Carolyn F. Deacon

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.

The metabolism of glucagon-like peptide-1 (GLP-1) has not been studied in detail, but it is known to be rapidly cleared from the circulation. Measurement by RIA is hampered by the fact that most antisera are side-viewing or C-terminally directed, and recognize both intact GLP-1 and biologically inactive. N-terminally truncated fragments. Using high pressure liquid chromatography in combination with RIAs, methodology allowing specific determination of both intact GLP-1 and its metabolites was developed. Human plasma was shown to degrade GLP-1-(7-36)amide, forming an N-terminally truncated peptide with a t1/2 of 20.4 +/- 1.4 min at 37 C (n = 6). This was unaffected by EDTA or aprotinin. Inhibitors of dipeptidyl peptidase-IV or low temperature (4 C) completely prevented formation of the metabolite, which was confirmed to be GLP-1-(9-36)amide by mass spectrometry and sequence analysis. High pressure liquid chromatography revealed the concentration of GLP-1-(9-36)amide to be 53.5 +/- 13.7% of the concentration of endogenous intact GLP-1 in the fasted state, which increased to 130.8 +/- 10.0% (P < 0.01; n = 6) 1 h postprandially. Metabolism at the C-terminus was not observed. This study suggests that dipeptidyl peptidase-IV is the primary mechanism for GLP-1 degradation in human plasma in vitro and may have a role in inactivating the peptide in vivo.

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 rapidly degraded 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.

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 revealed specific DPP IV staining in the brush border epithelium as well as in the capillary endothelium. Double staining showed juxtapositioning of DPP IV-positive capillaries and GLP-1-containing L cells. From these results, we suggest that GLP-1 is degraded as it enters the DPP IV containing blood vessels draining the intestinal mucosa.