Jeppe Sturis

Small-molecule agonists for the glucagon-like peptide 1 receptor.

The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists.

Estimation of Insulin Secretion Rates from C-Peptide Levels: Comparison of Individual and Standard Kinetic Parameters for C-Peptide Clearance

Insulin secretion rates can be accurately estimated from plasma C-peptide levels with a two-compartment model for C-peptide distribution and degradation. In previous studies, the kinetic parameters of C-peptide clearance were derived in each subject from the decay curve observed after bolus intravenous injection of biosynthetic human C-peptide. To determine whether standard parameters for C-peptide clearance could be defined and used to calculate insulin secretion without obtaining a decay curve in each subject, we analyzed 200 decay curves of biosynthetic human C-peptide obtained in normal, obese, and non-insulindependent diabetes mellitus subjects studied in ourlaboratory. This analysis showed that the volume of distribution and kinetic parameters of C-peptide distribution and metabolism vary by <30% in a population highly heterogenous in terms of age, sex, degree of obesity, and degree of glucose tolerance. The volume of distribution correlated with the degree of obesity as quantified by body surface area (BSA). This dependence of C-peptide distribution volume on BSA was more marked in men than in women. The long half-life was slightly longer in elderly subjects than in younger adults. When effects of BSA, sex, and age were taken into account, the parameters of C-peptide kinetics were very similar in normal, obese, and diabetic subjects. Based on these findings, a simple procedure to derive standard parameters for C-peptide clearance taking into account degree of obesity, sex, and age was defined. These standard parameters resulted in estimations of mean insulin secretion rates, which differed in each subject by only 10-12% from those obtained with individual parameters. The approach of using standard ratherthan individual parameters did not systematically underestimate or overestimate insulin secretion so that group values for the fasting secretion rate, the mean 24-h secretion rate, and the number and the amplitude of secretory pulses obtained with standard parameters differed by only 1–2% from the values obtained with individual parameters. Furthermore, the accuracy of measurements based on standard parameters was not different from that associated with replicate determinations of the parameters of C-peptide clearance in the same subject. We conclude that it is possible to estimate insulin secretion rates from plasma C-peptide levels with standard parameters for C-peptide clearance rather than individually derived parameters without significant loss of accuracy.

One Week’s Treatment With the Long-Acting Glucagon-Like Peptide 1 Derivative Liraglutide (NN2211) Markedly Improves 24-h Glycemia and α- and β-Cell Function and Reduces Endogenous Glucose Release in Patients with Type 2 Diabetes

Glucagon-like peptide 1 (GLP-1) is potentially a very attractive agent for treating type 2 diabetes. We explored the effect of short-term (1 week) treatment with a GLP-1 derivative, liraglutide (NN2211), on 24-h dynamics in glycemia and circulating free fatty acids, islet cell hormone profiles, and gastric emptying during meals using acetaminophen. Furthermore, fasting endogenous glucose release and gluconeogenesis (3-(3)H-glucose infusion and (2)H(2)O ingestion, respectively) were determined, and aspects of pancreatic islet cell function were elucidated on the subsequent day using homeostasis model assessment and first- and second-phase insulin response during a hyperglycemic clamp (plasma glucose approximately 16 mmol/l), and, finally, on top of hyperglycemia, an arginine stimulation test was performed. For accomplishing this, 13 patients with type 2 diabetes were examined in a double-blind, placebo-controlled crossover design. Liraglutide (6 micro g/kg) was administered subcutaneously once daily. Liraglutide significantly reduced the 24-h area under the curve for glucose (P = 0.01) and glucagon (P = 0.04), whereas the area under the curve for circulating free fatty acids was unaltered. Twenty-four-hour insulin secretion rates as assessed by deconvolution of serum C-peptide concentrations were unchanged, indicating a relative increase. Gastric emptying was not influenced at the dose of liraglutide used. Fasting endogenous glucose release was decreased (P = 0.04) as a result of a reduced glycogenolysis (P = 0.01), whereas gluconeogenesis was unaltered. First-phase insulin response and the insulin response to an arginine stimulation test with the presence of hyperglycemia were markedly increased (P < 0.001), whereas the proinsulin/insulin ratio fell (P = 0.001). The disposition index (peak insulin concentration after intravenous bolus of glucose multiplied by insulin sensitivity as assessed by homeostasis model assessment) almost doubled during liraglutide treatment (P < 0.01). Both during hyperglycemia per se and after arginine exposure, the glucagon responses were reduced during liraglutide administration (P < 0.01 and P = 0.01). Thus, 1 weeks treatment with a single daily dose of the GLP-1 derivative liraglutide, operating through several different mechanisms including an ameliorated pancreatic islet cell function in individuals with type 2 diabetes, improves glycemic control throughout 24 h of daily living, i.e., prandial and nocturnal periods. This study further emphasizes GLP-1 and its derivatives as a promising novel concept for treatment of type 2 diabetes.

Insulin secretory defects in polycystic ovary syndrome. Relationship to insulin sensitivity and family history of non-insulin-dependent diabetes mellitus.

The increased prevalence of non-insulin-dependent diabetes mellitus (NIDDM) among women with polycystic ovary syndrome (PCOS) has been ascribed to the insulin resistance characteristic of PCOS. This study was undertaken to determine the role of defects in insulin secretion as well as familial factors to the predisposition to NIDDM seen in PCOS. We studied three groups of women: PCOS with a family history of NIDDM (PCOS FHx POS; n = 11), PCOS without a family history of NIDDM (PCOS FHx NEG; n = 13), and women without PCOS who have a family history of NIDDM (NON-PCOS FHx POS; n = 8). Beta cell function was evaluated during a frequently sampled intravenous glucose tolerance test, by a low dose graded glucose infusion, and by the ability of the beta cell to be entrained by an oscillatory glucose infusion. PCOS FHx POS women were significantly less likely to demonstrate appropriate beta cell compensation for the degree of insulin resistance. The ability of the beta cell to entrain, as judged by the spectral power for insulin secretion rate, was significantly reduced in PCOS FHx POS subjects. In conclusion, a history of NIDDM in a first-degree relative appears to define a subset of PCOS subjects with a greater prevalence of insulin secretory defects. The risk of developing NIDDM imparted by insulin resistance in PCOS may be enhanced by these defects in insulin secretion.

Evolution of β-Cell Dysfunction in the Male Zucker Diabetic Fatty Rat

The molecular basis for the β-cell dysfunction that characterizes non-insulin-dependent diabetes mellitus (NIDDM) is unknown. The Zucker diabetic fatty (ZDF) male rat is a rodent model of NIDDM with a predictable progression from the prediabetic to the diabetic state. We are using this model to study β-cell function during the development of diabetes with the goal of identifying genes that play a key role in regulating insulin secretion and, thus, may be potential targets for therapeutic intervention aimed at preserving or improving β-cell function. As a first step, we have characterized morphology, insulin secretion, and pattern of gene expression in islets from prediabetic and diabetic ZDF rats. The development of diabetes was associated with changes in islet morphology, and the islets of diabetic animals were markedly hypertrophic with multiple irregular projections into the surrounding exocrine pancreas. In addition, there were multiple defects in the normal pattern of insulin secretion. The islets of prediabetic ZDF rats secreted significantly more insulin at each glucose concentration tested and showed a leftward shift in the dose-response curve relating glucose concentration and insulin secretion. Islets of prediabetic animals also demonstrated defects in the normal oscillatory pattern of insulin secretion, indicating the presence of impairment of the normal feedback control between glucose and insulin secretion. The islets from diabetic animals showed further impairment in the ability to respond to a glucose stimulus. Changes in gene expression were also evident in islets from prediabetic and diabetic ZDF rats compared with age-matched control animals. In prediabetic animals, there was no change in insulin mRNA levels. However, there was a significant 30–70% reduction in the levels of a large number of other islet mRNAs including glucokinase, mitochondrial glycerol-3-phosphate dehydrogenase, voltage-dependent Ca2+ and K+ channels, Ca2+-ATPase, and transcription factor Islet-1 mRNAs. In addition, there was a 40–50% increase in the levels of glucose-6-phosphatase and 12-lipoxygenase mRNAs. There were further changes in gene expression in the islets from diabetic ZDF rats, including a decrease in insulin mRNA levels that was associated with reduced islet insulin levels. Our results indicate that multiple defects in β-cell function can be detected in islets of prediabetic animals well before the development of hyperglycemia and suggest that changes in the normal pattern of gene expression contribute to the development of β-cell dysfunction.

Insulin secretory abnormalities in subjects with hyperglycemia due to glucokinase mutations.

Pancreatic beta-cell function was studied in six subjects with mutations in the enzyme glucokinase (GCK) who were found to have elevated fasting and postprandial glucose levels in comparison to six normoglycemic controls. Insulin secretion rates (ISRs) were estimated by deconvolution of peripheral C-peptide values using a two-compartment model and individual C-peptide kinetics obtained after bolus intravenous injections of biosynthetic human C-peptide. First-phase insulin secretory responses to intravenous glucose and insulin secretion rates over a 24-h period on a weight maintenance diet were not different in subjects with GCK mutations and controls. However, the dose-response curve relating glucose and ISR obtained during graded intravenous glucose infusions was shifted to the right in the subjects with GCK mutations and average ISRs over a glucose range between 5 and 9 mM were 61% lower than those in controls. In the controls, the beta cell was most sensitive to an increase in glucose at concentrations between 5.5 and 6.0 mM, whereas in the patients with GCK mutations the point of maximal responsiveness was increased to between 6.5 and 7.5 mM. Even mutations that resulted in mild impairment of in vitro enzyme activity were associated with a > 50% reduction in ISR. The responsiveness of the beta cell to glucose was increased by 45% in the subjects with mutations after a 42-h intravenous glucose infusion at a rate of 4-6 mg/kg per min. During oscillatory glucose infusion with a period of 144 min, profiles from the subjects with mutations revealed reduced spectral power at 144 min for glucose and ISR compared with controls, indicating decreased ability to entrain the beta cell with exogenous glucose. In conclusion, subjects with mutations in GCK demonstrate decreased responsiveness of the beta cell to glucose manifest by a shift in the glucose ISR dose-response curve to the right and reduced ability to entrain the ultradian oscillations of insulin secretion with exogenous glucose. These results support a key role for the enzyme GCK in determining the in vivo glucose/ISR dose-response relationships and define the alterations in beta-cell responsiveness that occur in subjects with GCK mutations.

Altered Insulin Secretory Responses to Glucose in Diabetic and Nondiabetic Subjects With Mutations in the Diabetes Susceptibility Gene MODY3 on Chromosome 12

One form of maturity-onset diabetes of the young (MODY) results from mutations in a gene, designated MODY3, located on chromosome 12 in band q24. The present study was undertaken to define the interactions between glucose and insulin secretion rate (ISR) in subjects with mutations in MODY3. Of the 13 MODY3 subjects, six subjects with normal fasting glucose and glycosylated hemoglobin and seven overtly diabetic subjects were studied as were six nondiabetic control subjects. Each subject received graded intravenous glucose infusions on two occasions separated by a 42-h continuous intravenous glucose infusion designed to prime the β-cell to secrete more insulin in response to glucose. ISRs were derived by deconvolution of peripheral C-peptide levels. Basal glucose levels were higher and insulin levels were lower in MODY3 subjects with diabetes compared with nondiabetic subjects or with normal healthy control subjects. In response to the graded glucose infusion, ISRs were significantly lower in the diabetic subjects over a broad range of glucose concentrations. ISRs in the nondiabetic MODY3 subjects were not significantly different from those of the control subjects at plasma glucose levels <8 mmol/l. As glucose rose above this level, however, the increase in insulin secretion in these subjects was significantly reduced. Administration of glucose by intravenous infusion for 42 h resulted in a significant increase in the amount of insulin secreted over the 5–9 mmol/l glucose concentration range in the control subjects and nondiabetic MODY3 subjects (by 38 and 35%, respectively), but no significant change was observed in the diabetic MODY3 subjects. In conclusion, in nondiabetic MODY3 subjects insulin secretion demonstrates a diminished ability to respond when blood glucose exceeds 8 mmol/l. The priming effect of glucose on insulin secretion is preserved. Thus, β-cell dysfunction is present before the onset of overt hyperglycemia in this form of MODY. The defect in insulin secretion in the nondiabetic MODY3 subjects differs from that reported previously in nondiabetic MODY1 or mildly diabetic MODY2 subjects.

GLP-1 derivative liraglutide in rats with β-cell deficiencies: influence of metabolic state on β-cell mass dynamics

Liraglutide is a long‐acting GLP‐1 derivative, designed for once daily administration in type II diabetic patients. To investigate the effects of liraglutide on glycemic control and β‐cell mass in rat models of β‐cell deficiencies, studies were performed in male Zucker diabetic fatty (ZDF) rats and in 60% pancreatectomized rats. When liraglutide was dosed s.c. at 150 μg kg−1 b.i.d. for 6 weeks in ZDF rats 6–8 weeks of age at study start, diabetes development was markedly attenuated. Blood glucose was approximately 12 mM lower compared to vehicle (P<0.0002), and plasma insulin was 2–3‐fold higher during a normal 24‐h feeding period (P<0.001). Judged by pair feeding, approximately 53% of the antihyperglycemic effect observed on 24‐h glucose profiles was mediated by a reduction in food intake, which persisted throughout the study and averaged 16% (P<0.02). Histological analyses revealed that β‐cell mass and proliferation were significantly lower in prediabetic animals still normoglycemic after 2 weeks treatment compared to vehicle‐treated animals that had begun to develop diabetes. When the treatment period was 6 weeks, the liraglutide‐treated animals were no longer completely normoglycemic and the β‐cell mass was significantly increased compared to overtly diabetic vehicle‐treated animals, while β‐cell proliferation was unaffected. In the experiments with 60% pancreatectomized rats, 8 days treatment with liraglutide resulted in a significantly lower glucose excursion in response to oral glucose compared to vehicle treatment. Again, part of the antihyperglycemic effect was due to reduced food intake. No effect of liraglutide on β‐cell mass was observed in these virtually normoglycemic animals. In conclusion, treatment with liraglutide has marked antihyperglycemic effects in rodent models of β‐cell deficiencies, and the in vivo effect of liraglutide on β‐cell mass may in part depend on the metabolic state of the animals.

Defects in Insulin Secretion and Action in Women With a History of Gestational Diabetes

Gestational diabetes mellitus (GDM) is associated with defects in insulin secretion and insulin action, and women with a history of GDM carry a high risk for the development of non-insulin-dependent diabetes mellitus (NIDDM). Assessment of subjects with a history of GDM who are currently normoglycemic should help elucidate some of the underlying defects in insulin secretion or action in the evolution of NIDDM. We have studied 14 women with normal oral glucose tolerance who had a history of GDM. They were compared with a group of control subjects who were matched for both body mass index (BMI) and waist-to-hip ratio (WHR). All subjects underwent tests for the determination of oral glucose tolerance, ultradian oscillations in insulin secretion during a 28-h glucose infusion, insulin secretion in response to intravenous glucose, glucose disappearance after intravenous glucose (Kg), and insulin sensitivity (SI) as measured by the Bergman minimal model method. The BMI in the post-GDM women was similar to that in the control subjects (24.9 ± 1.2 vs. 25.4 ± 1.4 kg/m2, respectively), as was the WHR ratio (0.80 ± 0.01 vs. 0.76 ± 0.01, respectively). The post-GDM women were slightly older (35.2 ± 0.9 vs. 32.1 ± 1.4 years, P = 0.04). The fasting plasma glucose levels were significantly higher in the post-GDM group than in the control group (4.9 ± 0.1 vs. 4.4 ± 0.1 mmol/l, respectively, P < 0.001) and remained higher at each of the subsequent determinations during the oral glucose tolerance test, although none had a result indicative of either diabetes or impaired glucose tolerance. All measures of ultradian insulin secretory oscillations in post-GDM subjects were indistinguishable from those in the control subjects. The first-phase insulin release to intravenous glucose was lower in the post-GDM group. SI was also impaired in the post-GDM group compared with the control subjects (4.6 ± 0.5 vs. 6.8 ± 1.0·10−4·min−1· ³U−1·ml, respectively, P < 0.05). Kg was reduced in the post-GDM women compared with the control subjects (1.3 ± 0.1 vs. 2.7 ± 0.4%, P < 0.01). When the subjects were divided according to their BMI, lean post-GDM subjects (<24.2, n = 8) were more insulin resistant than the lean control subjects: SI 5.3 ± 0.6 vs. 8.8 ± 1 · 1·10−4 min −1· ³U−1·ml, P = 0.02, whereas obese post-GDM (>24.2 kg/m2, n = 6) and control subjects had a lower SI than the lean subjects, but they were not different from each other (3.6 ± 0.7 vs. 4.2 ± 1.2· 10−4· min−1 · ³U−1 · ml, respectively, P = 0.67). The acute insulin responses to glucose (0–10 min) within these groups showed that the lean post-GDM group had a significantly lower insulin response compared with control subjects (1,205 ± 179 vs. 2,404 ± 416 pmol· 1−1 min, respectively, P = 0.007), whereas the obese groups had similar responses (2,777 ± 1,112 vs. 3,114 ± 847 pmol ·1−·min, post-GDM vs. control subjects, P = 0.8). We have found defects in insulin secretion and action in post-GDM subjects who are at high risk for the development of NIDDM at a time that oral glucose tolerance is normal. These defects are present in the absence of obesity. Ultradian insulin secretory oscillations during constant glucose infusion are normal in these post-GDM subjects predisposed to NIDDM. We conclude that defects in both insulin secretion and insulin action are present before the development of hyperglycemia in women with a history of GDM.

Altered Insulin Secretory Responses to Glucose in Subjects with a Mutation in the MODY1 Gene on Chromosome 20

This study was undertaken to test the hypothesis that the diabetes susceptibility gene on chromosome 20q12 responsible for maturity-onset diabetes of the young (MODY) in a large kindred, the RW family, results in characteristic alterations in the dose-response relationships between plasma glucose concentration and insulin secretion rate (ISR) that differentiate this form of MODY from MODY in subjects with glucokinase mutations. Ten marker-positive subjects and six matched nondiabetic marker-negative subjects from the RW family received graded intravenous glucose infusions on two occasions separated by a 42-h continuous intravenous glucose infusion designed to prime the β-cell to secrete more insulin in response to glucose. ISR was derived by deconvolution of peripheral C-peptide levels. Basal glucose and insulin levels were similar in marker-negative and marker-positive groups (5.3 ± 0.2 vs. 5.0 ± 0.2 mmol/l, P > 0.2, and 86.1 ± 3.9 vs. 63.7 ± 12.1 pmol/l, P > 0.1, respectively). However, the marker-positive subjects had defective insulin secretory responses to an increase in plasma glucose concentrations. Thus, as the glucose concentration was raised above 7 mmol/l, the slope of the curve relating glucose and ISR was significantly blunted in the marker-positive subjects (13 ± 4 vs. 68 ± 8 pmol · min−1 · mmol−1 · 1, P < 0.0001). The reduced insulin secretory responses in the marker-positive subjects were most evident at higher plasma glucose concentrations >7 mmol/1, and differences between the two groups were not significant at lower glucose levels between 5 and 7 mmol/1. After a 42-h glucose infusion, the amount of insulin secreted over the glucose concentration range 5–9 mmol/1 increased by 54 ± 16% in the markernegative subjects. This priming effect of glucose on insulin secretion was not seen in 9 of the 10 markerpositive subjects. In contrast, previous results in MODY subjects with glucokinase mutations showed persistence of the glucose-priming effect on ISR and continued increases, although subnormal, of ISR as plasma glucose concentration rises from 7–12 mmol/1. In conclusion, subjects from the RW family who have inherited the at-risk allele of the MODY1 gene appear to have a characteristic pattern of altered insulin secretory responses to glucose. These alterations are present before the onset of hyperglycemia, suggesting a unique mechanism of (β-cell dysfunction different from the defect in MODY subjects with glucokinase mutations.