Secretion, Degradation, and Elimination of Glucagon-Like Peptide 1 and Gastric Inhibitory Polypeptide in Patients with Chronic Renal Insufficiency and Healthy Control Subjects

The study protocol was approved by the ethics committee of the Ruhr-University of Bochum on 20 January 2000 (registration number 1,417) before the study. Written informed consent was obtained from all participants.

Two groups of subjects/patients were studied: 1) 10 subjects with normal renal function: subjects were included when they had a serum creatinine concentration <1.1 mg/dl (normal range, 0.5–1.1 mg/dl) and exhibited no other clinical signs of renal insufficiency; and 2) 10 patients with CRI: patients were included when they had a serum creatinine concentration >1.5 mg/dl. All patients with impaired or diabetic oral glucose tolerance were excluded.

From all participants, blood was drawn in the fasting state for measurements of standard hematologic and clinical chemistry parameters. Urine was collected over 24 h for the determination of albumin and protein by standard methods. Patients with anemia (hemoglobin <10 g/dl) and elevation in liver enzymes (alanine aminotransferase, aspartate aminotransferase, AP, γ-glutamine transferase) to higher activities than double the respective normal value were excluded. The participant characteristics are presented in Table 1.

The diagnoses leading to renal insufficiency included immunoglobulin A nephropathy (Berger’s disease) in three cases, amyloidosis in one case, cystic kidney disease in two cases, secondary nephroangiosclerosis in two cases, and hereditary renal dysplasia in two cases. Five CRI patients and four control subjects who had participated in the screening were excluded because they had impaired glucose tolerance. Nine patients with renal insufficiency received one or more antihypertensive drugs: angiotensin-converting enzyme inhibitors in seven, angiotensin II receptor antagonists in four, β-blocking agents in three, diuretics in five, calcium antagonists in three, and α-blocking agents in two cases. In contrast, none of the control subjects received antihypertensive medication.

Synthetic human GLP-1 was a gift from Restoragen (Lincoln, NE; lot number [pharmaceutical grade] 0340298). Synthetic human GIP was purchased from PolyPeptide Laboratories (Wolfenbüttel, Germany; lot number [pharmaceutical grade] E-0517). High-performance liquid chromatography profiles (provided by the manufacturer) showed that the preparation was >95% pure (single peak co-eluting with appropriate standards). The peptides were dissolved in 0.9% NaCl/1% human serum albumin (Behring, salt poor, Marburg, Germany), filtered through 0.2-μm nitrocellulose filters (Sartorius, Göttingen, Germany) and stored frozen at −28°C. Samples were analyzed for bacterial growth (standard culture techniques) and for pyrogens (laboratory of Dr. Balfanz, Münster, Germany). No bacterial contamination was detected. Endotoxin concentrations in samples from the stock solutions were 0.08 IU/ml for both GLP-1 and GIP.

At a screening visit, venous blood samples for the determination of standard hematologic and clinical chemistry parameters were drawn and a clinical examination was performed. When the subjects met the inclusion criteria, they were recruited for the following tests. All subjects were examined on three occasions: 1) oral glucose tolerance test: after basal venous blood samples were drawn (−15 and 0 min), 75 g of oral glucose (OGTT; Roche Diagnostics, Mannheim, Germany) was ingested within 5 min; venous blood samples were drawn after 30, 60, 90, and 120 min; 2) GLP-1 infusion: after basal venous and capillary blood samples were drawn twice, at t = 0 min, synthetic human GLP-1 (7–36 amide) was infused over 30 min with an infusion rate of 0.5 pmol · kg⁻¹ · min⁻¹; venous blood samples were obtained at 15, 30, 32, 35, 40, 50, 60, 90, 120, 150, and 180 min; and 3) GIP infusion: after basal venous blood samples were drawn twice, at t = 0 min, synthetic human GIP (1–42) was infused over 30 min with an infusion rate of 1.0 pmol · kg⁻¹ · min⁻¹; venous blood samples were obtained at 15, 30, 32, 35, 40, 50, 60, 90, 120, 150, and 180 min.

The tests were performed in the morning after an overnight fast with the subjects in a sitting position throughout the experiments. Two forearm veins were punctured with Teflon cannulas (Moskito 123, 18 gauge; Vygon, Aachen, Germany) and kept patent using 0.9% NaCl (for blood sampling and for GIP or GLP-1 administration, respectively). At least 48 h had to pass between the tests to avoid carryover effects.

Venous blood was drawn into chilled tubes containing EDTA and aprotinin (Trasylol; 20,000 KIU/ml, 200 μl/10 ml blood; Bayer AG, Leverkusen, Germany) and kept on ice. A sample (∼100 μl) was stored in NaF (Microvette CB 300; Sarstedt, Nümbrecht, Germany) for the immediate measurement of glucose. After centrifugation at 4°C, plasma for hormone analyses was kept frozen at −28°C. This procedure has previously been shown to prevent in vitro degradation of incretin hormones in human plasma samples (14).

Glucose was measured as described (19) using a glucose oxidase method with a Glucose Analyzer 2 (Beckman Instruments, Munich, Germany). Insulin was measured using an insulin microparticle enzyme immunoassay (IMx Insulin; Abbott Laboratories, Wiesbaden, Germany). Intra-assay coefficients of variation were ∼4%. C-peptide was measured using an enzyme-linked immunoabsorbent assay from DAKO (Cambridgeshire, U.K.). Intra-assay coefficients of variation were 3.3–5.7%, and interassay variation was 4.6–5.7%.

GLP-1 immunoreactivity was determined using two different assays specific for either the COOH-terminus or the NH₂-terminus of the peptide. The COOH-terminal assay measures the sum of the intact peptide plus the primary metabolite GLP-1 (9–36 amide) using the antiserum 89390 and synthetic GLP-1 (7–36 amide) as standard. This assay cross-reacts <0.01% with COOH-terminally truncated fragments and 83% with GLP-1 (9–36 amide). The detection limit was 3 pmol/l. The NH₂-terminal assay measures the concentration of intact GLP-1 (7–36 amide) using antiserum no. 93242, which cross-reacts ∼10% with GLP-1 (1–36 amide) and <0.1% with GLP-1 (8–36 amide) and GLP-1 (9–36 amide). The detection limit was 3 pmol/l. For both assays, intra-assay and interassay coefficients of variation were <6 and 15%, respectively, at 40 pmol/l (20).

GIP immunoreactivity was determined using two different assays specific for either the COOH-terminus or the NH₂-terminus of the peptide as well (6). The COOH-terminal assay using antiserum R65 fully reacts with intact GIP (1–42) and the truncated metabolite (3–42) but not with the so-called 8-kDa GIP, of which the chemical nature and relation to GIP secretion is uncertain. The assay has a detection limit of <2 pmol/l and an intra-assay variation of ∼6%. The NH₂-terminal assay measures the concentration of intact GIP (1–42), using antiserum 98171. The cross-reactivity with GIP (3–42) was <0.1%. The lower detection limit of the assay is ∼5 pmol/l. Intra-assay variation was <6%, and interassay variations were ∼8 and 12% for 20 and 80 pmol/l standards, respectively. For both assays, human GIP (Peninsula Laboratories, Europe) was used as standard, and radiolabeled GIP was obtained from Amersham Pharmacia Biotech (Aylesbury, U.K.). Valine-pyrrolidide (0.01 mmol/l, final concentration) was added to the assay buffers to prevent NH₂-terminal degradation of GIP during the assay incubation. The concentrations of the metabolites GIP (3–42 amide) and GLP-1 (9–36 amide) were calculated as the concentration differences between the total GIP/GLP-1 and the intact GIP/GLP-1.

Cystatin C plasma concentrations were determined for the assessment of the glomerular filtration rate, because this parameter was postulated to be even more accurate than creatinine clearance (21,22). Cystatin C concentrations were measured using a commercially available assay (Behringwerke Diagnostica, Marburg, Germany), as described (23). Intra-assay variation was <3.3%, and interassay variation was <4.1%.

Plasma half-lives were calculated by log_e linear regression analysis of the peptide concentrations in the samples collected after the end of the infusion period. For integrated incremental responses of glucose, insulin, and C-peptide, the positive or negative area under the curve was calculated using the trapezoidal method (baseline subtracted). Metabolic clearance rates were calculated from the total amount of peptide infused divided by the integrated incremental plasma concentrations. The distribution space (DS) was calculated using the formula DS = MCR/k, where MCR is the metabolic clearance rate and k is the fractional clearance rate (= 0.693/t_1/2). The homeostasis model assessment model was used as an estimate of insulin resistance and B-cell function (24). Creatinine clearance was calculated according to the formula by Cockcroft and Gault (25).

Results are reported as mean ± SE. All statistical calculations were carried out using repeated-measures ANOVA using Statistica Version 5.0 (Statsoft Europe, Hamburg, Germany). This analysis provides P values for differences between groups/experiments, differences over time, and for the interaction of group/experiment with time. When a significant interaction of treatment and time was documented (P < 0.05), values at single time points were compared by one-way ANOVA. P < 0.05 was taken to indicate significant differences.

The groups were well matched for age, sex, and obesity (Table 1). There were no differences in the HbA_1c levels or in the fasting glucose concentrations between both groups (P = 0.38 and P = 0.24, respectively). Serum creatinine concentrations and serum urea concentrations were significantly higher in the CRI patients compared with the control subjects (P < 0.001; Table 1). Cystatin C plasma concentrations, used as a marker of the glomerular filtration rate, were significantly higher in the CRI patients (P < 0.0001). Similarly, the creatinine clearance was significantly reduced in the patients with renal insufficiency (P < 0.0001). There was a high degree of correlation between the cystatin C concentrations and the creatinine clearance (r = 0.81; P < 0.0001; details not shown). Urinary albumin and total protein excretion were higher in the patients only by trend but without a statistically significant difference between the groups (Table 1). Hemoglobin concentrations were lower in the patients than in the control subjects (P = 0.00033; Table 1).

After the ingestion of 75 g of oral glucose, plasma glucose concentrations increased significantly in both groups (P < 0.001; Fig. 1A) but without any differences between the patients and the control subjects (P = 0.61). Insulin and C-peptide concentrations rose significantly in both groups (P < 0.001). There was a trend toward higher rises in C-peptide concentrations but lower insulin levels in the CRI patients (Fig. 1B and C). As a result, the molar C-peptide-to-insulin ratio calculated from the total integrated plasma concentrations after oral glucose ingestion was significantly higher in the patients than in the control subjects (11.2 ± 1.0 vs. 7.5 ± 1.4, respectively; P = 0.045).

Plasma concentrations of intact GLP-1 (7–36 amide) only marginally increased after glucose ingestion. There were no differences between the patients with renal insufficiency and the control subjects (P = 0.31; Fig. 2B). In contrast, plasma concentrations of the GLP-1 metabolite (9–36 amide) significantly increased in response to the oral glucose load (P < 0.0001). The plasma levels of GLP-1 (9–36 amide) in the CRI patients significantly exceeded those reached in the control subjects (P = 0.027).

Plasma concentrations of intact GIP (1–42) increased after the oral glucose load (P < 0.001), but no differences occurred between both groups (P = 0.87; Fig. 3B). There was a marked increase in the GIP metabolite (3–42) in both groups (P < 0.001). The rise in GIP (3–42) levels after glucose ingestion was greater in the renal insufficiency patients than in the control subjects (P = 0.0021).

During the intravenous infusion of GLP-1, plasma glucose concentrations were lowered significantly in both groups (P < 0.001; Fig. 4A). This was accompanied by a significant rise in insulin and C-peptide secretion (P < 0.001; Fig. 4). There were no differences in glucose, insulin, or C-peptide concentrations between the CRI patients and the control subjects (P = 0.55, P = 0.87, and P = 0.96, respectively; Fig. 4).

Plasma concentrations of intact GLP-1 (7–36 amide) increased during GLP-1 infusion in both groups (P < 0.001; Fig. 5A). There were no differences in the intact GLP-1 levels between the patients with renal insufficiency and the control subjects (P = 0.29; Fig. 5B). In contrast, plasma concentrations of the GLP-1 metabolite (9–36 amide) were significantly higher in the patients compared with the control subjects during and after the infusion (P < 0.001; Fig. 5C).

The incremental area under the curve as well as the plasma half-lives and the metabolic clearance rates for intact GLP-1 were similar in the patients and the control subjects (P = 0.67, P = 0.13, and P = 0.92, respectively; Table 2). However, calculation of the same parameters for the GLP-1 metabolite (9–36 amide) revealed significantly higher values in the CRI patients than in the control subjects (P < 0.05; Table 2).

During the intravenous infusion of GIP, plasma glucose concentrations were only slightly but significantly lowered in both groups (P < 0.001; Fig. 6A). Insulin and C-peptide concentrations increased during GIP infusion (P < 0.001; Fig. 6). There were no differences between the CRI patients and the control subjects (P = 0.33 for glucose, P = 0.062 for insulin, and P = 0.62 for C-peptide; Fig. 6).

Plasma concentrations of intact GIP (1–42) increased significantly during the infusion in both groups (P < 0.001; Fig. 7B), but the plasma concentrations reached during and after the infusion period were similar in both groups (P = 0.27). Different from the intact peptide, the GIP metabolite (3–42) reached significantly higher plasma concentrations in the CRI patients during and after the infusion period (P = 0.0033; Fig. 7C).

The metabolic clearance rates, the plasma half-lives, and the area under the curve for intact GIP (1–42) were similar in both groups (Table 2). For the GIP metabolite (3–42), a significantly higher area under the curve and a longer plasma half-life was calculated in the patients (P = 0.031 and P = 0.032, respectively; Table 2). The same trend was obvious for the metabolic clearance rates (P = 0.088; Table 2).

The incretin hormones GIP and GLP-1 and their derivatives are currently being discussed as a potential new treatment of type 2 diabetes because of their glucose-lowering potential (3,4,26). Because diabetes is frequently complicated by the development of renal insufficiency (17), it was important to assess the pharmacokinetic properties of GIP and GLP-1 in patients with impaired renal function.

The present data indicate that the kidneys are not the primary site of the DPP IV-mediated metabolism of GIP and GLP-1. There was only an insignificant trend toward higher concentrations of both intact hormones in the CRI patients after the oral glucose load as well as during the peptide infusions (Figs. 2, 3, 5, and 7). This is in good agreement with previous studies in pigs showing a high degree of NH₂-terminal degradation of both GLP-1 and GIP in the hepato-portal bed and throughout the extremities (27), where DPP IV is found in high concentrations associated with hepatocytes and endothelial cells (28,29). The kidneys were found to extract ∼70% of intact GLP-1 and only 25% of intact GIP (15,27), but because the kidneys receive only ∼25% of the cardiac output, they would be expected to contribute to the total body DPP IV-mediated degradation of the intact incretin hormones by only 10–20%. This probably explains the modest increases in intact GIP and GLP-1 observed in the present experiments. It is interesting that renal extraction of intact GIP can still be detected after DPP IV inhibition, suggesting that glomerular filtration and possibly peritubular uptake are at least in part involved in the extraction of GIP (15). Studies in isolated tubules have also suggested that the renal extraction of GLP-1 involves both glomerular filtration and tubular uptake and catabolism (13), whereas detailed studies of the renal handling of the related peptide glucagon have pointed to the involvement of peritubular uptake in addition to glomerular filtration (30,31).

Although DPP IV-mediated degradation of GIP and GLP-1 takes place in different tissues, the present data demonstrate that the kidneys are the major site of extraction of GIP (3–42) and GLP-1 (9–36 amide). Accordingly, plasma levels of the incretin metabolites were found to be increased in response to oral glucose as well as during exogenous infusion in the patients with renal insufficiency (Figs. 2, 3, 5, and 7).

Considering derivatives of incretin hormones as a therapeutic approach for type 2 diabetes or obesity, the consequences resulting from a slowed elimination in patients with impairments in renal function should be taken into account. Although in the present study intact hormone levels were only marginally affected by impairments in renal function, it cannot be excluded that elevated plasma levels of the metabolites GIP (3–42) and GLP-1 (9–36 amide) may antagonize the respective intact peptides at their receptors. However, antagonistic properties of GIP (3–42) and GLP-1 (9–36 amide) have been shown only during the administration of supraphysiological plasma levels in some (7,32,33) but not all (34,35) studies. In the present experiments, there was only a trend toward lower plasma levels of insulin after oral glucose ingestion as well as during the infusion of GIP and GLP-1 in the patients (Figs. 1, 4, and 6).

The low responses of intact GLP-1 plasma concentrations to oral glucose ingestion compared with the higher rises in total GLP-1 levels probably partly reflect the much higher clearance rate of the intact hormone versus its metabolite. This finding is in agreement with previous studies (18,36). The lower levels of intact hormone may also be explained by the high rate of conversion of intact GLP-1 into its metabolite immediately after secretion, catalyzed by the high concentrations of DPP IV in the capillaries of the gut mucosa (37). However, there is evidence that under physiological circumstances, GLP-1 can interact with sensory nerve fibers before it enters the circulation and is degraded by DPP IV (38). In this way, L-cell secretion may have biological effects, e.g., on insulin secretion, without changes in the circulating levels of intact GLP-1.

Previous studies based on immunoassays that were unable to distinguish between the intact and degraded forms of GLP-1 and GIP have revealed plasma half-lives between 3 and 11 min for GLP-1 (39–41) and ∼20 min for GIP (42,43). However, on the basis of the same immunoassays that were used in the present study, Vilsbøll et al. (44) recently reported half-lives of ∼2 min for intact GLP-1 and between 4 and 5 min for the GLP-1 metabolite after the intravenous bolus administration of different GLP-1 doses in healthy volunteers. The investigators also found similar elimination rates in patients with type 2 diabetes and in healthy control subjects (44). For GIP, we have previously calculated a plasma half-life of 7.4 ± 0.4 min after intravenous infusion in healthy volunteers (6).

In the present study, the metabolic clearance rates of both GIP (3–42) and GLP-1 (9–36 amide) were lower than those calculated for the respective intact hormones (Table 2). A similar observation was reported recently for the elimination of GLP-2, which is also subject to DPP IV degradation (45). However, this probably reflects the overall greater contribution of DPP IV-mediated degradation in the elimination of the intact hormones, rather than a lower effectiveness of the kidneys. It is interesting that despite a high degree of sequence homology between GLP-1 and GLP-2, the latter is metabolized much more slowly (45).

The secretion of incretin hormones from endocrine K- and L-cells in the gut is controlled by the ingestion of nutrients (46). It is unclear whether increased plasma concentrations of incretin hormones can inhibit their own secretion via feedback mechanisms. In the present experiments, despite higher rises in the plasma concentrations of GIP (3–42) and GLP-1 (9–36 amide), there was no suppression of incretin secretion, as judged from the intact hormone concentrations. However, because only the plasma levels of metabolites were significantly affected by impaired renal function, it cannot be excluded that increased plasma levels of intact GIP or GLP-1 would lead to a feedback suppression of their own secretion. The latter hypothesis is supported by the observation of reduced total hormone levels and higher rises of intact incretin hormone levels after DPP IV inhibition in dogs (35).

Disturbances of glucose homeostasis have previously been characterized in patients with renal insufficiency (47). Glucose intolerance frequently develops as a result of a high degree of insulin resistance, as well as elevated plasma levels of glucagon, growth hormone, and catecholamines (47). In the present study, patients with impaired or diabetic oral glucose tolerance were excluded. As a result, glucose, insulin, and C-peptide concentrations were similar after glucose ingestion in both healthy subjects and patients (Fig. 1), and homeostasis model assessment analysis indicated no differences in insulin sensitivity or B-cell function (Table 1). However, the significant difference in the C-peptide-to-insulin ratio calculated from the integrated areas under the curves does suggest a reduced elimination of C-peptide in CRI patients.

In conclusion, the present data provide evidence that the kidneys are less important for the initial degradation (inactivation) of GIP and GLP-1. In contrast, the extraction of the metabolites GIP (3–42) and GLP-1 (9–36 amide) primarily depends on renal function. Therefore, altered elimination of DPP-IV-resistant analogues of GLP-1 (which are likely to be cleared with similar kinetics as the primary metabolite) in patients with renal impairment should be taken into consideration if these analogues are used for therapy.

This study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (Na 203/6-1) and the Forschungsförderung Ruhr-Universität Bochum Medizinische Fakultät (FoRUM) (F233/00).

We kindly acknowledge the expertise of Dr. Cedrik Meier for the calculations of the pharmacokinetic parameters and statistical analyses. The excellent technical assistance of Birgit Baller and Lone Bagger is greatly acknowledged.