Carolyn F. Deacon

Reduced postprandial concentrations of intact biologically active glucagon-like peptide 1 in type 2 diabetic patients.

Incretin hormones importantly enhance postprandial insulin secretion but are rapidly degraded to inactive metabolites by ubiquitous dipeptidyl peptidase IV. The concentrations of the intact biologically active hormones remain largely unknown. Using newly developed assays for intact glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP), we measured plasma concentrations after a mixed breakfast meal (566 kcal) in 12 type 2 diabetic patients (age 57 years [range 49-67], BMI 31 kg/m2 [27-38], and HbA1c 9.2% [7.0-12.5]) and 12 matched healthy subjects. The patients had fasting hyperglycemia (10.7 mmol/l [8.0-14.8]) increasing to 14.6 mmol/l (11.5-21.5) 75 min after meal ingestion. Fasting levels of insulin and C-peptide were similar to those of the healthy subjects, but the postprandial responses were reduced and delayed. Fasting levels and meal responses were similar between patients and healthy subjects for total GIP (intact + metabolite) as well as intact GIP, except for a small decrease in the patients at 120 min; integrated areas for intact hormone (area under the curve [AUC]INT) averaged 52 +/- 4% (for patients) versus 56 +/- 3% (for control subjects) of total hormone AUC (AUC(TOT)). AUC(INT) for GLP-1 averaged 48 +/- 2% (for patients) versus 51 +/- 5% (for control subjects) of AUC(TOT). AUC(TOT) for GLP-1 as well as AUC(INT) tended to be reduced in the patients (P = 0.2 and 0.07, respectively); but the profile of the intact GLP-1 response was characterized by a small early rise (30-45 min) and a significantly reduced late phase (75-150 min) (P < 0.02). The measurement of intact incretin hormones revealed that total as well as intact GIP responses were minimally decreased in patients with type 2 diabetes, whereas the late intact GLP-1 response was strongly reduced, supporting the hypothesis that an impaired function of GLP-1 as a transmitter in the enteroinsular axis contributes to the inappropriate insulin secretion in type 2 diabetes.

Both Subcutaneously and Intravenously Administered Glucagon-Like Peptide I Are Rapidly Degraded From the NH2-Terminus in Type II Diabetic Patients and in Healthy Subjects

To fate of exogenous glucagon-like peptide I (GLP-I)(7–36) amide was studied in nondiabetic and type II diabetic subjects using a combination of high-pressure liquid chromatography (HPLC), specific radioimmunoassays (RIAs), and a sensitive enzyme-linked immunosorbent assay (ELISA), whereby intact biologically active GLP-I and its metabolites could be determined. After GLP-I administration, the intact peptide could be measured using an NH2-terminally directed RIA or ELISA,while the difference in concentration between these assays and a COOH-terminal–specific RIA allowed determination of NH2-terminally truncated metabolites. Subcutaneous GLP-I was rapidlydegraded in a time-dependent manner, forming a metabolite, which co-eluted on HPLC with GLP-I(9–36) amide and had the same immunoreactive profile. Thirty minutes after subcutaneous GLP-I administration to diabetic patients (n = 8), the metabolite accounted for 88.5 ± 1.9% of the increase in plasma immunoreactivity determined by the COOH-terminal RIA, which was higher than the levels measured in healthy subjects (78.4 ± 3.2%; n = 8; P < 0.05). Intravenously infused GLP-I was also extensively degraded, but no significant differences were seen between the two groups. Intact GLP-I accounted for only 19.9 ± 3.4% of the increase in immunoreactivity measured with the COOH-terminal RIA in normal subjects (n = 8), and 25.0 ± 4.8% of the increase in diabetic subjects (n = 8), the remainder being the NH2-terminally truncated metabolite.

Glucagon-Like Peptide-1-(7–36)Amide Is Transformed to Glucagon-Like Peptide-1-(9–36)Amide by Dipeptidyl Peptidase IV in the Capillaries Supplying the L Cells of the Porcine Intestine

The insulinotropic hormone glucagon-like peptide-1 (GLP-1) is stored in the intestinal L cell in an active form, GLP-1-(7–36)amide, but more than half of the endogenous peptide circulates in an inactive, N-terminally truncated form, GLP-1-(9–36)amide. This study examined the GLP-1 newly secreted from the porcine ileum, in vitro (isolated perfused preparation) and in vivo (anesthetized pig), to determine where this conversion occurs. Although the GLP-1 extractable from the porcine ileum is predominantly the intact peptide (94.6 ± 1.7%), a large proportion of the GLP-1 that is secreted has already been degraded to the truncated form both in vitro (53.8± 0.9% intact) and in vivo (32.9 ± 10.8% intact). In the presence of a specific dipeptidyl peptidase IV (DPP IV) inhibitor (valine-pyrrolidide), the proportion of intact GLP-1 released from the perfused ileum was increased under both basal (99% intact; P < 0.05) and stimulated (86–101% intact; P < 0.05) conditions. Immunohistochemical and histochemical studies...

The incretin system and its role in type 2 diabetes mellitus

The incretin hormones are released during meals from gut endocrine cells. They potentiate glucose-induced insulin secretion and may be responsible for up to 70% of postprandial insulin secretion. The incretin hormones include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), both of which may also promote proliferation/neogenesis of beta cells and prevent their decay (apoptosis). Both hormones contribute to insulin secretion from the beginning of a meal and their effects are progressively amplified as plasma glucose concentrations rise. The current interest in the incretin hormones is due to the fact that the incretin effect is severely reduced or absent in patients with type 2 diabetes mellitus (T2DM). In addition, there is hyperglucagonaemia, which is not suppressible by glucose. In such patients, the secretion of GIP is near normal, but its effect on insulin secretion, particularly the late phase, is severely impaired. The loss of GIP action is probably a consequence of diabetes, since it is also observed in patients with diabetes secondary to chronic pancreatitis, in whom the incretin effect is also lost. GLP-1 secretion, on the other hand, is also impaired, but its insulinotropic and glucagon-suppressive actions are preserved, although the potency of GLP-1 in this respect is decreased compared to healthy subjects. However, in supraphysiological doses, GLP-1 administration may completely normalize beta as well as alpha cell sensitivity to glucose. The impaired action of GLP-1 and GIP in T2DM may be at least partly restored by improved glycaemic control, as shown in studies involving 4 weeks of intensive insulin therapy. The reduced incretin effect is believed to contribute to impaired regulation of insulin and glucagon secretion in T2DM, and, in support of this, exogenous GLP-1 administration may restore blood glucose regulation to near normal levels. Thus, the pathogenesis of T2DM seems to involve a dysfunction of both incretins. Enhancement of incretin action may therefore represent a therapeutic solution. Clinical strategies therefore include the development of metabolically stable activators of the GLP-1 receptor; and inhibition of DPP-4, the enzyme that destroys native GLP-1 almost immediately. Orally active DPP-4 inhibitors and the metabolically stable activators, exenatide (Byetta), are now on the market, and numerous clinical studies have shown that both principles are associated with durable antidiabetic activity.

Dipeptidyl peptidase-4 inhibitors in the treatment of type 2 diabetes: a comparative review.

The dipeptidyl peptidase (DPP)‐4 inhibitors are a new class of antihyperglycaemic agents which were developed for the treatment of type 2 diabetes by rational drug design, based on an understanding of the underlying mechanism of action and knowledge of the structure of the target enzyme. Although they differ in terms of their chemistry, they are all small molecules which are orally available. There are some differences between them in terms of their absorption, distribution, metabolism and elimination, as well as in their potency and duration of action, but their efficacy, both in terms of inhibiting plasma DPP‐4 activity and as antidiabetic agents, appears to be similar. They improve glycaemic control, reducing both fasting and postprandial glucose levels to lower HbA1c levels, without weight gain and with an apparently benign adverse event profile. At present, there seems to be little to distinguish between the different inhibitors in terms of their efficacy as antidiabetic agents and their safety. Long‐term accumulated clinical experience will reveal whether compound‐related characteristics lead to any clinically relevant differences.

Secretion of glucagon-like peptide-1 (GLP-1) in type 2 diabetes: what is up, what is down?

The incretin hormones gastric inhibitory polypeptide and especially glucagon-like peptide (GLP) have an important physiological function in augmenting postprandial insulin secretion. Since GLP-1 may play a role in the pathophysiology and treatment of type 2 diabetes, assessment of meal-related GLP-1 secretory responses in type 2 diabetic patients vs healthy individuals is of great interest. A common view states that GLP-1 secretion in patients with type 2 diabetes is deficient and that this applies to a lesser degree in individuals with impaired glucose tolerance. Such a deficiency is the rationale for replacing endogenous incretins with GLP-1 receptor agonists or re-normalising active GLP-1 concentrations with dipeptidyl peptidase-4 inhibitors. This review summarises the literature on this topic, including a meta-analysis of published studies on GLP-1 secretion in individuals with and without diabetes after oral glucose and mixed meals. Our analysis does not support the contention of a generalised defect in nutrient-related GLP-1 secretory responses in type 2 diabetes patients. Rather, factors are identified that may determine individual incretin secretory responses and explain some of the variations in published findings of group differences in GLP-1 responses to nutrient intake.

Dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1 which have extended metabolic stability and improved biological activity.

Summary Glucagon-like peptide 1 (GLP-1) has great potential in diabetes therapy due to its glucose-dependent stimulation of insulin secretion, but this is limited by its rapid degradation, primarily by dipeptidyl peptidase IV. Four analogues, N-terminally substituted with threonine, glycine, serine or α-aminoisobutyric acid, were synthesised and tested for metabolic stability. All were more resistant to dipeptidyl peptidase IV in porcine plasma in vitro, ranging from a t1/2 of 159 min (Gly8 analogue) to undetectable degradation after 6 h (Aib8 analogue; t1/2 for GLP-1 (7–36) amide, 28 min). During i. v. infusion in anaesthetised pigs, over 50 % of each analogue remained undegraded compared to 22.7 % for GLP-1 (7–36) amide. In vivo, analogues had longer N-terminal t1/2 (intact peptides: means, 3.3–3.9 min) than GLP-1 (7–36) amide (0.9 min; p < 0.01), but these did not exceed the C-terminal t1/2 (intact plus metabolite: analogues, 3.5–4.4 min; GLP-1 (7–36) amide, 4.1 min). Analogues were assessed for receptor binding using a cell line expressing the cloned receptor, and for ability to stimulate insulin or inhibit glucagon secretion from the isolated perfused porcine pancreas. All bound to the receptor, but only the Aib8 and Gly8 analogues had similar affinities to GLP-1 (7–36) amide (IC50; Aib8 = 0.45 nmol/l; Gly8 = 2.8 nmol/l; GLP-1 (7–36) amide = 0.78 nmol/l). All analogues were active in the isolated pancreas, with the potency order reflecting receptor affinities (Aib8 > Gly8 > Ser8 > Thr8). N-terminal modification of GLP-1 confers resistance to dipeptidyl peptidase IV degradation. Such analogues are biologically active and have prolonged metabolic stability in vivo, which, if associated with greater potency and duration of action, may help to realise the potential of GLP-1 in diabetes therapy. [Diabetologia (1998) 41: 271–278]

Glucagon-like peptide-1 mediates the therapeutic actions of DPP-IV inhibitors

Our opponents in this debate, Nauck and El-Ouaghlidi, have challenged the standard view that the therapeutic actions of the dipeptidyl peptidase-IV (DPP-IV) inhibitors are mediated by glucagon-like peptide-1 (GLP-1) [1]. Based on the findings that exogenously administered GLP-1 is rapidly and extensively degraded by DPP-IV [2], and that this degradation appears to be mediated by DPP-IV [3], it was proposed that DPP-IV inhibitors could enhance the survival of intact, biologically active GLP-1 and thus be of use in the treatment of type 2 diabetes mellitus [2, 4]. Although the kinetic studies performed by Mentlein et al. clearly demonstrated that DPP-IVmay have substrates in addition toGLP1 [5, 6], the effect of this enzyme on GLP-1 was considered to be of particular importance because: (1) GLP-1 is extremely susceptible to rapid degradation by DPP-IV, as compared with the majority of other substrates; (2) DPP-IV degradation is the primary route of GLP-1 inactivation; (3) GLP-1 is perhaps the most potent and efficacious insulinotropic hormone in the body [7], whereas gastric inhibitory peptide (GIP), the other major incretin hormone, has no insulinotropic effects in patients with type 2 diabetes [8]; and (4) DPP-IV inhibitors have no metabolic effect in mice with a targeted deletion of the GLP-1 and GIP receptor genes [9]. The authors of the previous article have listed five arguments that have caused them to doubt that GLP-1 is the only, or at least the major, mediator of the remarkable hypoglycaemic effects of DPP-IV inhibitors observed in clinical studies [10–12].Wewould certainly agree that there are other potential substrates for DPP-IV whose extended survival might contribute to the therapeutic effects of these inhibitors; nonetheless, we believe that the protection of GLP-1 against degradation is the major mechanism involved. In the remainder of this paper, we shall respond to each of the five arguments in turn.

What do we know about the secretion and degradation of incretin hormones

The incretin hormones, glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted from endocrine cells located in the intestinal mucosa, and act to enhance meal-induced insulin secretion. GIP and GLP-1 concentrations in the plasma rise rapidly after food ingestion, and the presence of unabsorbed nutrients in the intestinal lumen is a strong stimulus for their secretion. Nutrients can stimulate release of both hormones by direct contact with the K-cell (GIP) and L-cell (GLP-1), and this may be the most important signal. However, nutrients also stimulate GLP-1 and GIP secretion indirectly via other mechanisms. Incretin hormone secretion can be modulated neurally, with cholinergic muscarinic, beta-adrenergic and peptidergic (gastrin-releasing peptide, GRP) fibres generally having positive effects, while secretion is restrained by alpha-adrenergic and somatostatinergic fibres. Hormonal factors may also influence incretin hormone secretion. Somatostatin exerts a local inhibitory effect on the activity of both K- and L-cells via a paracrine mechanism, while, in rodents at least, GIP from the proximal intestine has a stimulatory effect on GLP-1 secretion, possibly mediated via a neural loop involving GRP. Once they have been released, both GLP-1 and GIP are subject to rapid degradation. The ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV) cleaves N-terminally, removing a dipeptide and thereby inactivating both peptides, because the N-terminus is crucial for receptor binding. Subsequently, the peptides may be degraded by other enzymes and extracted in an organ-specific manner. The intact peptides are inactivated during passage across the hepatic bed and further metabolised by the peripheral tissues, while the kidney is important for the final elimination of the metabolites.

Inhibitors of dipeptidyl peptidase IV: a novel approach for the prevention and treatment of Type 2 diabetes?

Inhibitors of the enzyme dipeptidyl peptidase IV (DPP IV) are of increasing interest to both diabetologists and the pharmaceutical industry alike, as they may become established as the next member of the oral antidiabetic class of therapeutic agents, designed to lower blood glucose and, possibly, prevent the progressive impairment of glucose metabolism in patients with impaired glucose tolerance and Type 2 diabetes. DPP IV has become a focus of attention for drug design, as it has a pivotal role in the rapid degradation of at least two of the hormones released during food ingestion, a property that has warranted the design of inhibitor-based drugs. At the molecular level, DPP IV cleaves two amino acids from the N-terminus of the intact, biologically active forms of both so-called incretin hormones, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide (formerly known as gastric inhibitory polypeptide), resulting in truncated metabolites, which are largely inactive. Inhibition of the enzyme, therefore, is thought to increase levels of the active forms of both incretin hormones, culminating in an increase in insulin release after a meal, in a fully glucose-dependant manner. DPP IV inhibitors combine several features of interest to the drug design process. They can be readily optimised for their target and be designed as low molecular weight, orally active entities compatible with once-daily administration.