Macroalgal protein hydrolysates from Palmaria palmata influence the ‘incretin effect’ in vitro via DPP-4 inhibition and upregulation of insulin, GLP-1 and GIP secretion

Purpose This study investigated metabolic benefits of protein hydrolysates from the macroalgae Palmaria palmata, previously shown to inhibit dipeptidylpeptidase-4 (DPP-4) activity in vitro. Methods Previously, Alcalase/Flavourzyme-produced P. palmata protein hydrolysate (PPPH) improved glycaemia and insulin production in streptozotocin-induced diabetic mice. Here the PPPH, was compared to alternative Alcalase, bromelain and Promod-derived hydrolysates and an unhydrolysed control. All PPPH’s underwent simulated gastrointestinal digestion (SGID) to establish oral bioavailability. PPPH’s and their SGID counterparts were tested in pancreatic, clonal BRIN-BD11 cells to assess their insulinotropic effect and associated intracellular mechanisms. PPPH actions on the incretin effect were assessed via measurement of DPP-4 activity, coupled with GLP-1 and GIP release from GLUTag and STC-1 cells, respectively. Acute in vivo effects of Alcalase/Flavourzyme PPPH administration on glucose tolerance and satiety were assessed in overnight-fasted mice. Results PPPH’s (0.02–2.5 mg/ml) elicited varying insulinotropic effects (p < 0.05–0.001). SGID of the unhydrolysed protein control, bromelain and Promod PPPH’s retained, or improved, bioactivity regarding insulin secretion, DPP-4 inhibition and GIP release. Insulinotropic effects were retained for all SGID-hydrolysates at higher PPPH concentrations. DPP-4 inhibitory effects were confirmed for all PPPH’s and SGID counterparts (p < 0.05–0.001). PPPH’s were shown to directly influence the incretin effect via upregulated GLP-1 and GIP (p < 0.01–0.001) secretion in vitro, largely retained after SGID. Alcalase/Flavourzyme PPPH produced the greatest elevation in cAMP (p < 0.001, 1.7-fold), which was fully retained post-SGID. This hydrolysate elicited elevations in intracellular calcium (p < 0.01) and membrane potential (p < 0.001). In acute in vivo settings, Alcalase/Flavourzyme PPPH improved glucose tolerance (p < 0.01–0.001) and satiety (p < 0.05–0.001). Conclusion Bioavailable PPPH peptides may be useful for the management of T2DM and obesity.

The role of incretins on insulin function and glucose homeostasis

The incretin effect – the amplification of insulin secretion after oral versus intravenous administration of glucose as a mean to improve glucose tolerance – was suspected even before insulin was discovered, and today we know that the effect is due to the secretion of two insulinotropic peptides, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). But how important is it? Physiological experiments have shown that, because of the incretin effect, we can ingest increasing amounts of amounts of glucose (carbohydrates) without increasing postprandial glucose excursions, which otherwise might have severe consequences. The mechanism behind this is incretin-stimulated insulin secretion. The availability of antagonists for GLP-1 and most recently also for GIP has made it possible to directly estimate the individual contributions to postprandial insulin secretion of a) glucose itself: 26%; b) GIP: 45%; and c) GLP-1: 29%. Thus, in healthy individuals, GIP is the champion. When the action of both incretins is prevented, glucose tolerance is pathologically impaired. Thus, after 100 years of research, we now know that insulinotropic hormones from the gut are indispensable for normal glucose tolerance. The loss of the incretin effect in type 2 diabetes, therefore, contributes greatly to the impaired postprandial glucose control.

The impact of low dose gliclazide on the incretin effect and indices of beta cell function

Aims/Hypothesis Studies in permanent neonatal diabetes suggest that sulphonylureas lower blood glucose without causing hypoglycaemia, in part by augmenting the incretin effect. This mechanism has not previously been attributed to sulphonylureas in patients with type 2 diabetes (T2DM). We therefore aimed to evaluate the impact of low dose gliclazide on beta-cell function and incretin action in patients with T2DM. Methods Paired oral glucose tolerance tests and isoglycaemic infusions were performed to evaluate the difference in the classical incretin effect in the presence and absence of low dose gliclazide in 16 subjects with T2DM (HbA1c &lt;64mmol/mol, 8.0%) treated with diet or metformin monotherapy. Beta-cell function modelling was undertaken to describe the relationship between insulin secretion and glucose concentration. Results A single dose of 20mg gliclazide reduced mean glucose during the OGTT from 12.01 ± 0.56 to 10.82 ± 0.5 mmol/l (p=0.0006) (mean ± SEM). The classical incretin effect was augmented by 20mg gliclazide, from 35.5% (LQ 27.3, UQ 61.2) to 54.99% (34.8, 72.8) (p=0.049). Gliclazide increased beta-cell glucose sensitivity by 46% (Control 22.61 ± 3.94, Gliclazide 33.11 ± 7.83 (p=0.01)) as well as late-phase incretin potentiation (Control 0.92 ± 0.05, Gliclazide 1.285 ± 0.14 (p=0.038). Conclusions/Interpretation Low dose gliclazide reduces plasma glucose in response to oral glucose load, with concomitant augmentation of the classical incretin effect. Beta-cell modelling shows that low plasma concentrations of gliclazide potentiate late phase insulin secretion and increase glucose sensitivity by 50%. Further studies are merited to explore whether low dose gliclazide, by enhancing incretin action, could effectively lower blood glucose without risk of hypoglycaemia.

No detectable effect of a type 2 diabetes-associated TCF7L2 genotype on the incretin effect

The T allele of TCF7L2 rs7903146 is a common genetic variant associated with type 2 diabetes (T2D), possibly by modulation of incretin action. In this study, we evaluated the effect of the TCF7L2 rs7903146 T allele on the incretin effect and other glucometabolic parameters in normal glucose tolerant individuals (NGT) and participants with T2D. The rs7903146 variant was genotyped in cohorts of 61 NGT individuals (23 were heterozygous (CT) or homozygous (TT) T allele carriers) and 43 participants with T2D (20 with CT/TT). Participants were previously examined by an oral glucose tolerance test (OGTT) and a subsequent isoglycemic intravenous glucose infusion (IIGI). The incretin effect was assessed by quantification of the difference in integrated beta cell secretory responses during the OGTT and IIGI. Glucose and hormonal levels were measured during experimental days, and from these, indices of beta cell function and insulin sensitivity were calculated. No genotype-specific differences in the incretin effect were observed in the NGT group (P = 0.70) or the T2D group (P = 0.68). NGT T allele carriers displayed diminished glucose-dependent insulinotropic polypeptide response during OGTT (P = 0.01) while T allele carriers with T2D were characterized by lower C-peptide AUC after OGTT (P = 0.04) and elevated glucose AUC after OGTT (P = 0.04). In conclusion, our findings do not exclude that this specific TCF7L2 variant increases the risk of developing T2D via diminished incretin effect, but genotype-related defects were not detectable in these cohorts.

Incretin as a Pathophysiological Component and Target for Treatment in Youth Type 2 Diabetes (T2D)

Incretin hormones have recently been considered an important pathophysiological factor for T2D in adults and youth due to their role in augmenting insulin secretion (Michaliszyn et al., 2014). It is recognized that glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are glucose-dependent hormones released from the gut that stimulate insulin release from pancreatic β-cells (Muscelli et al., 2006). Thus, the insulin response to oral glucose is significantly greater than the intravenous (IV) glucose administration response; this is known as the incretin effect (Michaliszyn et al., 2014). To date, adults with T2D demonstrate a remarkable decrease in the incretin effect (Nauck et al., 1986), whereas there is lack of evidence in pediatric populations. The incretin effect is also associated with β-cell glucose sensitivity (βCGS), attesting incretins as a high-promising target for T2D treatment (Michaliszyn et al., 2014). Utilizing GLP-1 receptor agonists can be advantageous as a therapeutic option for both adults and youth (Tamborlane et al., 2019; Vanderheiden et al., 2016; Yeow et al., 2017). While metformin and insulin were the sole treatment options for Y-T2D prior to FDA approval (in 2019) for the use of GLP-1 agonists, a recent RISE (Restoring Insulin Secretion) clinical trial reported disappointing results that both metformin alone and insulin glargine for three months followed by metformin for nine months were not effective in restoring/preserving β-cell function in youth with prediabetes and T2D (RISE Consortium & RISE Consortium Investigators, 2019). On the contrary, the role of GLP-1 and the efficacy of the GLP-1 receptors in T2D signifies a strong target for potential treatment. Additional clinical trials are warranted to see if monotherapy of GLP-1 agonist vs. combined (metformin + liraglutide) therapy is effective to reserve Y-T2D to prediabetes and/or normal state. More importantly, future research should focus on disease prevention rather than treatment to avoid aggressive complications and metabolic degradations of Y-T2D (RISE Consortium & RISE Consortium Investigators, 2019). Altogether, it would be germane to investigate whether lifestyle changes (diet, physical activity, exercise medicine) can improve the incretin effect in conjunction with glycemic control in youth.

A reduced incretin effect mediated by the rs7903146 variant in the TCF7L2 Gene is an early marker of beta-cell dysfunction in obese youth

<b>Background.</b> The risk genotype for the common variant <i>rs7903146 </i>of the transcription factor-7-like-2 gene (<i>TCF7L2</i>) has been found to affect the incretin response in healthy and obese adults, however, whether a similar functional defect is also present in obese adolescents remains unexplored. Herein, we examined the functional effect of the <i>rs7903146</i> variant in the TCF7L2 gene on the incretin effect and determined its translational metabolic manifestation by performing deep phenotyping of the incretin system, beta-cell function relative to insulin sensitivity, the Gastrointestinal Induced Glucose Disposal (GIGD) in obese youths with normal and impaired glucose tolerance. <p><b>Methods</b> Thirty nine non-diabetic obese adolescents (15[14,18] years; BMI 37[33, 43]kg/m²) were genotyped for the <i>rs7903146 </i>of <i>TCF7L2</i> and underwent a 3-hour OGTT followed by an iso-glycemic intravenous glucose infusion (iso-IVGTT) to match the plasma glucose concentrations during the OGTT and a hyperglycemic clamp with arginine stimulation.</p> <p>The incretin effect was measured as 100*(AUC-SR_OGTT– AUC-SR_iso-IVGTT)/AUC-SR_OGTT[AUC-SR=AUC of C-peptide secretion rate]. Participants were grouped into tertiles according to the percentage incretin effect (High-, Moderate- and Low-incretin effect) to describe their metabolic phenotype.</p> <p><b>Results </b>The presence of T risk allele for <i>TCF7L2</i> was associated with a markedly reduced </p> <p>incretin effect compared to the wild type genotype(0.3[-7.2,14] vs 37.8[12.5-52.4], p<0.002) When the cohort was stratified by incretin effect, the High-, Moderate- and Low-incretin groups did not differ with respect to anthropometric features, while the Low-incretin group exhibited higher 1-h glucose (p=0.015), a reduced disposition index, insulin sensitivity and insulin clearance, compared with the High-incretin group. Gastrointestinal induced glucose disposal (GIGD) was reduced in the Low-incretin group (p=0.001). The three groups did not differ with respect to intravenous glucose-induced insulin secretion and arginine response during the hyperglycemic clamp. </p> <p><b>Conclusion </b>A<b> </b> reduced incretin effect and its association with the <i>TCF7L2</i> variant rs7903146 identify an early metabolic phenotype in obese non-diabetic youths, featured by a higher plasma glucose peak at 1hr, lower insulin secretion, sensitivity and clearance, and gastrointestinal glucose disposal. </p>