Incretin hormone responses to carbohydrate and protein/fat are preserved in adults with sulfonylurea‐treated KCNJ11 neonatal diabetes

Abstract The incretin hormones glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP), are thought to be the main drivers of insulin secretion in individuals with sulfonylurea (SU)‐treated KCNJ11 permanent neonatal diabetes. The aim of this study was to assess for the first time the incretin hormone response to carbohydrate and protein/fat in adults with sulfonylurea‐treated KCNJ11 permanent neonatal diabetes compared with that of controls without diabetes. Participants were given a breakfast high in carbohydrate and an isocaloric breakfast high in protein/fat on two different mornings. Incremental area under the curve and total area under the curve (0‐240 minutes) for total GLP‐1 and GIP were compared between groups, using non‐parametric statistical methods. Post‐meal GLP‐1 and GIP secretion were similar in cases and controls, suggesting this process is adenosine triphosphate‐sensitive potassium channel‐independent. Future research will investigate whether treatments targeting the incretin pathway are effective in individuals with KCNJ11 permanent neonatal diabetes who do not have good glycemic control on sulfonylurea alone.


INTRODUCTION
Heterozygous activating mutations in the KCNJ11 gene cause permanent neonatal diabetes (PNDM) by rendering the Kir6.2 subunit of the pancreatic adenosine triphosphate (ATP)sensitive (K ATP ) channel insensitive to ATP 1 .The molecular defect can be overcome by sulfonylurea (SU) drugs, which still bind mutant K ATP channels and facilitate endogenous insulin secretion 2 .Affected individuals can be treated successfully with high doses of SU instead of insulin injections, maintaining excellent glycemic control long term 3 .
Regulation of insulin secretion in SU-treated KCNJ11-PNDM cannot occur through the classical ATP pathway, and is thought to be driven predominantly by the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) 2 .We previously described physiological responses to carbohydrate and protein/fat meals in affected adults; insulin secretion was similar after the two meals, despite higher glucose levels after carbohydrate than protein/fat.This pattern was not seen in controls, who matched insulin secretion to the content of the meal 4 .
Understanding the unique physiology of people with SUtreated KCNJ11-PNDM is important both scientifically and therapeutically.Affected individuals represent human models for the study of non-K ATP -channel pathways, including those relating to the secretion of GLP-1 and GIP from L cells and K cells.In addition, drugs targeting incretin pathways might be a potential adjunct to treatment for the minority of patients with KCNJ11-PNDM who do not achieve good glycemic control on SU monotherapy 5,6 .Despite these implications, no studies have assessed the incretin responses to different foods in these patients.
Our aim was to assess the GLP-1 and GIP responses to carbohydrate and protein/fat meals in adults with KCNJ11-PNDM, and to compare these with adults without diabetes.

Clinical studies
Sample collection took place as part of a previously reported study 4 .Five cases aged >18 years with SU-treated KCNJ11-PNDM, and five age-, sex-and body mass index-matched controls without diabetes (Table S1) fasted overnight, then consumed a high carbohydrate meal and an isocaloric high protein/fat meal (in random sequence) on two different mornings.Cases took their prescribed SU medication with each meal.Cases had an additional visit, during which they took SU without food.Blood samples were taken at baseline (-5 and 0 minutes), 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min post-meal, and spun and frozen at -80°C.
Minor deviations from the meal and sampling protocol were previously reported 4 .One case had an increase in prescribed gliclazide between the second and third visits (from 200 to 320 mg daily).

Biochemical analysis
Plasma concentrations of total GIP and total GLP-1 were measured with commercially available sandwich enzyme-linked immunosorbent assay kits (cat no: 10-1258-01 and 10-1278-01; Mercodia, Uppsala, Sweden).The detection limit for the GIP enzyme-linked immunosorbent assay is 1.62 pmol/L, and for the GLP-1 enzyme-linked immunosorbent assay the detection limit is 0.65 pmol/L.

Statistical analysis
Data were analyzed in StataSE Version 17 (StataCorp, College Station, TX, USA).Incremental area under the curve (iAUC 0-240 min ); that is, the change from baseline on the day of each visit, and total area under the curve (tAUC 0-240 min ) over 240 min, were calculated for GIP and GLP-1 after each meal (and without food for cases).Values were compared between cases and controls, and between meals for each group.Non-parametric statistical methods were used (Wilcoxon signed-rank test for paired continuous data and Mann-Whitney test for unpaired continuous data [independent samples]).Data are presented as the median (range), unless otherwise stated.

DISCUSSION
We have shown that the incretin response is similar in adults with SU-treated KCNJ11-PNDM when compared with healthy controls after protein/fat and carbohydrate meals.Incremental GLP-1 and GIP secretion is of similar magnitude in both groups.In addition, protein/fat results in greater GLP-1 secretion than carbohydrate, in keeping with previous research 7 .
The preserved incretin response in adults with KCNJ11-PNDM contrasts with the reduced post-meal GLP-1 that is often seen in adults with type 2 diabetes 8 .Although GIP secretion can remain near-normal in individuals with type 2 diabetes, the insulin response to GIP is markedly impaired; in contrast, the insulin response to GLP-1 is preserved 9 .The adults in our study had lower insulin secretion after a carbohydrate meal despite higher glucose values in comparison with controls 4 , which is most likely a result of impaired ATP sensitivity at the level of the pancreatic K ATP channel.Further research is needed to investigate whether the insulin response to GIP is affected in these individuals.
The present research provides mechanistic insights.Preserved incretin hormone secretion in individuals with mutant K ATP channels suggests that GLP-1 and GIP secretion from L cells and K cells is independent of K ATP channels, consistent with previous rodent and human studies 2,10 .
The present study also has therapeutic implications.Although long-term SU monotherapy is effective in the majority of individuals with KCNJ11-PNDM, a proportion of patients require adjunctive medication to optimize glycemic control, including the reintroduction of insulin 3 .Our data support consideration of the use of second-line treatments that further augment the incretin pathway, consistent with two previous case reports 5,6 .Dual GIP and GLP-1 receptor agonists might have future therapeutic potential 11 .
However, an important consideration is the possible direct effects of sulfonylureas on incretin hormone secretion or action.Previous studies in perfused rat pancreas suggest that high-dose sulfonylureas cause dissociation of the insulinotropic effects of GLP-1 from glucose levels; at 3 mmol/L glucose, tolbutamide alone resulted in increased insulin secretion that was not seen with GLP-1 alone, whereas administration of both GLP-1 and tolbutamide augmented insulin secretion much more markedly 12 .More recently, studies in humans with type 2 diabetes have shown that low-dose gliclazide, which does not stimulate insulin secretion at low glucose levels, augments insulin secretion in response to oral, but not intravenous, glucose 13 .The latter scenario might mimic more closely the mechanism of action of sulfonylureas in the context of KCNJ11-related neonatal diabetes, as although high doses are used, they are thought to act largely permissively in allowing b-cells to respond to incretins, rather than being directly stimulatory 2 .Our data show very limited effects of sulfonylureas alone on insulin 4 or incretin secretion, but a drop in glucose was still observed in the context of sulfonylurea with no food 4 .Further research is needed to investigate the mechanism of action of sulfonylureas and interactions with drugs that augment the incretin pathway in this physiologically unique group of patients.
The present study had limitations.First, the sample size was small.However, to our knowledge, this is the only study measuring GIP and GLP-1 levels in individuals with KCNJ11-PNDM, in response to different nutrients.Second, the study was limited to individuals aged >18 years.More research is needed to investigate incretin hormone secretion in children with KCNJ11-PNDM.An additional limitation is that there was no comparison with a control group on sulfonylurea treatment; for example, adults with type 2 diabetes.This will be an important area to address in future research studies.
In conclusion, we have shown that incretin hormone responses are preserved in adults with SU-treated KCNJ11-PNDM after carbohydrate and protein/fat meals, in comparison with healthy controls.This suggests that K ATP channels do not play a role in GIP and GLP-1 secretion.Future research will establish the utility of GLP-1 agonists and DPP-IV inhibitors as adjunctive treatments in individuals with KCNJ11-PNDM who have suboptimal glycemic control on SU monotherapy.