Introduction: It is generally regarded that insulin and glucagon exert opposing actions on plasma glucose: insulin lowers glucose by suppressing endogenous glucose production (EGP) and stimulating glucose uptake, while glucagon increases glucose by stimulating EGP. Protein and glucose co-ingestion stimulates both insulin and glucagon secretion, however glucose excursions are typically reduced compared to ingestion of a matched amount of glucose alone. Despite stimulating glucagon secretion, it is unclear how protein lowers glucose excursions. Therefore, this study addressed this by measuring postprandial glucose fluxes via the triple stable isotope glucose tracer technique following ingestion of either glucose (alone), or glucose and whey protein combined.

Methods: The study was approved by Deakin University Human Research Ethics Committee (2022-181). Eleven adults (5M/6F, 27±1.5 years, BMI: 23.4±0.6 kg/m²) underwent three trials in random order, ingesting either 25g glucose (25G;~100 kcal), 50g glucose (50G;~200kcal) or 25g glucose plus 25g whey protein (WG;~200kcal). This allowed group comparisons where both glucose (WG vs 25G) and calories (WG vs 50G) were matched. Each trial utilised three stable isotope glucose tracers: [6,6-²H] and [U-¹³C] for variable infusions, and [1-²H] for ingestion. Arterialised blood samples were obtained for 4h post-meal, to determine tracer enrichments (gas chromatography-mass spectrometry), insulin, glucagon, GLP-1, GIP, and C-peptide (ELISA). Glucose fluxes (EGP and rates of exogenous glucose appearance (meal Ra) and glucose disappearance (Rd)) were calculated using Steele’s non-steady-state model. Data were reported as mean±SEM. One-way or two-way ANOVA were used for analyses where appropriate, followed by a Holm-Sidak’s post hoc test (Graphpad Prism), with statistical significance set at p<0.05.

Results: The integrated postprandial glucose response (incremental area under the curve; iAUC) was markedly lower for WG (78±13 mmol.240min/L) compared to both 25G (182±27 mmol.240min/L, p<0.001) and 50G (310±45 mmol.240min/L, p<0.001). As expected, WG increased glucagon concentrations (~3-fold basal), while both 25G and 50G reduced glucagon levels. Insulin total iAUC was higher for WG vs 25G (p=0.01). Comparing WG vs 50G (calories matched), WG produced higher peak insulin concentrations (630±131 pmol/L vs 442±82 pmol/L, p=0.04) and tended to produce a higher insulin response over the initial 30min postprandial period (iAUC_0-30min, p=0.07). Despite the enhanced early insulin response, WG and 50G produced comparable GIP and GLP-1 responses (p>0.05). EGP suppression was less pronounced for WG (~48% suppressed) compared to 25G (~70% suppressed) or 50G (79% suppressed), p<0.001. WG vs 25G resulted in comparable Rd iAUC_0-60min (p>0.05) but tended to lower meal Ra iAUC_0-60min (254±35 mg.60min/kg vs 294±36 mg.60min/kg, p=0.07).

Conclusion: The findings confirm that whey/glucose co-ingestion, compared to the same glucose dose alone, reduces postprandial glucose excursions, enhances the insulin response, and induces a robust glucagon response. Additionally, we show that for the same caloric content but half glucose amount, whey/glucose co-ingestion enhances the early insulin response compared to glucose alone, which could be driven by amino acids and/or glucagon but not GLP-1 or GIP. We also provide novel mechanistic data, revealing that the addition of whey protein lowers glycaemic excursions despite less EGP suppression, with the net glycaemic benefit coming from reduced glucose absorption rates, not enhanced Rd.