 9.1a

<0.0001

<0.0001

NS

100.2 ± 8.8

103.5 ± 9.1

101.5 ± 7.8

122.0 ± 10.0

114.0 ± 8.1

NS

NS

<0.04

77.8 ± 8.1b

127.5 ± 8.3a

113.8 ± 6.4a

123.5 ± 9.0a

123.0 ± 8.1a

<0.0001

NS

NS

87.4 ± 6.1b

117.2 ± 6.3a

108.6 ± 5.0a

122.9 ± 6.4a

119.1 ± 5.7a

<0.0001

NS

0.04

405.0 ± 42.2ab

444.0 ± 64.6ab

322.7 ± 27.0b

512.3 ± 95.7a

407.4 ± 61.8ab

<0.05

NS

NS

304.1 ± 30.3ab

340.5 ± 32.0ab

215.6 ± 21.6b

431.7 ± 78.1a

328.0 ± 35.1ab

<0.002

NS

NS

347.4 ± 26.0bc

384.8 ± 33.6ab

261.5 ± 18.2c

466.2 ± 60.3a

362.0 ± 33.3abc

0.0001

0.0006

NS

2

meal

Post3

Total4 GIP

Pre-

(pg/mL)

meal Post-

Total PYY

Pre-

(pg/ mL)

meal Post-

Total CCK

Pre-

(ng/mL)

meal Postmeal Total

Ghrelin

Pre-

(pg/mL)

meal

CE

meal

ED

meal

NU

meal

T

20 g Glucose

SC

(pg/mL)

Pre-

10 g Glucose

MA

GLP-1

Control

PT

Biomarkers

AC

P (Two-way ANOVA)

<0.0001

NS

Postmeal Total

27

ACCEPTED MANUSCRIPT 1

All values are ± SEM. n = 8. Data were analyzed for pre-meal, post-meal and total for preload ,

time, and preload x time interaction by 2-factor ANOVA (Proc Mixed) and significance was

Pre-meal values are means of all plasma concentrations before the test meal and calculated from

RI P

2

T

assessed using Tukey's post hoc, P < 0.05 for all, NS (not significant)

0-30 min

Post-meal values are means of all plasma concentrations after the test meal and calculated from

SC

3

4

NU

60-230 min

Total values are means of all plasma concentrations before and after the test meal and

AC

CE

PT

ED

MA

calculated from 0-230 min

28

ACCEPTED MANUSCRIPT FIGURE LEGENDS

480

FIGURE 1 Mean (± SEM) pre- and post-meal plasma concentrations of glucose (A), insulin (B),

481

C-peptide (C) and amylin (D) in 8 healthy men after intake of water (····Ӂ····), 10 g glucose

482

(····Δ····), 20 g glucose (····▲····), 10 g whey protein (—○—), and 20 g whey protein (—●—).

483

Different superscripts at each measured time are different between preloads (one-way ANOVA,

484

Proc Mixed, followed by Tukey‘s post hoc, P < 0.05).

SC

RI P

T

479

NU

485

FIGURE 2 Mean (± SEM) pre-meal, post-meal and total insulin secretion rate (pmol/kg/min)

487

calculated from plasma C-peptide concentrations (pmol/L) after the whey protein and glucose

488

preload consumption (n = 8). Two-factor repeated-measures ANOVA followed by Tukey‘s post

489

hoc was used to compare the effect of preloads (means with different superscripts at pre-meal (0-

490

30 min), post-meal (50-230 min) and total (0-230 min) are different, P < 0.0001).

PT

ED

MA

486

491

FIGURE 3 Mean (± SEM) pre-and post-meal plasma concentrations of GLP-1 (A) and GIP (B)

493

in 8 healthy men after intake of water (····Ӂ····), 10 g glucose (····Δ····), 20 g glucose (····▲····), 10

494

g whey protein (—○—), and 20 g whey protein (—●—). Different superscripts at each measured

495

time are different between preloads (one-way ANOVA, Proc Mixed, followed by Tukey‘s post

496

hoc, P < 0.05).

AC

CE

492

497 498

FIGURE 4 Mean (± SEM) pre-meal plasma concentrations of paracetamol after the whey

499

protein and glucose preload consumption (n = 8). Two-factor repeated-measures ANOVA

500

followed by Tukey‘s post hoc was used to compare the effect of preloads (means with different

29

ACCEPTED MANUSCRIPT 501

superscripts at pre-meal (0-30 min) are different, P < 0.0001) and time (P < 0.0001), but no

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interaction effect.

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FIGURE 5 Mean (± SEM) ratio of pre-meal and post-meal plasma concentrations of C-

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peptide/insulin after the whey protein and glucose preload consumption (n = 8). Two-factor

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repeated-measures ANOVA followed by Tukey‘s post hoc was used to compare the effect of

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preloads (means with different superscripts at pre-meal (0-30 min) and post-meal (50-230 min)

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are different, P < 0.05).

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