Karin Filipsson

PACAP and PACAP receptors in insulin producing tissues: localization and effects

We have studied the localization, receptor occupancy and potency of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) in insulin-producing tissues. Immunocytochemistry showed that PACAP-like immunoreactivity (PACAP-IR) was localized to pancreatic nerves with accumulation in intrapancreatic ganglia in both mouse and rat. In contrast, PACAP-IR could not be demonstrated in endocrine cells. Furthermore, in situ hybridization, using oligodeoxyribonucleotide probes recognizing mRNA for PACAP receptors, demonstrated that mouse and rat pancreas, and the insulinoma cell lines HIT-T15 and RINm5F, expressed both the PACAP type 1 and the VIP2/PACAP receptors. Moreover, both PACAP27 and PACAP38 dose-dependently (0.1 nM to 100 nM) and equipotently stimulated insulin secretion in isolated mouse and rat islets and in HIT-T15 and RINm5F cells. Furthermore, in mouse islets, vasoactive intestinal polypeptide (VIP) was of equal potency as PACAP at stimulating insulin secretion. In mouse, PACAP also stimulated insulin secretion in a subfraction of the isolated islets also at the low dose of 1 fM. Thus, (1) PACAP is exclusively a neuropeptide in the pancreas, (2) insulin-producing cells express PACAP type 1 and VIP2/PACAP receptors and (3) the two forms of PACAP equipotently stimulate insulin secretion. Based on these results, we suggest that PACAP is involved in the neural regulation of insulin secretion.

PACAP stimulates insulin secretion but inhibits insulin sensitivity in mice

Although pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates insulin secretion, its net influence on glucose homeostasis in vivo has not been established. We therefore examined the action of PACAP-27 and PACAP-38 on insulin secretion, insulin sensitivity, and glucose disposal as derived from the minimal model of glucose disappearance during an intravenous glucose tolerance test in anesthetized mice. PACAP-27 and PACAP-38 markedly and equipotently potentiated glucose-stimulated insulin secretion, with a half-maximal effect at 33 pmol/kg. After PACAP-27 or PACAP-38 (1.3 nmol/kg), the acute (1-5 min) insulin response was 3.8 ± 0.4 nmol/l (PACAP-27) and 3.3 ± 0.3 nmol/l (PACAP-38), respectively, vs. 1.4 ± 0.1 nmol/l after glucose alone ( P < 0.001), and the total area under the curve for insulin (AUCinsulin) was potentiated by 60% ( P < 0.001). In contrast, PACAP-27 and PACAP-38 reduced the insulin sensitivity index (SI) [0.23 ± 0.04 10-4min-1/(pmol/l) for PACAP-27 and 0.29 ± 0.06 10-4min-1/(pmol/l) for PACAP-38 vs. 0.46 ± 0.02 10-4min-1/(pmol/l) for controls ( P < 0.01)]. Furthermore, PACAP-27 or PACAP-38 did not affect glucose elimination determined as glucose half-time or the glucose elimination rate after glucose injection or the area under the curve for glucose. Moreover, glucose effectiveness and the global disposition index (AUCinsulin times SI) were not affected by PACAP-27 or PACAP-38. Finally, when given together with glucose, PACAP-27 did not alter plasma glucagon or norepinephrine levels but significantly increased plasma epinephrine levels. We conclude that PACAP, besides its marked stimulation of insulin secretion, also inhibits insulin sensitivity in mice, the latter possibly explained by increased epinephrine. This complex action explains why the peptide does not enhance glucose disposal.

Intraperitoneal PACAP Administration Decreases Blood Glucose in GK Rats, and in Normal and High Fat Diet Mice

PACAP is an islet peptide that serves as an endogenous amplifier of glucose induced insulin secretion. Furthermore, we has recently found that PACAP also potentiates insulin stimulated glucose uptake in adipocytes. Therefore, an antidiabetic action of PACAP is possible. In the present study, we examined the effect of PACAP treatment of the hyperglycemia in GK rats, an animal model of type 2 diabetes, and in high fat fed C47BL/6J mice, an animal model for glucose intolerance. GK rats housed with normal diet exhibited a normal level of blood glucose until three weeks old but significant hyperglycemia at eight weeks. When GK rats were treated with daily PACAP38 (i.p. injection, 6 pmol/kg) from age three weeks, development of hyperglycemia was prevented. In high fat fed mice, i.p. administration of PACAP27 for five (25 nmol/kg twice daily) reduced plasma glucose levels to 6.9 +/- 0.2 mmol/l compared to 8.1 +/- 0.2 mmol/l in saline injected animals (p < 0.001) without altering baseline insulin levels. We conclude that PACAP reduces circulating glucose in animal models of type 2 diabetes and glucose intolerance. The mechanism of this action needs to be established.

Metabolite Profiling Reveals Normal Metabolic Control in Carriers of Mutations in the Glucokinase Gene (MODY2).

Mutations in the gene encoding glucokinase (GCK) cause a mild hereditary form of diabetes termed maturity-onset diabetes of the young (MODY)2 or GCK-MODY. The disease does not progress over time, and diabetes complications rarely develop. It has therefore been suggested that GCK-MODY represents a metabolically compensated condition, but experimental support for this notion is lacking. Here, we profiled metabolites in serum from patients with MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), and type 2 diabetes and from healthy individuals to characterize metabolic perturbations caused by specific mutations. Analysis of four GCK-MODY patients revealed a metabolite pattern similar to that of healthy individuals, while other forms of diabetes differed markedly in their metabolite profiles. Furthermore, despite elevated glucose concentrations, carriers of GCK mutations showed lower levels of free fatty acids and triglycerides than healthy control subjects. The metabolite profiling was confirmed by enzymatic assays and replicated in a cohort of 11 GCK-MODY patients. Elevated levels of fatty acids are known to associate with β-cell dysfunction, insulin resistance, and increased incidence of late complications. Our results show that GCK-MODY represents a metabolically normal condition, which may contribute to the lack of late complications and the nonprogressive nature of the disease.

Liraglutide in people treated for type 2 diabetes with multiple daily insulin injections: randomised clinical trial (MDI Liraglutide trial).

Study question What are the effects of liraglutide, an incretin based treatment, on glycaemic control in people with type 2 diabetes treated with multiple daily insulin injections? Methods The study was a randomised, double blind, placebo controlled trial with a parallel group design carried out at 13 hospital based outpatient clinics and one primary care unit in Sweden. Patients were considered eligible for inclusion if they had type 2 diabetes and inadequate glycaemic control (HbA1c concentrations ≥58 mmol/mol (7.5%) and ≤102 mmol/mol (11.5%)), a body mass index of 27.5-45 kg/m2, and required multiple daily insulin injections. Overall, 124 participants were randomised 1:1 to subcutaneous liraglutide or placebo by minimisation allocation. The main outcome measure was change in HbA1c level from baseline to week 24. Study answer and limitations Liraglutide was associated with a significant reduction of 16.9 mmol/mol (1.5%) in HbA1c versus 4.6 mmol/mol (0.4%) for placebo, difference −12.3 mmol/mol (95% confidence interval −15.8 to −8.8 mmol/mol; −1.13%, −1.45 to −0.81 mmol/mol). Body weight was significantly reduced in participants in the liraglutide compared with placebo group (3.8 v 0.0 kg, difference −3.8, −4.9 to −2.8 kg), and total daily insulin doses were significantly reduced, by 18.1 units and 2.3 units (difference −15.8, −23.1 to −8.5 units). Reductions in mean and standard deviation of glucose levels estimated by masked continuous glucose monitoring were significantly greater in the liraglutide group than placebo group (−1.9 and −0.5 mmol/L). Neither group experienced severe hypoglycaemic events nor were there any significant differences in symptomatic or asymptomatic non-severe hypoglycaemia (<4.0 or <3.0 mmol/L). The mean number of non-severe symptomatic hypoglycaemic events (<4.0 mmol/L) during follow-up was 1.29 in the liraglutide group and 1.24 in the placebo group (P=0.96). One of the study’s limitations was its relatively short duration. Sustained effects of liraglutide have, however, been found over lengthier periods in connection with other treatment regimens. Cardiovascular safety and potential adverse events during longer exposure to liraglutide need to be evaluated. Nausea was experienced by 21 (32.8%) participants in the liraglutide group and 5 (7.8%) in the placebo group and 3 (5%) and 4 (7%) participants in these groups, respectively, had any serious adverse event. What this study adds Adding liraglutide to multiple daily insulin injections in people with type 2 diabetes improves glycaemic control without an increased risk of hypoglycaemia, reduces body weight, and enables patients to lower their insulin doses. Funding, competing interests, data sharing This study was an investigator initiated trial, supported in part by Novo Nordisk and InfuCare. Potential competing interests have been reported and are available on thebmj.com. Study registration EudraCT 2012-001941-42.

Metabolite profile deviations in an oral glucose tolerance test-A comparison between lean and obese individuals.

While impaired glucose tolerance diagnosed by the oral glucose tolerance test (OGTT) is a common trait in obese individuals, less is known about changes in levels of other metabolites. The aim was to reveal the complex alterations in metabolite levels provoked by an OGTT and its perturbation in obese individuals.

The effects of PACAP on insulin secretion and glucose disposal are altered by adrenalectomy in mice.

Abstract: We previously showed that pituitary adenylate cyclase‐activating polypeptide (PACAP) potently stimulates insulin secretion in vivo in mice without altering glucose disposal. Such a combination of results would be explained if epinephrine released by PACAP counteracts the action of insulin and, therefore, that the glucose disposal after PACAP administration is altered by adrenalectomy. In the study reported in this paper, we examined the influence of PACAP27 (1.3 nmol/kg i.v.) on insulin secretion and glucose disposal during an intravenous glucose (1 g/kg) tolerance test in mice subjected to bilateral adrenalectomy 48 h prior to the tolerance test. We found that in control mice, PACAP potentiated glucose‐stimulated insulin secretion threefold without affecting glucose disposal. Adrenalectomy potentiated the augmentation by PACAP27 of glucose‐stimulated insulin secretion, and in adrenalectomized mice, PACAP27 simultaneously augmented glucose disposal (elimination rate 2.30 ± 0.07%/min vs. 2.56 ± 0.05%/min; p=0.011). Furthermore, PACAP27 augmented glucose elimination stimulated by i.v. insulin administration only in adrenalectomized, but not in control mice. We, therefore, conclude that under in vivo conditions, epinephrine released by PACAP from the adrenals prevents the marked insulinotropic action of the peptide from augmenting glucose disposal.

Glycogen metabolism in the glucose‐sensing and supply‐driven β‐cell

Glycogen metabolism in β‐cells may affect downstream metabolic pathways controlling insulin release. We examined glycogen metabolism in human islets and in the rodent‐derived INS‐1 832/13 β‐cells and found them to express the same isoforms of key enzymes required for glycogen metabolism. Our findings indicate that glycogenesis is insulin‐independent but influenced by extracellular glucose concentrations. Levels of glycogen synthase decrease with increasing glucose concentrations, paralleling accumulation of glycogen. We did not find cAMP‐elicited glycogenolysis and insulin secretion to be causally related. In conclusion, our results reveal regulated glycogen metabolism in human islets and insulin‐secreting cells. Whether glycogen metabolism affects insulin secretion under physiological conditions remains to be determined.

PACAP27 Sensitizes Glucose Induced Insulin Secretion in INS-1 Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized in nerves in the endocrine pancreas, and PACAP receptors are expressed in the islets. 1 Furthermore, both PACAP27 and PACAP38 stimulate insulin secretion 1–3 through an effect largely mediated by cAMP. 4,5 In anterior pituitary cells, PACAP sensitizes the action of gonadotropin releasing hormone on secretion of luteinizing hormone and follicle stimulating hormone, 6 since the release of these hormones is potentiated by preincubation with PACAP. Similarly, insulin secretion is enhanced after priming with glucagon-like peptide-1 (GLP-1) by an effect thought to be mediated by cAMP. 7 Therefore, we examined the potential priming effect of PACAP on glucose-induced insulin secretion in clonal rat insulin producing cell line INS-1, which shows resemblances to normal B-cells. 8

Variables associated with HbA1c and weight reductions when adding liraglutide to multiple daily insulin injections in persons with type 2 diabetes (MDI Liraglutide trial 3)

Objective To evaluate variables associated with hemoglobin A1c (HbA1c) and weight reduction when adding liraglutide to persons with type 2 diabetes treated with multiple daily insulin injections (MDI). Research design and methods This was a reanalysis of a previous trial where 124 patients were enrolled in a double-blind, placebo-controlled, multicenter randomized trial carried out over 24 weeks. Predictors for effect on change in HbA1c and weight were analyzed within the treatment group and with concurrent interaction analyses. Correlation analyses for change in HbA1c and weight from baseline to week 24 were made. Results The mean age at baseline was 63.7 years, 64.8% were men, the mean number of insulin injections was 4.4 per day, the mean daily insulin dose was 105 units and the mean HbA1c was 74.5 mmol/mol (9.0%). The mean HbA1c and weight reductions were 12.3 mmol/mol (1.13%; P<0.001) and 3.8 kg (P<0.001) greater in liraglutide than placebo-treated persons. There was no significant predictor for greater effect on HbA1c that existed in all analyses (univariate, multivariate and interaction analyses against controls). For a greater weight reduction when adding liraglutide, a lower HbA1c level at baseline was a predictor (liraglutide group P=0.002, P=0.020 for liraglutide group vs placebo). During follow-up in the liraglutide group, no significant correlation was found between change in weight and change in HbA1c (r=0.09, P=0.46), whereas a correlation existed between weight and insulin dose reduction (r=0.44, P<0.001). Conclusion Weight reduction becomes greater when adding liraglutide in patients with type 2 diabetes treated with MDI who had a lower HbA1c level compared with those with a higher HbA1c level. There was no correlation between reductions in HbA1c and weight when liraglutide was added, that is, different patient groups responded with HbA1c and weight reductions. Trial registration number EudraCT nr: 2012-001941-42.