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Glucose-Dependent Insulinotropic Hormone Potentiates the Hypoglycemic Effect of Glibenclamide in Healthy Volunteers: Evidence for an Effect on Insulin Extraction¹

Department of Endocrinology and Diabetology, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden

Address all correspondence and requests for reprints to: Henrik Kindmark, M.D., Ph.D., Department of Endocrinology and Diabetology D2:02, Karolinska Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: henrik.kindmark{at}ks.sc

	Abstract

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Glucose-dependent insulinotropic hormone (GIP) is an intestinal hormone considered to be an important mediator of the incretin effect, i.e. the augmented insulin release observed in response to orally, compared with iv, administered glucose, despite isoglycemic glucose profiles. Stimulation of ß-cell secretion of insulin by GIP is seen both in vitro and in vivo at permissive extracellular glucose concentrations (>6 mmol/L). It has also been claimed that part of the incretin effect is due to decreased insulin extraction. We now show that an infusion of GIP in healthy volunteers in whom blood glucose levels were maintained at 5 mmol/L, increased glibenclamide-stimulated levels of plasma insulin without significantly changing the C peptide profile. The increased plasma insulin levels necessitated extra glucose infusion to maintain euglycemia, demonstrating the biological significance of the elevated insulin levels. Infusion of GIP alone caused neither glucose changes nor elevation of C peptide or insulin levels. Hence, our results show that at a blood glucose concentration of 5 mmol/L, GIP augments the increase in plasma insulin levels stimulated by glibenclamide, possibly acting through a mechanism involving decreased insulin extraction in the liver or peripheral tissues, thus increasing insulin availability.

	Introduction

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IT IS WELL known that under conditions of comparable hyperglycemic profiles, orally administered glucose results in significantly higher plasma insulin levels compared with glucose administered via the iv route. The mechanism behind the potentiated secretion of insulin in response to orally administered glucose is believed to be the release of gastrointestinal hormones, incretins, that stimulate insulin secretion. The term incretin was coined as early as 1927 (1) and refers primarily to a mechanism, rather than to a specific factor. Several gastrointestinal hormones are known to contribute to the incretin effect, the two most important being glucose-dependent insulinotropic peptide (GIP; formerly known as gastric inhibitory peptide) and glucagon-like peptide-1 (GLP-1). GIP is present in epithelial K cells in the upper portion of the small intestine and is released in response to nutrient stimuli (2). In both animals and humans, GIP stimulates insulin release as long as the blood glucose concentration is greater than 6 mmol/L (2). The ability of GIP to stimulate insulin secretion is reported in some studies to be reduced in noninsulin-dependent diabetes mellitus (NIDDM) (3, 4). GIP release has been shown to be exaggerated in obese subjects during oral glucose tolerance tests (5) According to other reports, GIP release is reduced in response to a test meal in both obese subjects with normal glucose tolerance and lean subjects with NIDDM (6). It has been claimed that the main mechanism by which GIP increases insulin levels is by stimulating ß-cell secretion of insulin (7). However, evidence from earlier indirect studies in healthy subjects have suggested that part of the incretin effect is due to decreased hepatic insulin extraction (8, 9, 10), providing another mechanism by which the intestinal hormone could increase plasma insulin concentrations. The present study was performed to assess the effects of GIP on insulin secretion and insulin handling after stimulation with the sulfonyl urea compound glibenclamide. The study was performed in healthy volunteers at blood glucose levels clamped at 5 mmol/L.

	Subjects and Methods

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Subjects

Six subjects, five men and one woman, from the pool of volunteers at the Department of Endocrinology were recruited. All subjects had normal oral glucose tolerance according to WHO criteria (11). The mean age of the subjects was 34.2 ± 9.6 yr; they had a mean body mass index of 23.5 ± 1.1 kg/m² and a fasting blood glucose of 4.8 ± 0.5 mmol/L.

Protocols

The study was conducted according to Declaration of Helsinki principles. Protocols were approved by the ethical committee of the Karolinska Hospital. Consent to participate in the studies was obtained from subjects after the nature of the procedure had been explained. All studies were performed after overnight fast. Subjects were studied using three protocols performed in random order. On each occasion, a euglycemic clamp with a target blood glucose of 5 mmol/L was established. In-dwelling catheters (Venflon, Viggo, Helsingborg, Sweden) were inserted into veins in both arms. One cannula was used for the sampling of blood, and another was used for the administration of glucose, glibenclamide, and/or GIP. Arterialization of venous blood was achieved by heating (50 C) the forearm and hand of the arm used for sample collection in a thermoregulated sleeve. On one of the study occasions, protocol 1, 1 mg glibenclamide (Hoechst, Stockholm, Sweden) was administered as an iv bolus injection 1 h after the start of the study. On a second study occasion, protocol 2, synthetic human GIP (Peninsula Laboratories, Inc., Belmont, CA) was infused iv at a rate of 8 ng/kg·min for 30 min beginning 50 min after the start of the study. In a third study, protocol 3, both glibenclamide and GIP were administered iv as described above. One subject was studied using only protocols 1 and 3, but the other subjects were studied using all three protocols. Blood samples for the determination of blood glucose, insulin, C peptide, and glucagon were drawn at the start of each study and then at regular intervals.

Assays

Blood glucose was determined with a glucose oxidase method using a glucose analyzer (YSI, Inc., Yellow Springs, OH). Immunoreactive insulin, C peptide, and glucagon were measured by RIA. For the insulin RIA, the interassay coefficient of variation (CV) was less than 3.9%, and the intraassay CV was less than 3.1%. Serum C peptide was measured with a commercially available kit (Novo Research, Bagsvaerd, Denmark). The interassay CV was 4.5%, and the intraassay CV was 3%. Plasma glucagon was measured as previously described (12) using the antibody 30K. The interassay CV was 14%, and the intraassay CV was 5%.

Statistical analysis

All values are given as the mean ± SE. Statistical analysis was performed with ANOVA and, where appropriate, was further assessed with two-tailed Student’s t test for paired data as indicated in the text.

	Results

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Blood glucose

Blood glucose was maintained close to 5 mmol/L in all three study protocols (Fig. 1A). In summary, the average blood glucose concentration was 4.44 ± 0.77 mmol/L (protocol 1), 4.57 ± 0.38 mmol/L (protocol 2) and 4.62 ± 0.61 mmol/L (protocol 3). The rate of infusion of 20% glucose needed to maintain euglycemia is shown in Fig. 1B. Analysis of average glucose infusion shows that statistically significantly higher quantities of glucose had to be infused to maintain euglycemia during combined infusion of glibenclamide and GIP compared with glibenclamide alone (Fig. 1C).

View larger version (19K): [in this window] [in a new window]   Figure 1. Blood glucose concentration and need for glucose infusion. A, Blood glucose levels measured at different time points during the protocols. B, Rate of glucose infusion necessary to maintain a blood glucose concentration of approximately 5 mmol/L throughout the studies. C, Average total quantity of glucose infused between the time points corresponding to -20 and 60 min in B. Where applicable, glibenclamide (SU) is added at 0 min (arrow), and GIP is infused from -10 to 20 min (horizontal bar), as indicated in A and B. Statistically significant difference (*, P < 0.005) between protocols 1 and 3 regarding total quantity of glucose infused (C), calculated by ANOVA and two-tailed, paired Student’s t test. , Glibenclamide added alone, protocol 1; , GIP infused alone, protocol 2; , GIP plus glibenclamide, protocol 3.

Changes in plasma insulin and C peptide levels are displayed in Fig. 2, A and B, respectively. The mean incremental insulin and C peptide levels during 20 min after glibenclamide injection, or the corresponding time point in protocol 2, are displayed in Fig. 3, A and B. Infusion of GIP alone did not significantly change plasma insulin or C peptide compared with basal levels. As expected, both infusion of glibenclamide alone and glibenclamide together with GIP increased insulin and C peptide levels compared with basal levels. The combination of glibenclamide and GIP resulted in statistically significantly higher insulin levels compared with injection of glibenclamide alone (Fig. 3A). However, there was no statistically significant difference between C peptide levels during stimulation with both glibenclamide and GIP compared with glibenclamide alone (Fig. 3B).

View larger version (17K): [in this window] [in a new window]   Figure 2. Plasma insulin (A) and C peptide (B) levels measured at different time points during the protocols. Where applicable, glibenclamide (SU; arrow) was added at 0 min, and GIP (horizontal bar) was infused from -10 to 20 min, as indicated. , Glibenclamide added alone, protocol 1; , GIP infused alone, protocol 2; , GIP plus glibenclamide, protocol 3.

View larger version (15K): [in this window] [in a new window]   Figure 3. Mean incremental insulin levels (A) and C peptide levels (B) per min during 0–20 min in the three study protocols. SU refers to protocol 1 in which glibenclamide is added alone. GIP + SU refers to protocol 3 in which the combination of GIP and glibenclamide is added. GIP refers to protocol 2 in which GIP is added alone. In A, GIP+SU is significantly different from SU (*, P < 0.05, by ANOVA, t test). No statistically significant differences between the protocols were detected in B.

Plasma glucagon remained unchanged during all three study protocols (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Figure 4. Plasma glucagon levels measured at different time points during the protocols. Where applicable, glibenclamide (SU; arrow) was added at 0 min, and GIP (horizontal bar) was infused from -10 to 20 min, as indicated. ANOVA of average plasma glucagon levels for each protocol during 0–60 min in A did not detect any statistically significant differences. , Glibenclamide added alone, protocol 1; , GIP infused alone, protocol 2; , GIP plus glibenclamide, protocol 3.

	Discussion

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Assessment of insulin extraction based on insulin and C peptide concentrations in peripheral blood has been used in the literature (8, 9, 14). This is an indirect procedure and can be misleading, especially during nonsteady state conditions (15). However, comparisons of insulin profiles in individuals exposed to different conditions during which the C peptide profile remains unchanged should be informative regarding changes in insulin extraction. Such comparisons, of course, assume that hepatic and peripheral C peptide metabolism remain unchanged under the various conditions tested.

The ability of GIP to stimulate insulin secretion when ambient glucose levels are at or above the threshold for stimulation by the sugar per se has been firmly established, both in vitro in isolated islets (16, 17) and in vivo (18). GIP acts to increase cytosolic concentrations of cAMP in the pancreatic ß-cell, and increased signaling through the cAMP/PKA pathway affects the secretory machinery of the ß-cell, stimulating exocytosis (19).

Regarding the possible actions of GIP on insulin extraction, there are conflicting reports. Studies in normal subjects have suggested that the hepatic extraction of insulin is lower with oral glucose delivery than with iv glucose administration, as estimated by diverging plasma C peptide and insulin increments (8). In another study of young normal subjects, who were given oral glucose loads or isoglycemic glucose infusions, Nauck et al. observed a discrepancy between the incretin effect calculated from peripheral insulin responses, on the one hand, and C peptide responses or calculated insulin secretion rates, on the other (10). It was concluded that oral glucose reduces fractional hepatic extraction of insulin compared with an isoglycemic iv glucose load and that lower hepatic extraction could be caused by gastrointestinal factors such as hormones or nerves (10). In another study of healthy subjects, it was claimed that a large part of the incretin effect is due to decreased clearance of insulin, calculated as the molar ratio of integrated C peptide to integrated insulin responses or from a formula stating that insulin clearance equals insulin secretion divided by integrated insulin responses (14). Furthermore, an inverse correlation between GIP levels and estimated hepatic extraction of insulin, using the insulin/C peptide ratio, has been demonstrated in guar gum-treated normal subjects (9). Also, Kogire et al. demonstrated increased portal venous blood flow in dogs injected with GIP (20), and this might secondarily affect insulin handling in these organs, including hepatic insulin extraction.

Other reports speak against decreased insulin extraction as a mechanism of action for GIP. Experiments using isolated perfused rat liver and in vivo studies in conscious dogs have suggested that GIP does not mediate variations in hepatic insulin extraction between the fed and fasted states (21). Studies in healthy human volunteers indicate that the main cause of increased peripheral levels of insulin observed after oral glucose is augmented insulin secretion rather than reduced hepatic extraction (22, 23). Furthermore, studies in normal volunteers suggest that higher rates of secretion of insulin are associated with decreased extraction of the hormone and that this phenomenon is independent of increased incretin levels (24). Finally, the incretin effect is not reduced in type I diabetic patients who have undergone combined pancreas-kidney transplantation compared with nondiabetic kidney recipients, despite the systemic venous drainage of the pancreas grafts (25). In this situation, the liver will be exposed to much lower insulin concentrations than with portal delivery of secreted insulin, which could be expected to affect hepatic extraction of the hormone, and yet the incretin effect is preserved.

With regard to the possible GIP-induced changes in insulin extraction, an effect on insulin handling in peripheral target tissues cannot be excluded. Specific binding sites for GIP have been detected in skeletal muscle (7), and GIP receptor messenger ribonucleic acid is expressed in adipose tissue (26).

Is GIP a candidate for use in the treatment of diabetes mellitus? A number of previous studies indicate a decreased effect of GIP on insulin levels in diabetes mellitus (3, 4, 27). In a review of the field it was concluded that there is a loss of the incretin effect on insulin secretion in the majority of type II diabetes patients, judging from the C peptide responses to isoglycemic curves during oral and iv glucose supply (2). Another study showed that in the presence of 8 mmol/L glucose, physiological concentrations of porcine GIP caused an impaired ß-cell response in insulin-dependent diabetes mellitus (type I diabetes) patients with residual ß-cell function and in NIDDM patients compared with normal subjects (3). Furthermore, in a third study, newly diagnosed, previously untreated patients with type II diabetes mellitus were given an infusion of GIP or control solution together with a mixed meal. Fasting and postprandial glucose, C peptide, and insulin levels were similar in both groups (27). In a fourth study, GIP, GLP-1, or placebo was administered to type II diabetic patients and controls under conditions of hyperglycemic clamp. The maximal GIP-induced insulin release was reduced by 54% in subjects with type II diabetes compared with controls (4). GLP-1-(7–36) amide administered to subjects with type II diabetes, on the other hand, resulted in a C peptide increment 71% of that seen in normal subjects, a statistically nonsignificant difference. Thus, the insulinotropic effect of GIP is reduced in patients with type II diabetes, which implies that this peptide, unlike GLP-1, has no significant antidiabetogenic activity. However, the present study shows that GIP potentiates the hypoglycemic effect of sulfonyl urea in normal subjects, possibly through a mechanism involving decreased insulin extraction and thus increased insulin availability. The possibility of using this mechanism in the treatment of diabetes mellitus could be of clinical interest.

	Acknowledgments

 
We thank nurses Annica Clark and Kerstin Waldelöf for expert technical assistance and dedicated care of the study subjects, and all volunteers for their participation in the study.

	Footnotes

 
¹ This work was supported by grants from the Swedish Medical Research Council (K99-72X-00034-35A) and the Novo Nordisk Insulin Foundation.

² Present address: Department of Anesthesia and Intensive Care, Karolinska Hospital/Institute, S-171 76 Stockholm, Sweden.

Received October 10, 2000.

Revised February 1, 2001.

Accepted February 7, 2001.

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