Introduction
In this thesis I have evaluated the postprandial effects on glucose metabolism of a GLP-1 receptor analogue (Exenatide) in subjects with increased postprandial hyperglycemia (ie IGT or newly diagnosed diabetes at screening OGTT). By using tracers infusion combined with Positron Emission Tomography (PET) and 18F-2- fluoro-2-deoxy-D-glucose (FDG), I have evaluated the effect of a single injection of Exeatide vs Placebo on hepatic glucose production, oral glucose absorption, peripheral glucose clearance as well as organ metabolism, eg postprandial brain and liver glucose metabolism.
Diabetes mellitus is a common condition and a serious global health problem and it is important to understand the pathophysiology of this disease in order to prevent the onset of disease as well as further associated complications. Over 382 million people worldwide are estimated to have diabetes that represents about 8.3% of adults (1).
This trend demonstrates that by 2035, some 592 million people, or one adult in 10, will have diabetes. It is demonstrated that diabetes is the leading cause of cardiovascular diseases (angina, myocardial infarction, stroke, peripheral artery disease, and congestive heart failure), blindness, kidney failure, and lower-limb amputation strictly associated and caused by peripheral neuropathy. IDF estimates that as many as 175 million people worldwide, or close to half of all people with diabetes, are unaware of their disease (1). Most of these cases are type 2 diabetes and many studies have found that people with undiagnosed diabetes already have complications, such as chronic kidney disease and heart failure, retinopathy and neuropathy (2-4).
Prediabetes is a condition of high-risk for diabetes and is defined by impaired glucose tolerance (IGT), along with impaired fasting glucose (IFG), that are intermediate states in the transition in glucose tolerance from normal to overt diabetes. IFG was originally introduced by the American Diabetes Association to be analogous to IGT (5). IFG is defined by a fasting glucose between 100 and 126 mg/dl (5.6 and 7.0 mmol/l) while IGT is detected as a postprandial glucose level between 140 and 200 mg/dl (7.8 and 11.1 mmol/l) after a standard glucose load of 75 g oral glucose tolerance testing (OGTT). The future risk of type 2 diabetes associated with IFG and IGT is additive. Thus, the risk of type 2 diabetes in subjects with IFG plus IGT is twofold greater compared with subjects with either state alone (6-11). Furthermore,
(12) IFG and IGT are associated with a modestly increased risk of cardiovascular disease.
Among the causes of impaired glucose tolerance there are increased insulin resistance and impaired insulin secretion (13). Moreover subjects with prediabetes have an impaired response of splanchnic hormones after meal ingestion, ie glucagon like peptide 1 (GLP-1) and GIP (14-18). Several clinical trials involed prediabetic individuals have been conducted to prevent or delay the overt type 2 diabetes either employing lifestyle intervention (19-25) or antihyperglycemic treatment (21,26-28).
GLP-1 receptor analogues are a new class of antihyperglycemic agents that have been shown to be effective in treating diabetic hyperglycemia.After discovering GLP-1 and GLP-1-receptors in the brain (29), subsequent studies have shown metabolic effects of GLP-1 intracerebroventricular administration (30). A recent study (31) employing 15O-water Positron Emission Tomography (PET), showed that the postprandial GLP-1 response is associated with activation of the left dorsolateral prefrontal cortex and the hypothalamus. PET in combination with FDG is considered the gold-standard method in the quantification of regional brain glucose uptake. The effect of GLP-1 on brain glucose uptake across the blood brain barrier was recently investigated using this method (32). The results of the study indicated a neuroprotective effect of GLP-1 in maintaining glucose balance and limiting glucose transport across the blood brain barrier. Another study reported that intravenous GLP- 1 infusion in non-diabetic male subjects under conditions of hyperglycemic pancreatic-pituitary clamp, the intracerebral glucose concentration was reduced significantly in multiple brain regions although the glucose clearance across the blood brain barrier, net glucose clearance and cerebral glucose metabolic rate were increased (33).
These studies although interesting, do not answer important questions, such as the effect of GLP-1 on cerebral glucose metabolism in postprandial state since GLP-1 is secreted in response to a meal while previously subjects were studied under euglycemic or hyperglycemic condition obtained with hyperinsulinemic or pancreatic clamp with the suppression of counter-regolatory hormonal secretion by somatostatin.
Thus the primary aim of this study was to measure brain glucose metabolism during an oral glucose load in subjects at risk of developing T2DM using [18F]FDG-PET.
There have been increasing experimental and clinical evidences suggesting that hypothalamic dysregulation may be one of the underlying mechanisms of abnormal
peripheral glucose metabolism. It has been recently shown that the intravenous continuous infusion of Exenatide in a group of obese non-diabetic subjects, was associated to a increase of functional hypothalamic connectivity (studied with fMRI) with the rest of the areas involved in the glucose homeostasis (34). Therefore the second aim of this study was to evaluate the hypothalamic glucose metabolism in postprandial state and its relation with peripheral glucose metabolism.
It has been suggested that brain GLP-1 signalling mediates peripheral glucose metabolism (35). Recently it has been shown that physiological manifestations of the GLP-1 incretin effect are potentiation of insulin secretion and glucose sensing(36), with a positive relationship between the appearance of oral glucose in peripheral blood and the release of GLP-1(36).
However, the above experiments did not allow to distinguish whether GLP-1 acts on glucose absorption or hepatic extraction. Thus, the third aim of the study was to relate changes in cerebral glucose metabolism with other effects of Exenatide injection, such as postprandial changes in glucose metabolism and in insulin and glucagon secretion. By using glucose stable isotope tracers we measured hepatic glucose production and extraction, oral glucose absorption and peripheral clearance. Hepatic glucose extraction, glucose production and oral absorption, peripheral clearance were assessed by coinfusing stable isotopes and [18F]FDG using a method recently developed (37). Finally we explored if changes in cerebral and peripheral glucose metabolism induced by Exenatide are partly explained by reduction in whole body lipolysis.
The thesis is organized in six chapters. In the first chapter I described the metabolic abnormalities that characterize the large spectrum that leads a normal glucose tolerance individuals to develop prediabetes and then overt type 2 diabetes. In the second chapter I evaluated the postprandial effects of a single Exenatide administration in prediabetic individuals following a standard oral glucose load. In the third chapter the role of the brain in the glucose homeostasis is described highlighting the importance of the gut-to-brain GLP-1 dependent axis and its role in the regulation of food intake and energy expenditure. In the forth chapter the Positron Emission Tomography (PET) technique is fully examined including a detailed description of the methods used to process and analyze PET images obtained during this study. In the fifth chapter I have evaluated the effects of a single Exenatide administration on postprandial cerebral glucose metabolism in prediabetic individuals. In the last
chapter I have examine the role of Exenatide in the hepatic glucose metabolism suggesting a cross-talk hypothesis between the liver and the brain. An appendix is included to provide a complete description of the study design and methods used during the study.
This intervention study was carried out at the University of Texas, Health Science Center, Research Imaging Institute at San Antonio, TX, USA.
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Gastroenterology. 2007 Feb;132(2):531-42
Chapter 1
Metabolic abnormalities from prediabetes toward overt type 2 diabetes
1.1 Introduction
The glucose system is highly homeostatic and in normal glucose tolerance state, plasma glucose concentration fluctuations rarely exceed 54 mg/dl. As the plasma glucose concentration represent a tight balance between the glucose entry into and the exit from the circulation via tissue metabolism or excretion, any excessive release and/or defective removal will result in increasing glucose levels. The knowledge of the mechanisms that underlie prediabetes development provides an extremely useful tool for improving a pharmacological treatment approach focused on T2DM prevention. Insulin resistance is a core defect in both prediabetes and T2DM (1-4) and involves several organs as liver (5) muscle (6), and adipose tissue(7)and it precedes the development of glucose intolerance and overt T2DM (8-10). IFG individuals have moderate hepatic insulin resistance and impaired early insulin response (0–30 min) during the OGTT (11-12). As such, they are characterized by elevated FPG and an excessive early rise (0–60 min) in PG during OGTT (13). However, because the late plasma insulin response (60–120 min) is intact and muscle insulin sensitivity is normal/near-normal, the 2-h PG returns to its starting FPG level (14). In contrast, IGT individuals have moderate-severe muscle insulin resistance and impaired early (0–30 min) and late (60–120 min) plasma insulin responses during the OGTT (14). Thus, although the FPG is not elevated, there is a progressive, sustained rise in PG during the OGTT and the 2-h PG remains well above the FPG level. Impaired first phase insulin secretion is characteristic of both IGT and IFG. However, they are distinguished by intact second phase insulin secretion in IFG and the tissues (liver in IFG vs. muscle in IGT) responsible for insulin resistance. In both states skeletal muscle is the main responsible for impaired insulin-mediated glucose uptake (it typically represents 40% if body weight (15). However, adipose tissue has a significant contribution to whole body glucose disposal (16) especially because adipocytes insulin resistance directly causes an excessive NEFA release into the
bloodstream that directly generate their preferential uptake over that of circulating glucose to insulin target tissue (Randle cycle) (17).
1.2 The role of β-cell dysfunction and insulin resistance
Although insulin resistance is a cardinal feature of IGT, ultimately, β-cell failure is responsible for the development of IGT and its progression to T2DM (18-19). In all ethnic groups the low plasma insulin response during the OGTT associated with the impairment in first phase insulin secretion (0-10 minute time period following an intravenous glucose challenge) are strong predictors of the progression from IGT to T2DM (20-21). When the FPG exceeds 90 mg/dl, first phase insulin secretion begins to deteriorate, and, when the FPG exceeds 110 mg/dl, first phase insulin secretion is almost completely lost (22). Genetic and acquired factors [glucotoxicity (23), lipotoxicity (24), incretin deficiency/resistance (25-27)] play a major role in the progressive deterioration of β-cell function. Individuals with IGT are maximally/near maximally insulin resistant and have lost 70–80% of β-cell function and even a small further decline in insulin secretion will result in a marked increase in fasting/postprandial blood glucose levels. Therefore, any intervention effective in retard/prevent β-cell failure will result in reverse/delay of the progression from IGT to T2DM.
1.3 The role of Incretin System
The incretin effect is characterized by a 2-3 fold increase in plasma insulin response following a glucose ingestion as compared to the same level of hyperglycemia obtained by an intravenous glucose administration (28-29). Together, GLP-1, released by L cells, and glucose-dependent insulinotropic polypeptide (GIP), released by K cells, account for 90% of the incretin effect (28-29). Both GLP-1 and GIP are potent insulin secretagogues. In addition to augmenting insulin secretion, GLP-1 also inhibits glucagon secretion, delays gastric emptying, and promotes weight loss by suppressing appetite and decreasing food intake (28,30).Because both GLP-1 and GIP are rapidly cleaved (half-life = 1–2 min) by dipeptidyl peptidase-IV, both peptides are inappropriate for therapeutic use in T2DM and IGT subjects. Liraglutide and exenatide are GLP-1 receptor agonists (GLP-1rA) that mimic the actions of GLP-
1 and are resistant to dipeptidyl peptidase-IV degradation (31-32). Similar to endogenous GLP-1, both agonists are potent insulin secretagogues (28-29)inhibit glucagon secretion, promote weight loss and effectively reduce plasma glucose levels in T2DM (29). In a long-term (3 years) extension study, exenatide produced a durable HbA1c reduction, improved β-cell function, and caused progressive weight loss in type 2 diabetic individuals(33).Hypoglycemia is uncommon with GLP-1 analogues because they only augment insulin secretion in the presence of hyperglycemia. Both GLP-1 and GIP mediate their β-cell effects via a mechanism that is completely distinct from hyperglycemia. Following binding to their respective receptors, GLP-1 and GIP activate adenylate cyclase which convert ATP to cAMP, which, in turn,
“amplifies” an insulin secretory system that has been activated by hyperglycemia (34). In the absence of hyperglycemia, neither GLP-1 nor GIP increases insulin secretion.
Subjects with IGT and T2DM are characterized by severely impaired β-cell function (35-36) and markedly reduced incretin effect in response to meal/glucose ingestion (29).Conversely most studies have reported no change or slightly impaired total GLP- 1 response to a mixed meal in IGT (25,37-38) although the early (0–10 min) GLP-1 response is diminished, indicating a phasic defect in GLP-1 secretion; GIP secretion is normal or slightly increased in prediabetes (29). However, GLP-1 analogs represent a candidates for type 2 diabetes prevention. It has been shown that the primary defect in IGT and T2DM is an inability of the β-cell to respond to glucose (18) and this β-cell
“blindness” to glucose, at least in part, can be restored by the incretin hormones (39- 40). These results indicate that pharmacological levels of plasma GLP-1 are capable of correcting β-cell “blindness” to glucose and restoring normal β-cell function to glucose in IGT and T2DM individuals. Although the stimulatory effect of the GLP-1 analogues on beta cell function weans rapidly upon washing out the drug, a recent study reported that, if exenatide is continued for 3 years, its ability to enhance β-cell responsiveness to glucose is partially retained (41). For the reasons, GLP-1 analogs may represent an ideal treatment option to delay or prevent the progression to T2DM in individuals with prediabetes.
1.4 The role of endogenous glucose production in postabsorptive state
In the postabsorptive state, the liver of healthy subjects produces glucose at the rate of
2.0 mg/kg/min (4,42). This glucose efflux is important to meet the needs of the brain and other neural tissues, which use glucose at a constant rate of approximately 1.0 to 1.2 mg/kg/min (43). Under fasting conditions, brain glucose uptake is insulin- independent and accounts for approximately 50% to 60% of glucose disposal (Figure 1).
Figure 1. : Percentages of contribution to glucose homeostasis after an 18 hours fast in human metabolism.
Also brain glucose uptake occurs at the same rate during absorptive and postabsorptive periods, and seems to be not altered in type 2 diabetes. As shown in the figure 1 the rest of the glucose uptake occurs principally in the skeletal muscle, adipose tissue, liver and kidneys while the majority of endogenous glucose production occur in the liver. The kidney possesses all of the gluconeogenesis enzymes required to produce glucose, and estimates of the renal contribution to total endogenous glucose production have ranged from 10% to 25% (44-45). One unconfirmed study suggests that the rate of renal gluconeogenesis is increased in type 2 diabetics with fasting hyperglycemia (46), but studies using the hepatic vein catheter technique have shown that all of the increase in total body endogenous glucose production (measured with [3-3H]glucose) in type 2 diabetics can be accounted for by increased hepatic glucose output (measured by the hepatic vein catheter technique) (47). In type 2 diabetics with mild fasting hyperglycemia (≤140 mg/dL), the postabsorptive level of hyperinsulinemia is sufficient to overcome the hepatic insulin resistance and maintain a normal basal rate of hepatic glucose output (4). In diabetic subjects with mild to moderate fasting hyperglycemia (140– 200 mg/dL, 7.8–11.1 mmol/L), however, basal
hepatic glucose production is increased by approximately 0.5 mg/kg/min (4,42). The increase in basal hepatic glucose production (HGP) is closely correlated with the severity of fasting hyperglycemia and this has been demonstrated in numerous studies (48). In conclusion, in type 2 diabetics with overt fasting hyperglycemia (≥140 mg/dL, 7.8 mmol/L), an excessive rate of hepatic glucose output is the major abnormality responsible for the elevated FPG concentration.
1.5 The role of hepatic insulin resistance
Under postabsorptive conditions, the fasting plasma insulin concentration in type 2 diabetics is 2- to 4-fold greater than in nondiabetic subjects (49). Given that hyperinsulinemia is a potent inhibitor of HGP, an hepatic resistance to the action of insulin must be present to explain the excessive output of glucose by the liver.
Because hyperglycemia per se exerts a powerful suppressive action on HGP, the liver also must be resistant to the inhibitory effect of hyperglycemia on hepatic glucose output. The dose response relationship between hepatic glucose production and the plasma insulin concentration has been examined with the euglycemic insulin clamp technique and radioisotopic glucose (Figure 2)(42).
Figure 2. Dose-response curve relating the plasma insulin concentration to the suppression of hepatic glucose production in control () and type 2 diabetic (o) subjects with moderately severe fasting hyperglycemia. * P < 0.05; ** P < 0.01 versus control subjects. (Reproduced form Groop LC, Bonadonna RC, DelPrato S, Ratheiser K, Zyck K, Ferrannini E, DeFronzo RA. Glucose and free fatty acid metabolism in non-insulin-dependent diabetes mellitus. Evidence for multiple sites of insulin resistance. J Clin Invest. 1989;84(1):205–13)
The figure 2 shows that the dose-response curve relating inhibition of HGP to the
plasma insulin level is very steep and in type 2 diabetics it is shifted to the right, indicating resistance to the inhibitory effect of insulin on hepatic glucose production.
Elevation of the plasma insulin concentration to the high physiologic range (≈100 UI/mL), however, can overcome the hepatic insulin resistance and cause a near normal suppression of HGP, also the severity of the hepatic insulin resistance is related to the level of glycemic control. In type 2 diabetics with mild fasting hyperglycemia, an increment in plasma insulin concentration of 100 UI/mL causes a complete suppression of HPG; however, in diabetic subjects with more severe fasting hyperglycemia, the ability of the same plasma insulin concentration to suppress HGP is impaired. These observations indicate that there is an acquired component of hepatic insulin resistance, which becomes progressively worse as the diabetic state decompensates over time. Hepatic glucose production can be derived from either glycogenolysis or gluconeogenesis (50)(Figure 3 ).
Figure 3. Hepatic and renal gluconeogenesis in healthy individuals (CT) and type 2 diabetic subjects (T2D) (Reproduced from Gastaldelli A, Baldi S, Pettiti M, Toschi E, Camastra S, Natali A, et al. Influence of gluconeogenesis and glucose output in humans:
Recent studies using 13C-labeled magnetic resonance imaging (51-52) have confirmed that in the liver the majority of endogenous glucose production can be accounted by gluconeogenesis while the rest is accounted by glycogenolysis. In the kidneys the totality of glucose production is accounted by gluconeogenesis. A variety of mechanisms has been shown to contribute to the increase in hepatic
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gluconeogenesis, including hyperglucagonemia, enhanced sensitivity to glucagon, increased circulating levels of gluconeogenic precursors (lactate, alanine, glycerol), increased FFA oxidation, and decreased sensitivity to insulin.
1.6 The role of Splanchnic (hepatic) glucose uptake and Glucagon
The splanchnic tissues, like the brain, are largely insensitive to insulin with respect to the stimulation of glucose uptake. Also there are no differences between diabetic and control subjects in the amount of glucose taken up by the splanchnic tissues as demonstrated in study performed using the hepatic vein catheterization technique in combination with the euglycemic insulin clamp (53). Under conditions of euglycemic hyperinsulinemia, very little infused glucose is taken up by the splanchnic (and therefore hepatic) tissues. Under postabsorptive conditions, approximately 50% of total hepatic glucose output is dependent on the maintenance of normal basal glucagon levels, and inhibition of basal glucagon secretion with somatostatin causes a reduction in hepatic glucose production and plasma glucose concentration (Figure 4).
Figure 4. Interrelation between plasma insulin and glucagon levels during postprandial state and postabsorbitive state.
As reported in the figure 4, in fasting state glucagon levels participate to glucose homeostasis by stimulating glycogenolysis, gluconeogenesis and ketogenesis. After a glucose load, glucagon secretion is inhibited by hyperinsulinemia, and the resultant
hypoglucagonemia contributes to the suppression of hepatic glucose production and maintenance of normal postprandial glucose tolerance. The route of glucose entry into the body also plays an important role in the distribution of administered glucose and overall glucose homeostasis (50, 54-55).Several studies have shown that basal plasma glucagon concentration is elevated in type 2 diabetic individuals (56-58). The important contribution of elevated fasting plasma glucagon levels to the increased basal rate of HGP in type 2 diabetic individuals was provided by Baron (59).
Compared with control subjects, diabetic individuals had a markedly elevated rate of basal HGP, which correlated closely with the increase in fasting plasma glucagon concentration. However, following a somatostatin infusion, plasma glucagon levels declined by 44% in association with a 58% decrease in basal HGP. These results conclusively demonstrate the pivotal role of hyperglucagonemia in the pathogenesis of fasting hyperglycemia in type 2 diabetes.
1.7 The role of adipocytes and fat metabolism
A deranged adipocyte metabolism and altered fat topography has an important role in the development of glucose intolerance in type 2 diabetes(24,60-61). Fat cells are resistant to insulin’s antilipolytic effect, leading to day-long elevation in the plasma NEFA concentration (62). Chronically increased plasma NEFA levels stimulate gluconeogenesis (63), induce hepatic/muscle insulin resistance (64), and impair insulin secretion (60,65). These NEFA-induced disturbances are referred to as lipotoxicity. Dysfunctional fat cells produce excessive amounts of insulin resistance–
inducing, inflammatory, and atherosclerotic provoking adipocytokines and fail to secrete normal amounts of insulin-sensitizing adipocytokines such as adiponectin (24). Enlarged fat cells are insulin resistant and have diminished capacity to store fat (66). When adipocyte storage capacity is exceeded, lipid “over-flows” into muscle, liver, and beta cells, causing muscle/ hepatic insulin resistance and impaired insulin secretion. In both type 2 diabetic and obese nondiabetic subjects, the ability of insulin to suppress the plasma FFA concentration and inhibit FFA turnover is significantly impaired compared with lean normal glucose tolerant control subjects at all plasma insulin concentrations (62).Many studies have shown that a physiological elevation in the plasma NEFA concentration stimulates HGP and impairs insulin-stimulated glucose uptake in liver (67) and muscle (68-69). Also NEFA have been shown to have
independent effects to inhibit glycogen synthase (70) and both glucose transport and glucose phosphorylation (71). Collectively, these findings provide strong support for lipotoxicity and adipocyte insulin resistance in the pathogenesis of type 2 diabetes.
1.8 Conclusions
Individuals with prediabetes manifest almost all the metabolic abnormalities that are present in type 2 diabetes. Insulin resistance (primarily affecting skeletal muscle, liver and adipose tissue) and islet dysfunction mostly characterized by β-cell failure and hyperglucagonemia play an important role for the progression from NGT to IGT to T2DM. The principal consequences of insulin resistance in metabolically important organs, as such as liver and adipose tissue, are characterized by an increase in hepatic insulin resistance and derangement of adipocyte metabolism. The net results is an increased endogenous glucose production (primarily from liver) and lipid “over- flows” from adipose tissue into muscle, liver, and beta cells, which in turn causes muscle/ hepatic insulin resistance and impaired insulin secretion, suggesting a typical vicious cycle. The incretin system is an important player for the control of glucose homeostasis and acts within the continuum of metabolic abnormalities that lead to overt type 2 diabetes. It has been shown that incretin effect is abnormal in prediabetic individual and GLP-1 administration can, at least in part, correct the metabolic abnormalities that characterize the glucose tolerance deterioration. In particular it has been shown that GLP-1 analogs can improve β-cell function by restoring beta cell glucose sensitivity, promote weight loss, reduce appetite and food intake and do not cause hypoglycemia. Therefore GLP-1 analogs are likely to be an ideal agent for decreasing the progression of IFG/IGT to T2DM and reverting glucose tolerance to normal. However, the effect of GLP-1 analogues on the progression of IGT to T2DM has yet to be tested in a large scale in long term outcome studies.
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Chapter 2
The acute effects of Exenatide on postprandial glucose metabolism in prediabetic individuals
2.1 Introduction
It has been shown that the effect of Exenatide on postprandial glucose metabolism is mainly acute and it is due mainly to the delay in gastric emptying (1-2). The acute effect of Exenatide in subjects with prediabetes or newly diagnosed type 2 diabetes, i.e., when the beta cell function is not completely lost, has not been studied previously. In the present study we evaluated the effect of acute Exenatide subcutaneous administration on postprandial glucose metabolism in 15 individuals of which 12 with prediabetes and 3 newly diagnosed type 2 diabetes at screening using oral glucose tolerance test (OGTT, 75g of glucose per os) combined with the infusion of a deuterated glucose tracer i.v. and 13C glucose added to the glucose load.
It has been established that Ex decreases postprandial hyperglycemia mainly by delaying gastric emptying. We have previously shown that this effect of Exenatide is acute, since after 14 days of treatment gastric emptying was delayed only when subjects were given Ex before the meal (1-2).The double tracer OGTT allows to fully evaluate the role of Ex in the modulation of postprandial glucose metabolism via the evaluation of the rate of glucose absorption, the endogenous glucose production and the peripheral glucose disposal.
Moreover we combined the OGTT with the injection of 2-deoxy-2-(18F)fluoro-D- glucose (FDG) and PET screening to study if the acute injection of exenatide could affect postprandial hepatic glucose uptake.
2.2 Protocol and Characteristics of the study subjects
We studied fifteen male subjects that had at screening OGTT impaired glucose tolerance (n=12) or newly diagnosed with type 2 diabetes mellitus (n=3). In this study we included 12 subjects with prediabetes and 3 patients with type 2 diabetes that was newly diagnosed at the time of the screening visit. We decided to include these
subjects in the study because their HbA1c was lower than prediabetic subjects (5.54%
vs. 5.7%) and fasting plasma glucose was similar. Moreover, these 3 diabetic subjects provided a previous recent OGTT done before screening visit and the beginning of the study in which a prediabetes was diagnosed. Individuals with prediabetes had FPG=111±3 mg/dl, 2h-PG=157±4 mg/dl, HbA1c=5.7±0.1% while those with newly diagnosed type 2 diabetes had FPG=116±4 mg/dl, 2h-PG=230±2 mg/dl, HbA1c=5.5±0.2%. Each enrolled subject underwent two study sessions combining oral glucose tolerance test (OGTT, 75g of glucose per os) with the infusion of a deuterated glucose tracer i.v. and 13C glucose added to the glucose load. Moreover at OGTT we injected of 5 uCi of 2-deoxy-2-(18F)fluoro-D-glucose (FDG) and PET screening for the quantification of regional organ glucose uptake (in this case liver). A 6,6-2H2-glucose infusion was commenced at T=-120 min and administered continuously for 6h throughout the study. Exenatide (Ex) (5mcg) or placebo (PLC) was administered in random double blind order 30min before the 75 g oral glucose load. Glucose enriched with U-13C6-glucose (1.5g) was added to the 75g of oral glucose that was administered at T=0 together with the FDG bolus i.v. infusion.
Figure 1 reports the diagram of the study protocol.
Figure 1: Protocol Design
Liver/Heart*scan!
Brain*scan!
Posi1oning*
+*Pre*scan! Post*FDG*scan*
(Heart*+*Brain)!
75*grams*glucose*load*with*13!C!!D!glucose!to*! quan1fy*glucose*absorp1on!
D!180!min! D30!min! 0*min! 180!min!
[!18F]FDG! !
injec1on!
60*min! 120*min!
PET*scan! Exena1de!or*!
Placebo*! injec1on!
240!min!
75 grams glucose load with 13C-glucoseto quantify glucose absorption
-180min -20min 0 min 180min
[18F]FDG injection
60 min 120 min PET scan
Exenatideor Placebo injection
240min Liver scan
Brain scan Positioning
+ Pre scan Post FDG scan
(Liver + Brain)
Primed-continuous 2H Glucose and 2H Glycerol infusion to quantify glucose production, glucose clearance and whole body lipolysis
Blood drawn for glucose, insulin, c-peptide and NEFA
The parameters of postprandial glucose metabolism were obtained from stable isotope tracer infusion, ie plasma glucose clearance (from 6,6-2H-glucose co-infusion) as an index of insulin sensitivity, endogenous glucose production and oral glucose absorption (from modelling analysis of 6,6-2H-glucose and U-13C-glucose tracer kinetics during OGTT) (Details are provided in the Appendix). Table 1 shows the characteristic of study subjects.
Table 1. Characteristic of study subjects. Data are presented as mean ± standard error mean (SEM).
Range indicates the minimum and the maximum of the parameter
2.3 The effect of Exenatide on Postprandial glucose metabolism
After Ex or PLC injection but before glucose load (ie, between -30 min and 0 min) plasma glucose concentrations were almost superimposable (mean plasma glucose:
105±8 vs. 105±3 mg/dl in Ex vs. PLC respectively; p=0.11; Figure 2A). Following the glucose load glucose concentration rose only in PLC until 120 min as a
Characteristics of participants (n=15)
Mean ±SEM Range
Age (years) 56±8 47-65
Glucose tolerance status 2 IGT- 10 IFG/IGT – 3 T2DM
BMI (Kg/m2) 29.4±0.9 24.8-37.6
Waist (cm) 103±3 91-128
FPG (mg/dl) 112±3 95-124
HbA1c (%) 5.7±0.1 5.2-6.7
Fasting Plasma Insulin (mUI/l) 9.6±1.8 3.1-18.3
Fasting Plasma Glucagon (pg/ml) 34.6±5.3 13-99
Fasting Plasma C-peptide (ng/ml) 3.1±1.0 1.4-5.2
Fasting NEFA (mEq/L) 0.77±0.27 0.53-1.51
Creatinine (mg/dl) 0.94±0.03 0.7-1.11
ALT (U/l) 33±4 17-67
AST (U/l) 28±3 18-58
Alkaline Phosphatase (U/l) 78±5 41-131
Total Cholesterol (mg/dl) 181±8 140-250
LDL cholesterol (mg/dl) 112±7 63-178
HDL cholesterol (mg/dl) 40±1 31-48
Triglycerides (mg/dl) 146±20 45-316
consequence of gut glucose absorption (Figure 2, panel A-B) and then slowly decreased reaching the fasting levels at 240 min. This triggered insulin release (Figure 3A) that increased peripheral glucose clearance (Figure 2C) and inhibited hepatic glucose production (Figure 2D). As consequence of insulin release, hepatic glucose production was significantly inhibited in both groups reaching stable values after 60 min (Figure 2D). In Ex plasma glucose remained close to the fasting levels for 240 min after the glucose load although minimal variations were observed during the first and the last part of the OGTT (slightly decreased at 0-30 min and increased at 150- 240 min compared to fasting level). EGP in Ex also decreased but to a lesser extent compared to PLC. As shown in the Figure 2B the rate of oral glucose absorption increased significantly in PLC reaching the maximum between 60 and 120 min and then decreased during the last part of the OGTT. The single Exenatide injection significantly delayed the postprandial glucose absorption (Figure 2B) two hours as compared to PLC although the rate of glucose absorption slightly increased during the last part of the OGTT. For this reason glucose concentrations in Ex remained close to the fasting levels for the four hours after the glucose load.
The Figure 2C depicts the glucose clearance in both groups. In PLC glucose clearance constantly increased after the glucose load although an initial minor reduction below the fasting level was observed during the first 30 min of the OGTT. In Ex glucose clearance constantly increased from the beginning of the OGTT reaching the highest values between 180 and 210 min.
Table 2A-B illustrates in details the glucose fluxes in fasting and postprandial states in Exenatide vs. Placebo. Fasting glucose levels were similar in both groups, however the area under the curve (AUC) was significantly lower in Ex as compared to PLC throughout the OGTT. The rate of oral glucose appearance was significantly lower in Ex vs. PLC between 0 and 120 min (p=0.0004), however it was similar between 120 and 240 min. Finally the AUC between 0 and 240 min remained significantly lower in Ex as compared to PLC. The total rate of glucose appearance which includes endogenous as well as exogenous glucose, was similar in fasting state and it was significantly reduced in Ex as compared to PLC as demonstrated by the AUC 0-120 and AUC 0-240.
Figure 2: Effect of Exenatide (close circle) vs. Placebo (open circle) on postprandial glucose metabolism. A. Plasma glucose concentrations B. Rate of oral glucose absorption C. Glucose clearance D. Endogenous glucose production. Data are presented as mean±SEM
The rate of glucose disappearance was similar at fasting state in both groups and it was significantly lower in Ex vs. PLC throughout the study. However, if the glucose disposal is expressed as glucose clearance, a significant reduction brought by Ex injection vs. PLC was observed only in the last part of OGTT (AUC 120-240:
55.2±4.8 vs. 66.6±3.1 ml/Kg*min; p=0.03 respectively) whereas it was similar during at 0-120 min and collectively at 0-240 min. The endogenous glucose production that was equal to the rate of glucose disappearance in the fasting state, was similar at fasting and it was similarly suppressed in both Ex and PLC at 0-120 after glucose load. At 120-240 endogenous glucose production was more suppressed in PLC than Ex causing an greater overall endogenous glucose suppression in PLC as compared to EX.
mg/dl
0 40 80 120 160 200 240
-30 0 30 60 90 120 150 180 210 240
[µmol/(Kg ! min)]
0 5 10 15 20 25
0 30 60 90 120 150 180 210 240
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5
0 30 60 90 120 150 180 210 240
[ml/(Kg ! min)]
Time (min)
0 2 4 6 8 10 12
0 30 60 90 120 150 180 210 240
Placebo Exenatide
[µmol/(Kg ! min)]
Time (min)
A B
C D
Plasma Glucose Rate of Oral Glucose Appearance
Endogenous Glucose Production Glucose Clearance
Table 2A. Fasting and postprandial metabolic parameters in Exenatide and Placebo . Data are presented as mean ± standard error mean (SEM).
Parameter Placebo Exenatide p
Plasma Glucose (mg/dl)
Fasting 107±3 104±2 0.34
AUC 0-120 min 18353±1038 12607±760 0.001
AUC 120-240 min 5532±501 4859±502 0.001
AUC 0-240 min 23884±1204 17466±902 0.0007
Rate of Oral Glucose Appearance (RaOr) (µmol/ Kg min)
Fasting Ra Or 0 0 -
AUC 0-120 1885±148 660±167 <0.001
AUC 120-240 1210±64 1256±198 0.54
AUC 0-240 3096±138 1917±277 0.001
Total Rate of Glucose Appearance (Ratot) (µmol/Kg min)
Fasting Ra tot 10.5±0.3 10.2±0.3 0.26
AUC 0-120 2400±122 1385±152 <0.001
AUC 120-240 1487±70 1782±185 0.12
AUC 0-240 3887±143 3167±271 0.03
Rate of Glucose Disappearance (µmol/(Kg min)
Fasting Rd 10.5±0.4 10.2±0.3 0.48
AUC 0-120 1835±108 1357±116 0.01
AUC 120-240 2281±90 1741±176 0.008
AUC 0-240 4116±163 3099±265 0.002
