PREDICTORS OF INCRETIN CONCENTRATIONS IN SUBJECTS WITH NORMAL, IMPAIRED, AND DIABETIC GLUCOSE

TOLERANCE

Kirsten Vollmer1, Jens J. Holst2, Birgit Baller1, Mark Ellrichmann1, Michael A. Nauck3, Wolfgang E. Schmidt1, Juris J. Meier1

1: Department of Medicine I, St. Josef-Hospital, Ruhr-University Bochum, Germany

2: Department of Medical Physiology, The Panum-Institute, University of Copenhagen, Denmark

3: Diabeteszentrum Bad Lauterberg, Germany

Corresponding Author: Dr. Juris J. Meier

Department of Medicine I, St. Josef-Hospital

Ruhr-University Bochum Gudrunstr. 56

44791 Bochum Germany

juris.meier@rub.de

Received for publication 10 August 2007 and accepted in revised form 20 November 2007.

Diabetes Publish Ahead of Print, published online December 5, 2007

Copyright American Diabetes Association, Inc., 2007

ABSTRACT Introduction: Defects in GLP-1 secretion have been reported in some patients with type 2 diabetes after meal ingestion. We addressed the questions: (1) Is the quantitative impairment in GLP-1 levels different after mixed meal or isolated glucose ingestion? (2) Which endogenous factors are associated with the concentrations of GLP-1, and in particular do elevated fasting glucose or glucagon levels diminish GLP-1 responses. Patients and methods: 17 patients with mild type 2 diabetes, 17 subjects with impaired glucose tolerance and 14 matched controls participated in an oral glucose tolerance test (75 g) and a mixed meal challenge (820 kcal), both carried out over 240 min on separate occasions. Plasma levels of glucose, insulin, C-peptide, glucagon, triglycerides, free fatty acids, GIP and GLP-1 were determined. Results: GIP and GLP-1 levels increased significantly in both experiments (p < 0.0001). In patients with type 2 diabetes, the initial GIP response was exaggerated compared to controls after mixed meal (p < 0.001), but not oral glucose ingestion (p = 0.98). GLP-1 levels were similar in all three groups in both experiments. GIP responses were 186 ± 17 % higher after mixed meal ingestion than after the oral glucose load (p < 0.0001), whereas GLP-1 levels were similar in both experiments. There was a strong negative association between fasting glucagon and integrated FFA levels and subsequent GLP-1 concentrations. In contrast, fasting FFA and integrated glucagon levels after glucose or meal ingestion as well as female gender were positively related to GLP-1 concentrations. Incretin levels were unrelated to measures of glucose control or insulin secretion. Conclusions: Deteriorations in glucose homeostasis can develop in the absence of any impairment in GIP or GLP-1 levels. This suggests that the defects in GLP-1 concentrations previously described in patients with long-standing type 2 diabetes are likely secondary to other hormonal and metabolic alterations, such as hyperglucagonaemia. GIP and GLP-1 concentrations appear to be regulated by different factors and are independent of each other.

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ostprandial glucose homeostasis is controlled not only by the direct stimulation of insulin release by the

absorbed nutrients, but also through the secretion of incretin hormones, namely gastric inhibitory polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) [1-4]. In healthy, non-diabetic subjects, the quantitative contribution of this incretin effect to the overall postprandial insulin secretion has been estimated to be 50 – 70 % [5, 6], depending on meal size and composition. In contrast, a marked reduction of the incretin effect is characteristic of patients with type 2 diabetes [7], thereby contributing to the excess postprandial glucose excursions in such patients. While the exact mechanisms underlying the loss of incretin activity in patients with type 2 diabetes are still not completely understood, two defects have been described: First, the insulinotropic effect of GIP is markedly reduced in patients with type 2 diabetes compared to healthy controls (~10-20% of the normal response) [8-10], whereas the stimulation of insulin secretion by GLP-1 is largely preserved (~60-70% of the normal response) [9, 11]. The reasons underlying the loss of GIP effect in type 2 diabetes are not entirely clear, and different possible explanations have been expounded [12, 13]. These include a defective expression of GIP receptors [14], downregulation of GIP signalling [15], or a general reduction of beta-cell function and mass [16]. However, as yet none of these hypotheses have been convincingly proven. The second defect in the entero-insular axis reported in patients with type 2 diabetes relates to the secretion of GLP-1. In particular, postprandial levels of GLP-1 have been found to be deficient by ~20-30% in some [17, 18], but not all studies [19, 20]. This observation has led the idea that raising endogenous GLP-1 levels through DPP-4 inhibition or by the exogenous administration of GLP-1 analogues/incretin mimetics might serve to substitute for a primary defect involved in the pathogenesis of type 2 diabetes. In contrast to GLP-1, GIP concentrations have

been found to be slightly decreased, normal or even increased in patients with type 2 diabetes [17, 21-24], and most groups agree that defects in GIP secretion are unlikely to play a significant role in the pathogenesis of type 2 diabetes [13, 25]. In order to examine, whether defects in GLP-1 secretion represent a primary defect, potentially predisposing to the development of type 2 diabetes, or whether they develop secondarily during the pathogenesis of type 2 diabetes, we and others have previously characterised incretin concentrations following oral glucose ingestion in first degree relatives of patients with type 2 diabetes as well as in normal glucose tolerant women with a history of gestational diabetes [26-28], both populations with a high empiric risk for developing type 2 diabetes during their later life. However, we were unable to detect any abnormalities in GIP or GLP-1 levels in any of these groups [26, 27]. We therefore concluded that the reduction of GLP-1 concentrations in patients with type 2 diabetes is most likely a consequence of other abnormalities in such patients, such as hyperglycaemia or hyperglucagonaemia [13]. An alternative explanation for the lack of impairment in GLP-1 levels in these studies would be that most prior studies in patients with type 2 diabetes had examined incretin levels after mixed meal ingestion [18, 19], whereas in our studies GLP-1 levels were measured after an oral glucose load [26, 27]. In fact, an increased secretion of GLP-1 has been reported after oral glucose ingestion in one group of patients with type 2 diabetes, although based on an assay with suboptimal specificity [19].

Therefore, in the present studies, we addressed the following questions: (1) Is the quantitative impairment in GLP-1 concentrations in patients with type 2 diabetes different after the ingestion of a mixed solid meal or an isolated liquid glucose load? (2) Which other endogenous factors are associated with the concentrations of GLP-1, and in particular

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do high fasting glucose or glucagon levels diminish the subsequent GLP-1 responses. SUBJECTS, MATERIALS AND METHODS Study protocol. The study protocol was approved by the ethics committee of the medical faculty of the Ruhr-University, Bochum prior to the experiments (registration no. 2013). Written informed consent was obtained from all participants. Subjects. A total of 48 subjects participated in the study. Amongst those, 17 patients had type 2 diabetes (WHO criteria) of relatively short duration (3.2 ± 2.8 years), 17 had impaired glucose tolerance (IGT), and 14 had a normal oral glucose tolerance. Type 2 diabetes was previously known in 16 of the patients, whereas in one case the diagnosis type 2 diabetes was made on occasion of the OGTT. Six patients with type 2 diabetes were treated with metformin, the other patients were on a dietary regimen. Subject characteristics are presented in table 1. Study design. At a screening visit, blood was drawn from all participants in the fasting state for measurements of standard hematological and clinical chemistry parameters and a general clinical examination was performed. Subjects with anemia (hemoglobin < 12 g/dl), elevation in liver enzymes (ALAT, ASAT, AP, γ-GT) to higher activities than double the respective normal value, or elevated creatinine concentrations (>1.5 mg/dl) were excluded. Body height and weight were determined and waist- and hip-circumference were measured in order to calculate body mass index and the waist-to-hip ratio, respectively. Blood pressure was determined according to the Riva-Rocci method [29]. A vibration perception threshold performed on both medial malleoli revealed no major impairment in pallesthesia in the patients with diabetes (5.4 ± 1.6, 5.9 ± 1.2, and 6.1 ± 1.6 times 1/8 in patients with type 2 diabetes, IGT subjects and controls, respectively; p = 0.43).

If subjects met the inclusion criteria, they were studied on two occasions: a) An oral glucose challenge: 75 g of glucose (O.G.T. Roche diagnostics,

Mannheim, Germany) were ingested in the overnight fasting state within five minutes. Blood samples were drawn twice in the fasting state and at 30 min- intervals over 240 min afterwards. b) A mixed meal challenge: A large Continental breakfast (two European bread rolls, 20 g of butter, 40 g of gouda cheese, 30 g of jam, 1 egg, 150 g of yogurt (3.5 % fat content), 200 ml of tee) was ingested over 15 min. The total caloric content of the test meal was 820 kcal (107 kcal from protein, 353 kcal from fat, 360 kcal from carbohydrates). Blood samples were drawn twice in the fasting state and at 30 min- intervals over 240 min afterwards. All antidiabetic treatment was withdrawn at least two days prior to study commencement. Experimental procedures. Both tests were performed in the morning after an overnight fast with subjects in a supine position throughout the experiments. Both ear lobes were made hyperemic using Finalgon® (Nonivamid 4 mg/g, Nicoboxil 25 mg/g). The experiments were started by the ingestion of the oral glucose load (a) or the mixed test meal (b), and venous blood samples were drawn at t = -30, 0, 30, 60, 90, 120, 150, 180, 210, and 240. In addition, capillary blood samples (approximately 100 µl) were added to NaF (Microvette CB 300; Sarstedt, Nümbrecht, Germany) for the immediate measurement of glucose. Laboratory determinations. Glucose concentrations were measured in capillary blood samples using a glucose oxidase method with a Glucose Analyser 2 (Beckman Instruments, Munich, Germany) as previously described [10]. Insulin was measured as described [10] using an insulin microparticle enzyme immunoassay (MEIA), IMx Insulin, Abbott Laboratories, Wiesbaden, Germany. Intra-assay coefficient of variation was ≈ 4 %.

C-peptide was measured as described [10] using an enzyme-linked immunoabsorbent assay (ELISA) from DAKO Ltd., Cambridgeshire, UK. Intra-assay coefficients of variation were 3.3 to 5.7 %, inter-assay variation was 4.6 to 5.7

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%. Human insulin and C-peptide were used as standards.

Total GLP-1 concentrations were measured using a radioimmunoassay (RIA) (antiserum no. 89390), which is specific for the C-terminal end of the GLP-1 molecule and reacts equally with intact GLP-1 and the primary (N-terminally truncated) metabolite as described [27].

Total GIP was measured, as described previously, using the C-terminally directed antiserum R65 [30], which reacts fully with intact GIP and the N-terminally truncated metabolite.

Glucagon was measured by a radioimmunoassay using antibody no. 4305 in ethanol-extracted plasma, as described [31]. The detection limit was < 1 pmol/l. Intra-assay coefficients of variation were < 5.0 %, inter-assay coefficients of variation were 15%.

Triglycerides were measured using an enzymatic colour test system (Olympus system reagent triglyceride OSR 6133). Free fatty acids were determined using reagents from Wako chemicals, Neuss, Germany, by spectrophotometric analysis. Calculations and statistical analyses. Results are reported as mean ± SEM. All statistical calculations were carried out using repeated-measures analysis of variance (ANOVA) using Statistica version 5.0 (Statsoft Europe, Hamburg, Germany). Values at single time points were compared by one-way ANOVA followed Duncan`s post hoc-test. A two-sided p-value < 0.05 was taken to indicate significant differences. Integrated incremental plasma concentrations of insulin, C-peptide, GIP, GLP-1, and triglycerides were calculated according to the trapezoidal rule (baseline subtracted). Integrated glucagon and free fatty acid levels were calculated as the negative (decremental) areas under the curve. Insulin resistance was calculated according to the HOMA model [32]. Linear regression analyses were performed using GraphPad Prism, version 4.0. Multivariate regression analyses regarding the concentrations of GIP and GLP-1, taking into account the factors age, sex, body

weight, glucose (basal and positive AUC), glucagon (basal and negative AUC), insulin (basal and positive AUC), HbA1c, and free fatty acids (basal and negative AUC), were carried-out using Statistica version 5.0. RESULTS Islet cell secretion and lipid profiles after oral glucose and meal ingestion. As expected, glucose concentrations were higher in the subjects with impaired glucose tolerance and type 2 diabetes both after the oral glucose load and following the mixed meal ingestion (p < 0.0001; fig. 1). Likewise, insulin and C-peptide concentrations were higher in IGT subjects and patients with diabetes compared to controls, indicating a higher degree of insulin resistance (Fig. 1). This impression was confirmed by HOMA analysis revealing higher levels of insulin resistance in the patients with type 2 diabetes (5.0 ± 0.9), and IGT subjects (3.0 ± 0.5), compared to controls (1.8 ± 0.3; p = 0.006).

Glucagon levels were significantly lowered after oral glucose and mixed meal ingestion (p < 0.001; Fig. 2). The time course of glucagon concentrations was significantly different between the groups after the test meal (p < 0.001), with higher glucagon levels in patients with type 2 diabetes during the first 90 min of the test. There were no differences in the time course of glucagon levels between the groups after the oral glucose load, even though glucagon concentrations tended to be higher in the diabetic patients during the first 90 min following glucose ingestion as well.

Free fatty acid concentrations were higher in IGT subjects and patients with type 2 diabetes during both experiments (p < 0.0001). These differences were most pronounced in the fasting state, whereas FFA levels were even lower in the patients towards the end of the experiments (significant only after oral glucose ingestion). As expected, triglyceride levels remained unchanged after oral glucose ingestion (p = 0.06) and increased significantly after the mixed test meal (p < 0.0001). There was a trend towards higher

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triglyceride levels in the patients with type 2 diabetes (Fig. 2). Plasma levels of incretin hormones after oral glucose and meal ingestion. Plasma concentrations of GIP and GLP-1 increased significantly after oral glucose and meal ingestion (p < 0.0001). Peak levels of GIP were reached 90 min after the oral glucose load and 60 min after mixed meal ingestion. The maximum GLP-1 concentrations were detected at t = 30 and t = 90 min, respectively (Fig. 3). There were no differences in the plasma concentrations of GIP between the groups after oral glucose ingestion (p = 0.98). In contrast, the initial GIP response 30 min after meal ingestion was significantly increased in the patients with type 2 diabetes, whereas the IGT subjects exhibited higher GIP levels from t = 180 to 240 min after the test meal (p < 0.001). However, despite these differences at individual time points, there was no difference in integrated GIP levels after the meal (p = 0.33).

GLP-1 levels were not different between the groups both after oral glucose and meal ingestion. GIP responses were 186 ± 17 % higher after mixed meal ingestion than after the oral glucose load (p < 0.0001; Fig. 4). In contrast, there were no differences in GLP-1 plasma concentrations between the experiments with oral glucose or mixed meal ingestion (p = 0.06; Fig. 4). The patterns of GIP and GLP-1 levels were not significantly different when the six patients pre-treated with metformin were excluded from the analyses (details not shown). Predictors of incretin concentrations after oral glucose and meal ingestion. There was a strong negative association between fasting glucagon levels and GLP-1 levels, as assessed by the incremental AUC’s, after oral glucose and meal ingestion (Table 2; Figure 5). In contrast, the integrated decremental glucagon levels after glucose or test meal ingestion were positively associated with the increases in GLP-1 levels. Conversely, there was a positive relationship between fasting free fatty acid levels and subsequent GLP-1 concentrations, whereas the integrated

decremental FFA levels were inversely related to the integrated GLP-1 levels. There also was a significant positive relationship between GLP-1 levels and increasing age, and a negative association with higher BMI levels. These associations were stronger after oral glucose ingestion than following mixed meal ingestion (Table 2). GIP levels were positively related to fasting FFA levels and integrated decremental glucagon concentrations, whereas integrated decremental FFA levels were negatively associated with GIP concentrations (Table 3). There also was a significant positive relationship between age and GIP levels.

GLP-1 plasma concentrations were significantly higher in female than in male subjects both after oral glucose (3491 ± 491 pmol . l-1 . min vs. 2061 ± 296 pmol . l-1 . min; p = 0.04) and meal ingestion (4153± 485 pmol . l-1 . min vs. 2639 ± 419 pmol . l-1 . min; p = 0.037), and mixed meal ingestion, whereas GIP levels were similar in both groups (8846 ± 618 pmol . l-1 . min vs. 8090 ± 865 pmol . l-1 . min, respectively after oral glucose ingestion, p = 0.47 and 22951 ± 1153 pmol . l-1 . min vs. 19327 ± 1524 pmol .

l-1 . min, respectively after mixed meal ingestion; p = 0.063). GLP-1 concentrations were similar in diabetic patients treated with metformin or with a dietary regimen (details not shown).

There was no detectable association between the measures of glucose control (fasting and 120 min glucose, HbA1c), insulin secretion (HOMA beta-cell function), or insulin sensitivity (HOMA-IR) and the plasma concentrations of GIP or GLP-1 (Tables 1 and 2).

In a multivariate regression analysis, the GLP-1 levels after the oral glucose load were positively related to age (p = 0.015), and the decremental AUCglucagon (p = 0.0012), whereas no significant association with GIP levels was detected. DISCUSSION

The present studies were undertaken to examine whether defects in the plasma levels of incretin hormones in patients with type 2 diabetes and IGT were more

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prominent after an oral glucose load than following mixed meal ingestion. To our own surprise, we did not find any alterations in GLP-1 concentrations after oral glucose or mixed meal ingestion in the individuals with impaired glucose tolerance (IGT) and type 2 diabetes. In contrast, GIP levels were similar in all groups after the oral glucose load, and even slightly higher in subjects with IGT and type 2 diabetes after the meal test.

The present results are at variance with some [17, 18], but not all [20] previous studies reporting incretin hormone levels in patients with type 2 diabetes. Toft-Nielsen and colleagues reported ~20 and ~30 % lower postprandial GLP-1 levels in IGT subjects and patients with type 2 diabetes compared to NGT subjects, respectively [17]. In line with these data, Vilsboll and colleagues found not only total, but also intact GLP-1 levels to be reduced in patients with type 2 diabetes [18]. However, in subsequent studies the same group of investigators failed to detect such differences in GLP-1 levels in another group of patients with type 2 diabetes [20]. In the present studies only total GIP and GLP-1 levels were measured meaning that differences in the degradation of incretin hormones cannot be excluded. In addition to these studies in patients with overt diabetes, we and others have found similar GIP and GLP-1 levels in high risk groups, such as first-degree relatives of patients with diabetes as well as in women with prior gestational diabetes [26-28]. The reasons for the dissimilar results in different cohorts of patients are difficult to explain, but since in all of these studies incretin levels were measured in the same laboratory, technical issues are rather unlikely. It is therefore important to compare the subject characteristics in more detail. Thus, in the present studies, patients with a rather short diabetes duration (3.2 ± 2.8 years), and in relatively good glycaemic control (HbA1c: 6.8 ± 0.9 %) were examined. In contrast, the patients studied by Vilsboll et al. and Toft-Nielsen et al. had a longer diabetes duration and exhibited higher HbA1c levels [17, 18]. It is therefore possible that defects in GLP-1

secretion develop later during the pathogenesis of type 2 diabetes. In this context, another factor with a potential impact on postprandial GLP-1 levels is the velocity of gastric emptying [33, 34]. Thus any deceleration of gastric emptying might blunt the subsequent incretin responses [35]. In the present studies patients with a relatively short duration of diabetes and no signs of diabetic neuropathy were examined. However, since gastric emptying is significantly delayed in some patients with long-standing type 2 diabetes [36], it is possible that the impairment in postprandial GLP-1 concentrations reported earlier was partly driven by a delay in gastric motility.

Another possible factor that might impact on GLP-1 secretion in diabetic patients is the presence of hyperglucagonaemia. In fact, in this as well as in previous studies, high glucagon levels were found to be associated with lower GLP-1 concentrations [37]. This might explain why in the present study with rather modest differences in glucagon levels between the groups, GLP-1 plasma concentrations were similar in patients with and without diabetes, whereas previous studies examining patients with a longer diabetes duration and with more pronounced differences in glucagon secretion had found a significant impairment in GLP-1 levels in diabetic patients [17]. Thus, it seems possible that in health GLP-1 secretion is tonically inhibited by glucagon, and that the postprandial decline in alpha-cell secretion, along with other nutrient signals, allows for the rise in GLP-1 levels. In patients with long-standing type 2 diabetes, the lack of glucagon suppression by glucose or meal ingestion might lead to an impairment in GLP-1 secretion [38, 39]. However, it is important to emphasize the relationship between glucagon und GLP-1 levels observed herein was purely associative and does not allow for safe conclusions regarding a direct interaction between both hormones. Thus, hyperglucagonemia and reduced GLP-1 levels might as well be independent conditions in patients with type 2 diabetes without a causal relationship to

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each other. Therefore, it appears worthwhile to directly examine the effects of elevated glucagon levels on GLP-1 secretion in more detail.

An alternative explanation is that the impairment in GLP-1 levels can only be provoked by certain test meals, depending of the respective caloric content and nutrient composition. In the present study, GLP-1 plasma concentrations in patients with diabetes were normal both after an oral glucose load and after a mixed test meal representative of a typical European continental breakfast. However, it cannot be excluded that incretin response in patients with diabetes and controls would be different after the ingestion of a rather high-fat containing test meal. Finally, it cannot be excluded that in our present studies minor differences in GLP-1 concentrations at certain time points were overlooked due to the 30-min sampling intervals.

On a cautionary note, six of the 17 patients with type 2 diabetes in this study were pre-treated with metformin, and even though metformin was withdrawn 2 days prior to the experiments, it cannot be fully ruled out that GLP-1 concentrations were affected by this treatment [40]. However, the patterns of incretin concentrations were similar when these six patients were excluded from the analyses, and in this relatively small group of subjects there were no significant differences in GLP-1 levels between patients treated with or without metformin.

Despite the somewhat discrepant results in different studies, the present data showing normal incretin responses in a group of patients with type 2 diabetes suggest that defects in GLP-1 secretion are probably not an important factor in the early pathogenesis of the disease. Along these lines it is worth noting that the differences in GLP-1 levels reported in the prior studies were only apparent in the late postprandial period (~120 to 240 min after meal ingestion) [17, 18], whereas defects in insulin secretion typically occur in the early postprandial phase [41].

Another objective of these studies was to uncover endogenous predictors of GLP-1 levels. Using linear regression analysis, we identified fasting glucagon levels as a strong negative predictor of subsequent GLP-1 concentrations both after oral glucose and mixed meal ingestion. However, despite this inverse relationship in the fasting state, there was a strong positive association between GLP-1 levels and postprandial glucagon concentrations. This observation is even more surprising since GLP-1 is known to exert glucagonostatic actions [42, 43]. Therefore, the question arises what is the driving force in this relationship. Given the inverse association between glucagon and GLP-1 levels in the fasting state, any stimulation of GLP-1 release by glucagon seems rather unlikely [37]. A more plausible explanation is that the positive relationship between postprandial GLP-1 and glucagon levels was indirectly mediated by the secretion of GLP-2 [44]. Indeed, both GLP-1 and GLP-2 are secreted at equimolar amounts from entero-endocrine L-cells [45], but owing to its slower degradation, circulating GLP-2 levels exceed those of GLP-1 by several-fold [46, 47]. It is therefore likely that the potent glucagonotropic actions of GLP-2 shown previously by our group have supervened the glucagonostatic actions of GLP-1 [44, 48]. Thus, under physiological conditions, the GLP-1 induced suppression of glucagon secretion might be outweighed by the glucagonotropic actions of GLP-2.

One question directly arising from this hypothesis is why DPP-4 inhibitors act to suppress rather than to increase glucagon levels in patients with type 2 diabetes, despite their actions of intact GIP and GLP-2 levels. While due to their correlative nature, the present studies cannot provide a direct explanation for this phenomenon, two possible reasons come to mind: First, the affinity of DPP-4 for GLP-1 is much higher than for GLP-2 and GIP, meaning that the impact of DPP-4 inhibition on the intact plasma levels of these hormones is different as well. This may lead to an over-proportional rise in intact GLP-1 levels [49].

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Second, the actions of both GIP and GLP-1 on glucagon secretion been shown to be strictly glucose-dependent [42, 50, 51]. It is therefore conceivable that in hyperglycaemic patients with type 2 diabetes the glucagonostatic effects of GLP-1 outweigh the actions of GIP and GLP-2 during DPP-4 inhibitor administration. In contrast, in non-diabetic subjects studied at euglycaemia glucagon release has been shown to be suppressed to an even lesser extent after oral than during intravenous glucose administration [37].

In addition to glucagon, fasting free fatty acid levels and age were identified as positive predictors of GLP-1 concentrations, while body weight and integrated FFA levels were inversely related. The present analyses also revealed significantly greater GLP-1 responses in female subjects, consistent with the findings of Toft-Nielesen and colleagues [17].

While the ingestion of the mixed test meal elicited a significantly greater response of GIP levels than the oral glucose load, GLP-1 levels were not different between both experiments. This suggests that (a) GIP and GLP-1 are not entirely co-secreted and (b) that different factors regulate the secretion of GIP and GLP-1. Since both meals differed with respect to the overall caloric as well as the nutrient composition, it is likely that the increased GIP concentrations following the mixed meal ingestion were largely due to the additional lipid content, which might have had no additional impact on GLP-1 secretion. An alternative explanation is that GLP-1

secretion was already stimulated to a maximum extent, thereby not allowing for an additional gain in GLP-1 levels with increasing amounts of nutrients ingested. Given the diverging actions of both incretin hormones on glucagon secretion [42, 51], gastric emptying [52, 53], and lipid homeostasis [16, 54], the distinct relative responses of GIP and GLP-1 secretion in response to different test meals might serve as a fine regulator of postprandial metabolism.

In conclusion, the present studies have shown that deteriorations in glucose homeostasis can develop in the absence of any impairments in GIP or GLP-1 levels. This suggests that the impairments in postprandial GLP-1 concentrations previously described in some patients with long-standing type 2 diabetes are likely secondary to other metabolic alterations, such as hyperglucagonaemia. The unequal dependency of GIP and GLP-1 responses on meal size and composition implies that both hormones are largely released independently of each other, and that their respective secretion is regulated by different factors. ACKNOWLEDGEMENTS

The excellent technical assistance of Elisabeth Frick and Lone Bagger is greatly acknowledged. This study was supported by unrestricted grants from the Ruhr-Universität Bochum (FoRUM grant F-515-06 to JJM), Novo Nordisk (to JJM), The Danish Medical Research Council (to JJH) and the Deutsche Forschungsgemeinschaft (grant Me 2096/5-1 to JJM).

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28. Nyholm B, Walker M, Gravholt CH, et al. Twenty-four-hour insulin secretion rates, circulating concentrations of fuel substrates and gut incretin hormones in healthy offspring of Type II (non-insulin-dependent) diabetic parents: evidence of several aberrations. Diabetologia 1999;42(11):1314-23.

29. Geddes LA. The first accurate measurement of systolic and diastolic blood pressure. IEEE Eng Med Biol Mag 2002;21:102–3.

30. Deacon CF, Nauck MA, Meier JJ, Hücking K, Holst JJ. Degradation of endogenous and exogenous gastric inhibitory polypeptide (GIP) in healthy and in Type 2 diabetic subjects as revealed using a new assay for the intact peptide. J Clin Endocrinol Metab 2000;85:3575-3581.

31. Holst JJ. Evidence that peak II GLI or enteroglucagon is identical to the C-terminal sequence (residues 33-69) of glicentin. Biochem J 1982;207:381-388.

32. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: Insulin resistance and ß-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412-419.

33. Horowitz M, Edelbroek MA, Wishart JM, Straathof JW. Relationship between oral glucose tolerance and gastric emptying in normal healthy subjects. Diabetologia 1993;36:857-62.

34. Horowitz M, O'Donovan D, Jones KL, Feinle C, Rayner CK, Samsom M. Gastric emptying in diabetes: clinical significance and treatment. Diabetic Med 2002;19:177-194.

35. Gentilcore D, Chaikomin R, Jones KL, et al. Effects of fat on gastric emptying of and the glycemic, insulin, and incretin responses to a carbohydrate meal in type 2 diabetes. J Clin Endocrinol Metab. 2006;91(6):2062-7. Epub 2006 Mar 14.

36. Horowitz M, Harding PE, Maddox AF, et al. Gastric and oesophageal emptying in patients with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia. 1989;32(3):151-9.

37. Meier JJ, Deacon CF, Schmidt WE, Holst JJ, Nauck MA. Suppression of glucagon secretion is lower after oral glucose administration than during intravenous glucose administration in human subjects. Diabetologia. 2007;50:806-13.

38. Muller WA, Faloona GR, Aguilar-Parada E, Unger RH. Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion. N Engl J Med. 1970;283(3):109-15.

39. Mitrakou A, Kelley D, Veneman T, et al. Contribution of abnormal muscle and liver glucose metabolism to postprandial hyperglycemia in NIDDM. Diabetes. 1990;39(11):1381-90.

11

40. Migoya EM, Miller J, Larson P, et al. Sitagliptin, a selective DPP-4 inhibitor, and metformin have complementary effects to increase active GLP-1 activity. Diabetes 2007;56 (Suppl. 1):A74.

41. Pfeifer MA, Halter JB, Porte D, Jr. Insulin secretion in diabetes mellitus. Am J Med. 1981;70(3):579-88.

42. Ørskov C, Holst JJ, Nielsen OV. Effect of truncated glucagon-like peptide-1 [proglucagon-(78-107) amide] on endocrine secretion from pig pancreas, antrum, and nonantral stomach. Endocrinology 1988;123:2009-2013.

43. Nauck MA, Kleine N, Ørskov C, Holst JJ, Willms B, Creutzfeldt W. Normalization of fasting hyperglycaemia by exogenous glucagon-like peptide 1 (7-36 amide) in type 2 (non-insulin-dependent) diabetic patients. Diabetologia 1993;36:741-744.

44. Meier JJ, Nauck MA, Pott A, et al. Glucagon-like peptide 2 stimulates glucagon secretion, enhances lipid absorption, and inhibits gastric acid secretion in humans. Gastroenterology. 2006;130(1):44-54.

45. Ørskov C, Knuhtsen S, Baldissera FG, Poulsen SS, Nielsen OV, Holst JJ. Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are secreted separately from pig small intestine but not pancreas. Endocrinology 1986;119:1467-75.

46. Hartmann B, Harr MB, Jeppesen PB, et al. In vivo and in vitro degradation of glucagon-like peptide-2 in humans. J Clin Endocrinol Metab 2000;85:2884-2888.

47. Hartmann B, Johnsen AH, Orskov C, Adelhorst K, Thim L, Holst JJ. Structure, measurement, and secretion of human glucagon-like peptide-2. Peptides 2000;21(1):73-80.

48. de Heer J, Pedersen J, Orskov C, Holst JJ. The alpha cell expresses glucagon-like peptide-2 receptors and glucagon-like peptide-2 stimulates glucagon secretion from the rat pancreas. Diabetologia 2007;4:4.

49. Mentlein R. Dipeptidyl-peptidase IV (CD26) - role in the inactivation of regulatory peptides. Regul Pept 1999;85:9-24.

50. Pederson RA, Brown JC. Interaction of gastric inhibitory polypeptide, glucose, and arginine on insulin and glucagon secreton from the perfused rat pancreas. Endocrinol 1978;103:610-615.

51. Meier JJ, Gallwitz B, Siepmann N, et al. Gastric inhibitory polypeptide (GIP) dose-dependently stimulates glucagon secretion in healthy human subjects at euglycaemia. Diabetologia 2003;46:798-801.

52. Meier JJ, Gallwitz B, Salmen S, et al. Normalization of glucose concentrations and deceleration of gastric emptying after solid meals during intravenous glucagon-like peptide 1 in patients with type 2 diabetes. J Clin Endocrinol Metab 2003;88:2719-2725.

53. Meier JJ, Goetze O, Anstipp J, et al. Gastric inhibitory polypeptide (GIP) does not inhibit gastric emptying in man. Am J Physiol (Endocrinol Metab) 2004;286:E621-E625.

54. Meier JJ, Gethmann A, Goetze O, et al. Glucagon-like peptide 1 (GLP-1) abolishes the postprandial rise in triglyceride concentrations and lowers free fatty acid levels in humans. Diabetologia 2006;49:452-8.

12

TABLE 1. Subject characteristics

Parameter Patients with Subjects with Control p-value

Type 2 diabetes IGT subjects

Age [years] 57.5 ± 8.0 60.1 ± 8.9 57.0 ± 6.3 0.49

Body mass index [kg/m2] 32.1 ± 6.9 29.5 ± 6.9 27.5 ± 3.3 0.14

Waist-hip-ratio 0.92 ± 0.08 0.91 ± 0.10 0.92 ± 0.12 0.95

HbA1c [%]1 6.8 ± 0.9* 5.9 ± 0.4 5.8 ± 0.3 < 0.0001

Triglycerides [mg/dl] 226 ± 181 147 ± 58 147 ± 69 0.10

Total cholesterol [mg/dl] 225 ± 43 234 ± 31 233 ± 32 0.68

HDL-cholesterol [mg/dl] 54 ± 24 61 ± 20 55 ± 18 0.51

LDL-cholesterol [mg/dl] 143 ± 39 155 ± 39 160 ± 34 0.28

Means ± SD1: Normal range: 4.8 – 6.0 % *: Significantly different (p < 0.05) versus controls

13

TABLE 2. Predictors of GLP-1 secretion (AUC GLP-1) after oral glucose and meal ingestion Oral glucose Test meal Parameter [unit] r p-value r p-value Age [years] 0.40 0.0052 0.26 0.078 Body weight [kg] -0.35 0.016 -0.31 0.032 HbA1c [%] -0.19 0.19 -0.17 0.24 HOMA Insulin resistance -0.21 0.15 -0.16 0.28 Fasting glucose [mg/dl] -0.10 0.31 -0.083 0.57 Fasting glucagon [pmol/l] -0.49 < 0.001 -0.38 0.0083 AUC glucagon [pmol . l-1 . min] 0.57 < 0.0001 0.26 0.08 Fasting insulin [mU/l] -0.19 0.20 -0.25 0.084 AUC insulin [mU . l-1 . min] -0.22 0.14 -0.20 0.16 Fasting FFA [mg/dl] 0.39 0.0055 0.28 0.052 AUC FFA [mg . dl-1 . min] -0.53 < 0.001 -0.32 0.026 p-values were calculated by linear regression analysis; r = correlation coefficient

14

TABLE 3. Predictors of GIP secretion (AUC GIP) after oral glucose and meal ingestion Oral glucose Test meal Parameter [unit] r p-value r p-value Age [years] 0.30 0.038 0.17 0.24 Body weight [kg] -0.11 0.46 -0.068 0.64 HbA1c [%] -0.12 0.43 0.027 0.85 HOMA Insulin resistance -0.035 0.82 0.18 0.23 Fasting glucose [mg/dl] -0.21 0.14 0.017 0.91 Fasting glucagon [pmol/l] -0.25 0.0913 -0.23 0.11 AUC glucagon [pmol . l-1 . min] 0.34 0.019 0.20 0.18 Fasting insulin [mU/l] 0.02 0.88 -0.016 0.91 AUC insulin [mU . l-1 . min] 0.24 0.09 0.03 0.82 Fasting FFA [mg/dl] 0.29 0.043 0.31 0.033 Net integral FFA [mg . dl-1 . min] -0.37 0.01 -0.34 0.02 p-values were calculated by linear regression analysis; r = correlation coefficient

15

FIGURE LEGENDS

Figure 1. Plasma concentrations of glucose (A, B), insulin (C, D), and C-peptide (E, F) after ingestion of 75g oral glucose (A, C, E) or a mixed test meal (B, D, F) in 17 patients with type 2 diabetes, 17 subjects with impaired glucose tolerance (IGT) and 14 subjects with normal glucose tolerance (NGT). Means ± SEM. Statistics were carried-out using repeated measures ANOVA and denote A: differences between the experiments, B: differences over time and AB: differences due to the interaction of experiment and time. Asterisks indicate significant (p < 0.05) differences versus controls at individual time points, daggers indicate significant differences versus patients with type 2 diabetes (one-way ANOVA). Figure 2. Plasma concentrations of glucagon (A, B), free fatty acids (C, D), and triglycerides (E, F) after ingestion of 75g oral glucose (A, C, E) or a mixed test meal (B, D, F) in 17 patients with type 2 diabetes, 17 subjects with impaired glucose tolerance (IGT) and 14 subjects with normal glucose tolerance (NGT). Means ± SEM. Statistics were carried-out using repeated measures ANOVA and denote A: differences between the experiments, B: differences over time and AB: differences due to the interaction of experiment and time. Asterisks indicate significant (p < 0.05) differences at individual time points versus controls, daggers indicate significant differences versus patients with type 2 diabetes (one-way ANOVA). Figure 3. Plasma concentrations of GIP (A, B), and GLP-1 (C, D) after ingestion of 75g oral glucose (A, C) or a mixed test meal (B, D) in 17 patients with type 2 diabetes, 17 subjects with impaired glucose tolerance (IGT) and 14 subjects with normal glucose tolerance (NGT). Means ± SEM. Statistics were carried-out using repeated measures ANOVA and denote A: differences between the experiments, B: differences over time and AB: differences due to the interaction of experiment and time. Asterisks indicate significant (p < 0.05) differences at individual time points versus controls, daggers indicate significant differences versus patients with type 2 diabetes (one-way ANOVA). Figure 4. Integrated incremental plasma concentrations of GIP (A), and GLP-1 (B) after ingestion of 75g oral glucose (filled circles) and a mixed test meal (open diamonds) in 17 patients with type 2 diabetes, 17 subjects with impaired glucose tolerance and 14 healthy control subjects. Statistics were carried-out using paired repeated t-tests. Figure 5. Linear regression analysis between integrated incremental plasma concentrations of GLP-1 after ingestion of 75g oral glucose and fasting glucagon levels (A), decremental integrated glucagon levels (B), fasting free fatty acid levels (C), decremental free fatty acid levels (D), age (E) and body weight (F) in 17 patients with type 2 diabetes, 17 subjects with impaired glucose tolerance and 14 healthy control subjects. Dashed lines indicate the respective upper and lower 95 % confidence intervals. r = correleation coefficient.

16

FIGURE 1

0 60 120 180 2400

20

40

60

80

100

120 A: p = 0.34B: p < 0.0001AB: p = 0.044

†

*

Insu

lin [m

U/l]

0 60 120 180 2400

20

40

60

80

100

120 A: p = 0.12B: p < 0.0001AB: p = 0.09

Insu

lin [m

U/l]

Testmeal

0 60 120 180 24002468

10121416

A: p = 0.07B: p < 0.0001AB: p = 0.001†

**†

** †

**

†

**

Time [min]

C-p

eptid

e [n

g/m

l]

*

0 60 120 180 24002468

10121416 A: p = 0.066

B: p < 0.0001AB: p = 0.0032

*

†

*

†

Time [min]

C-p

eptid

e [n

g/m

l]

* *

A

DC

B

0 60 120 180 2400

100

200

300

400 A: p < 0.0001B: p < 0.0001AB: p < 0.0001

Plas

ma

gluc

ose

[mg/

dl] 75 g oral

glucose

* ***

***

*

**

††††

††

†

†

††

†*†**†

† †

††*

†††

*NGTIGTType 2 diabetes

0 60 120 180 2400

100

200

300

400A: p < 0.0001B: p < 0.0001AB: p < 0.0001

†*†**†

† †††

†† †

† ††††††

†††

* ******

** *

Testmeal

Plas

ma

gluc

ose

[mg/

dl]

FE

17

FIGURE 2

0 60 120 180 2404

6

8

10

12

14

16 NGTIGTType 2 diabetes

A: p = 0.38B: p < 0.0001AB: p = 0.098

75 g oralglucose

Glu

cago

n [p

mol

/l]

0 60 120 180 2404

6

8

10

12

14

16 A: p = 0.62B: p < 0.0001AB: p < 0.001

Glu

cago

n [p

mol

/l]

Test meal

0 60 120 180 2400

5

10

15

20

A: p = 0.66B: p < 0.0001AB: p < 0.000**

† *

†

*†*†

*†

*†

*

†† †

Free

fatty

aci

ds [m

g/dl

]

0 60 120 180 2400

5

10

15

20A: p = 0.87B: p < 0.0001AB: p < 0.001

Free

fatty

aci

ds [m

g/dl

]

0 60 120 180 2400

50

100

150

200

250

300

A: p = 0.08B: p = 0.06AB: p = 0.75

Time [min]

Tri

glyc

erid

es [m

g/dl

]

0 60 120 180 2400

50

100

150

200

250

300

A: p = 0.62B: p < 0.0001AB: p = 0.18

Time [min]

Tri

glyc

erid

es [m

g/dl

]

A

DC

B

E F

18

FIGURE 3

0 60 120 180 240

20

60

100

140

180NGTIGTType 2 diabetes

75 g oral glucose

A: p = 0.36B: p < 0.0001AB: p = 0.98

GIP

[pm

ol/l]

0 60 120 180 240

20

60

100

140

180 Test meal

A: p = 0.15B: p < 0.0001AB: p < 0.001

* ††*

†

*

GIP

[pm

ol/l]

†

0 60 120 180 2400

10

20

30

40

50

Time [min]

GL

P-1

[pm

ol/l]

A: p = 0.36B: p < 0.0001AB: p = 0.66

0 60 120 180 2400

10

20

30

40

50

Time [min]

GL

P-1

[pm

ol/l]

A: p = 0.46B: p < 0.0001AB: p = 0.73

A

DC

B

19

FIGURE 4

OGTT Meal0

10000

20000

30000

40000 p < 0.0001∫G

IP [p

mol

.l -1

. min

]

OGTT Meal0

2500

5000

7500

10000

12500

15000 p = 0.06

∫GL

P-1

[pm

ol .

l -1. m

in]

A B

20

FIGURE 5

0 5 10 15 200

2500

5000

7500

10000

12500 r = -0.49p < 0.001

Fasting glucagon[pmol/l]

∫GL

P-1 O

GTT

[pm

ol .

l -1. m

in]

-2000 -1500 -1000 -500 00

2500

5000

7500

10000

12500r = 0.57p < 0.0001

∫GlucagonOGTT

[pmol . l -1 . min]∫G

LP-

1 OG

TT [p

mol

.l -1

.m

in]

0 10 20 300

2500

5000

7500

10000

12500r = 0.39p = 0.006

Fasting free fatty acids[mg/dl]

∫GL

P-1 O

GTT

[pm

ol .

l -1 .

min

]

-4000 -2000 00

2500

5000

7500

10000

12500 r = -0.53p < 0.001

∫Free fatty acids OGTT

[mg . dl -1 . min]

∫GL

P-1 O

GTT

[pm

ol .

l -1 .

min

]

30 40 50 60 70 800

2500

5000

7500

10000

12500r = 0.4p = 0.0052

Age [years]

∫GL

P-1 O

GTT

[pm

ol .

l -1 .

min

]

40 60 80 100 120 1400

2500

5000

7500

10000

12500r = -0.35p = 0.16

Body weight [kg]

∫GL

P-1 O

GTT

[pm

ol .

l -1 .

min

]

A

DC

B

E F

21