Discussion
It has been controversial whether the impaired incretin effect observed in type 2 diabetes and obesity may result from a reduction in the release of incretin hormones and, if so, whether this reduction occurs prior to the development of type 2 diabetes and obesity. A general belief has been that impaired GLP-1 release in overt type 2 diabetes and obesity is a phenomenon secondary to other metabolic abnormalities. However, findings of reduced GLP-1 release in individuals with prediabetes have questioned this hypothesis, which is further supported by a high heritability of the GLP-1 response to oral glucose.
Our results, based on a large study population of 1,462 Danish adults, demonstrate that GLP-1 responses to an OGTT are up to 25% impaired in prediabetes and type 2 diabetes compared with NGT individuals and most pronounced in women compared with men. Furthermore, independent of glucose tolerance status, obese and overweight individuals had up to 20% impaired GLP-1 response compared with normal weight individuals. These findings indicate that alterations in incretin hormone release contribute to glucose and appetite dysregulation rather than being a consequence of type 2 diabetes or obesity. We also show that the early GLP-1 response was positively related to insulin sensitivity, β-cell function, and age but inversely associated with BMI and waist circumference in both sexes and that women in general had higher GLP-1 response than men.
Comparison With Other Studies
Our finding of a lower GLP-1 response in prediabetes and screen-detected type 2 diabetes in women is in agreement with previous studies of response to an OGTT in men and women with i-IGT, IFG&IGT, and type 2 diabetes. Differences in circulating GLP-1 levels were particularly observed after 120 min, which supports previous observations of reduced late GLP-1 response in diabetes and prediabetes. Among women, rAUC for GLP-1 was lower in prediabetes, but not in screen-detected diabetes, compared with the NGT group, which may suggest that a compensatory increase in GLP-1 release takes place with worsening of glucose tolerance above the prediabetic range. If this assumption is correct, the threshold for a compensatory increase in GLP-1 seems to be lower for men than for women, since a slight but not significant increase in rAUC for GLP-1 was observed in men with IFG&IGT but not in men with screen-detected diabetes. Such a compensatory mechanism could also explain why some studies find increased GLP-1 release in patients with newly diagnosed type 2 diabetes and others find reduced GLP-1 release in patients who are already in treatment for type 2 diabetes. A review and meta-analysis including a total of 400 individuals receiving oral glucose or a meal test showed that patients with type 2 diabetes and individuals without diabetes overall have similar GLP-1 responses. Another meta-analysis combining data from 22 trials during 29 different stimulation tests concluded the same. However, collecting data from smaller studies obtained by different stimulation tests performed on diverse patient and control groups with different distribution of sex, age, and BMI makes interpretations difficult. In addition, different analytical methods may also influence the results. We now demonstrate, in the largest study to date, that GLP-1 responses to oral glucose are affected by sex, glucose tolerance status, age, and BMI. This may explain the diverse results obtained in previously published studies, where a small number of study participants made such detailed analysis impossible.
BMI has been inversely associated with GLP-1 response in a number of studies. We have now confirmed in a large cohort of individuals with normal and impaired glucose regulation that obesity, independent of glucose tolerance, is an important determinant of GLP-1 release.
Potential Mechanisms
Glucose-stimulated GLP-1 levels were positively associated with first-phase insulin release, insulin sensitivity, and the DI. These findings are in agreement with the glucoregulatory effects of GLP-1 and with studies showing that infusion of the GLP-1 receptor antagonist exendin-(9–39) reduced first-phase insulin release and decreased insulin action in healthy individuals and in patients with type 2 diabetes. GLP-1 has also been shown to expand the microvascular surface area of skeletal muscles and may therefore improve the metabolic action of insulin. However, our finding that doubling of the GLP-1 response was associated with an up to 28% increase in the insulinogenic index and the DI, but only with a 4% increase in insulin sensitivity, underscores that the ability of GLP-1 to improve β-cell response is more predominant than the ability of GLP-1 to improve insulin sensitivity, which is in agreement with previous observations.
Some of the mechanisms behind the lower GLP-1 response in both prediabetes and overweight may include genetic variation in the regulation of GLP-1 synthesis, since twin studies have shown that GLP-1 response has a heritability of up to 67%. Lower GLP-1 response has also been observed in the twin with diabetes of twin pairs discordant for type 2 diabetes. Finally, the L cells may be sensitive to insulin resistance, since weight loss, and thereby reduction of insulin resistance, induces a marked increase in GLP-1 release.
Role of Sex and Age
Sex differences in the secretion of GLP-1 have not been studied in detail because of small and selected study populations. As women have lower fasting plasma glucose concentrations, higher 2-h plasma glucose concentrations, slower gastric emptying and glucose absorption rates, and higher insulin sensitivity than men, we chose to analyze our study population stratified by sex. Indeed, we found that the lower GLP-1 response in prediabetes and type 2 diabetes was most pronounced in women. However, this finding was partially due to the result of ~20% higher relative GLP-1 response in normoglycemic women than in their male counterparts, whereas the GLP-1 response in men and women with prediabetes or type 2 diabetes was comparable in absolute terms. Therefore, our results suggest that healthy women have a higher GLP-1 response than men, but when glucose tolerance is worsening, this sex difference is no longer apparent.
The role of age in the release of GLP-1 has not previously been clearly established. Our finding of a significantly positive association between age and GLP-1 response in the entire study population may reflect a general reduced renal clearance with increasing age, thereby increasing the GLP-1 concentration. It should be noted, however, that the effect size was rather small; a 5-year increase in age was associated with a 3% increase in GLP-1 response.
Study Strengths and Limitations
The main strength of this study is the large cohort with NGT, prediabetes, and screen-detected type 2 diabetes. We had the opportunity to examine individuals with screen-detected/epidemiological and, hence, untreated diabetes, which is not possible in studies based on clinically known diabetes cases. It is well documented that prediabetes, and especially IFG&IGT, is associated with a high risk for developing type 2 diabetes, and therefore we conclude that the changes in GLP-1 response observed in individuals with i-IFG, i-IGT, and IFG&IGT precede the development of overt type 2 diabetes, at least in women. Our finding of a relationship of GLP-1 release with insulin sensitivity, β-cell function, BMI, and waist circumference supports this notion. It should also be mentioned that any reduced GLP-1 response observed in our study population can reflect either an impaired release or an increased elimination of GLP-1. However, as other studies have found similar elimination rates and gastric emptying rates of GLP-1 in individuals with and without diabetes, our findings of reduced GLP-1 response in prediabetes and diabetes are likely to reflect an impaired release of GLP-1.
The GLP-1 response to 75-g oral glucose may be considered less physiologically relevant than a mixed-meal test. However, it is more difficult to standardize a mixed-meal test than an OGTT, and differences in GLP-1 response between individuals with and without diabetes seem to be more pronounced after a solid meal compared with an OGTT. Accordingly, confirmation of our findings in large studies using mixed-meal tests is important in terms of generalizability and application of the results to clinical practice.
We used a well-documented assay method with high specificity and sensitivity for analyzing the plasma samples. Many studies of GLP-1 secretion have been carried out with various commercially available kits, which have considerable variation in specificity and sensitivity. This should be taken into account for assay selection and when comparing data from different studies. Another important aspect of the measurement method is that we measured total GLP-1 and not active (intact) GLP-1. The reason for choosing this method is that measurement of total GLP-1 provides information not only about the release of GLP-1 but also about its probable biological effects. In contrast, measurement of peripheral circulating levels of the intact GLP-1 metabolite [GLP-1-(9–36)NH2/37] only reflects the small fraction of the hormone that reaches its targets via the classical endocrine route (possibly as little as 8% of what was released) after having exerted part of its action via the nervous system. Due to the large number of study participants, samples were only collected at three time points during the OGTT. However, since the response of GLP-1 to oral glucose has been shown to peak around 30 min independent of the degree of dysglycemia the estimated AUCs are likely to include the peak GLP-1 response.
Some individuals have lower plasma GLP-1 levels after 120 min than in the fasting state. Accordingly, calculation of the incremental AUC will result in negative numbers and therefore be missing after log transformation. In our data set, 5% of data would be missing if the incremental AUC were used instead of the relative AUC for GLP-1, and most of the missing variables were in the smaller groups with prediabetes and diabetes. The relative and incremental AUCs express the same (the change in GLP-1 release from baseline) but on a different scale (relative vs. absolute).
Conclusions and Perspectives
In the largest study population analyzed to date, comprising 1,462 individuals, we demonstrate that GLP-1 response to an OGTT is up to 25% impaired in prediabetes and screen-detected diabetes compared with NGT and most pronounced in women compared with men. Furthermore, independent of glucose tolerance status, obese and overweight individuals had up to 20% impaired GLP-1 response compared with normal weight individuals. Thus, we now show that a reduction in GLP-1 release occurs already before diabetes or obesity is manifest. This finding indicates that alterations in GLP-1 release contribute to dysregulation of glucose metabolism and appetite rather than being a consequence of type 2 diabetes or obesity.
Treatment with GLP-1 analogs has been shown to induce weight loss and reverse prediabetes to NGT. Since we found reduced endogenous GLP-1 levels and response in prediabetes, treatment with GLP-1 analogs could be relevant as part of a prevention strategy if weight loss attempts are unsuccessful, in agreement with recent findings from studies of obese individuals with prediabetes.