Antidiabetic actions of endogenous and exogenous GLP-1 in type 1 diabetic patients with and without residual β-cell function

OBJECTIVE

To investigate the effect of exogenous as well as endogenous glucagon-like peptide 1 (GLP-1) on postprandial glucose excursions and to characterize the secretion of incretin hormones in type 1 diabetic patients with and without residual β-cell function.

RESEARCH DESIGN AND METHODS

Eight type 1 diabetic patients with (T1D+), eight without (T1D−) residual β-cell function, and eight healthy matched control subjects were studied during a mixed meal with concomitant infusion of GLP-1 (1.2 pmol/kg/min), saline, or exendin 9-39 (300 pmol/kg/min). Before the meal, half dose of usual fast-acting insulin was injected. Plasma glucose (PG), glucagon, C-peptide, total GLP-1, intact glucose-dependent insulinotropic polypeptide (GIP), free fatty acids, triglycerides, and gastric emptying rate (GE) by plasma acetaminophen were measured.

RESULTS

Incretin responses did not differ between patients and control subjects. Infusion of GLP-1 decreased peak PG by 45% in both groups of type 1 diabetic patients. In T1D+ patients, postprandial PG decreased below fasting levels and was indistinguishable from control subjects infused with saline. In T1D− patients, postprandial PG remained at fasting levels. GLP-1 infusion reduced GE and glucagon levels in all groups and increased fasting C-peptide in T1D+ patients and control subjects. Blocking endogenous GLP-1 receptor action increased endogenous GLP-1 secretion in all groups and increased postprandial glucose, glucagon, and GE in T1D+ and T1D− patients. The insulinogenic index (the ratio of insulin to glucose) decreased in T1D+ patients during blockade of endogenous GLP-1 receptor action.

CONCLUSIONS

Type 1 diabetic patients have normal incretin responses to meals. In type 1 diabetic patients, exogenous GLP-1 decreases peak postprandial glucose by 45% regardless of residual β-cell function. Endogenous GLP-1 regulates postprandial glucose excursions by modulating glucagon levels, GE, and β-cell responsiveness to glucose. Long-term effects of GLP-1 in type 1 diabetic patients should be investigated in future clinical trials.At time of diagnosis and during the first year, prevalence of residual β-cell function in patients with type 1 diabetes is nearly 100% (1,2). After alleviation of initial hyperglycemia with exogenous insulin, patients enter a remission period with improved β-cell function, where insulin treatment can be paused in up to 20–30% of the patients without loss of target glycemic control (3). Persistence of residual insulin secretion is associated with reduced risk of ketosis (4), lower HbA_1c levels (5), lower insulin doses, less risk of hypoglycemia, and reduced long-term complications (2,6). However, after disease duration of 5–10 years, the prevalence of residual β-cell function has declined to about 15% (2). Even though lack of insulin is considered to be the most important factor for the hyperglycemia in type 1 diabetic patients, other metabolic disturbances may also play a role: the glucagon response to carbohydrate and protein ingestion has been shown to be abnormal (7) and there is evidence that postprandial hyperglycemia is because of lack of insulin as well as inappropriately elevated glucagon levels (8,9). The gut hormone, glucagon-like peptide 1 (GLP-1), reduces glucagon levels, increases insulin secretion (10), and inhibits gastric emptying rate (GE), thereby reducing postprandial glucose excursions (11). The insulinotropic and the glucagonostatic properties of GLP-1 are glucose dependent (12), and exogenous GLP-1, therefore, does not produce hypoglycemia. Several studies have found lowering of fasting and postprandial glucose by GLP-1 or GLP-1 agonists in type 1 diabetic patients with (13–15) as well as without (16–19) residual β-cell function. Some studies suggested that the glucose lowering effect was because of the enhancement of insulin sensitivity (19), whereas others concluded that delay of gastric emptying (13,14) or reduction of glucagon levels (17) was the most important mechanism. In animal studies, treatment with GLP-1 or GLP-1 agonists has been shown to delay diabetes development or reverse recent onset diabetes in NOD mice (20), ascribed to an improved function of existing β-cells rather than through increments in β-cell mass. However, there is also evidence that GLP-1, in combination with gastrin, increases β-cell mass and restores normoglycemia in recent onset diabetic NOD mice (21) and that GLP-1 combined with gastrin is able to expand β-cell mass of human islets implanted under the renal capsule of immunodeficient diabetic NOD mice (22). In freshly isolated human islets, GLP-1 has been reported to inhibit β-cell apoptosis (23). However, in C-peptide–positive subjects with longstanding type 1 diabetes treated with exenatide for 6 to 9 months with or without daclizumab, insulin dose was significantly reduced, primarily because of the reduction of prandial insulin, but β-cell function was not improved (15). Four weeks of treatment with vildagliptin (a DPP-4 inhibitor that increases endogenous GLP-1 levels) in 11 well-controlled type 1 diabetic patients with longstanding disease decreased postmeal glucagon and glucose levels (24), and in adolescents with minimal or no endogenous insulin secretion treated with exenatide, postprandial glucose excursions were reduced despite 20% reduction of insulin dose (25). Therefore, GLP-1–based therapies have potential for treatment of type 1 diabetes alone or—more likely—in combination with insulin.Because of controversies regarding secretion of incretin hormones in type 1 diabetes (26,27), assessment of the meal-related GLP-1 secretory responses in patients with diabetes is of interest (28). We therefore studied incretin secretion as well as the antidiabetic actions of both endogenously secreted and exogenously infused GLP-1 during a mixed meal in type 1 diabetic patients with and without residual β-cell function.     

Exaggerated Glucagon-Like Peptide 1 Response Is Important for Improved β-Cell Function and Glucose Tolerance After Roux-en-Y Gastric Bypass in Patients With Type 2 Diabetes

β-Cell function improves in patients with type 2 diabetes in response to an oral glucose stimulus after Roux-en-Y gastric bypass (RYGB) surgery. This has been linked to the exaggerated secretion of glucagon-like peptide 1 (GLP-1), but causality has not been established. The aim of this study was to investigate the role of GLP-1 in improving β-cell function and glucose tolerance and regulating glucagon release after RYGB using exendin(9-39) (Ex-9), a GLP-1 receptor (GLP-1R)–specific antagonist. Nine patients with type 2 diabetes were examined before and 1 week and 3 months after surgery. Each visit consisted of two experimental days, allowing a meal test with randomized infusion of saline or Ex-9. After RYGB, glucose tolerance improved, β-cell glucose sensitivity (β-GS) doubled, the GLP-1 response greatly increased, and glucagon secretion was augmented. GLP-1R blockade did not affect β-cell function or meal-induced glucagon release before the operation but did impair glucose tolerance. After RYGB, β-GS decreased to preoperative levels, glucagon secretion increased, and glucose tolerance was impaired by Ex-9 infusion. Thus, the exaggerated effect of GLP-1 after RYGB is of major importance for the improvement in β-cell function, control of glucagon release, and glucose tolerance in patients with type 2 diabetes.Hyperglycemia in patients with type 2 diabetes is resolved shortly after Roux-en-Y gastric bypass (RYGB), suggesting that mechanisms independent of weight loss contribute to the improvement in glycemic control (1–4).Within 1 month and as early as 5 days after RYGB, β-cell function in response to a meal improves in subjects with type 2 diabetes, and this is accompanied by an increased postprandial glucagon-like peptide (GLP)-1 response (3,5,6). In contrast, after intravenous infusion of glucose, which does not elicit the incretin effect, an improvement in β-cell function is absent (5,7,8). Therefore, it could be speculated that the early improvements in β-cell function after RYGB are due to the enhanced GLP-1 secretion related to eating a meal, but causality has not been established (9).In patients with type 2 diabetes, energy restriction per se is known to result in improved hepatic insulin sensitivity and decreased hepatic glucose production and, as a result, lowered fasting plasma glucose concentrations (10–12). Similar metabolic changes are seen after RYGB, when energy intake is limited (13,14), and this has led to the proposal that caloric restriction with a subsequent reduction in glucotoxicity, rather than an increased effect of GLP-1, is responsible for the improved β-cell function (14,15).The aim of this study was to investigate the role of GLP-1 in the improved β-cell function and glucose tolerance seen after RYGB in subjects with type 2 diabetes. This was accomplished by pharmacologically blocking the GLP-1 receptor (GLP-1R) during a liquid meal tolerance test before and after surgery using exendin(9-39) (Ex-9; Bachem AG, Bubendorf, Switzerland), a specific GLP-1R antagonist (16).Previous studies have documented increased meal-related glucagon secretion after RYGB despite improvements in insulin secretion and sensitivity and exaggerated GLP-1 release (3,17,18). This observation is surprising given the glucagonostatic properties of GLP-1 and insulin (19,20). Therefore, a further aim of this study was to evaluate the interaction between GLP-1 and glucagon release after RYGB in both the fasting and postprandial states.     

Insulin Reciprocally Regulates Glucagon Secretion in Humans

OBJECTIVE

We tested the hypothesis that an increase in insulin per se, i.e., in the absence of zinc, suppresses glucagon secretion during euglycemia and that a decrease in insulin per se stimulates glucagon secretion during hypoglycemia in humans.

RESEARCH DESIGN AND METHODS

We measured plasma glucagon concentrations in patients with type 1 diabetes infused with the zinc-free insulin glulisine on three occasions. Glulisine was infused with clamped euglycemia (∼95 mg/dl [5.3 mmol/l]) from 0 to 60 min on all three occasions. Then, glulisine was discontinued with clamped euglycemia or with clamped hypoglycemia (∼55 mg/dl [3.0 mmol/l]) or continued with clamped hypoglycemia from 60 to 180 min.

RESULTS

Plasma glucagon concentrations were suppressed by −13 ± 3, −9 ± 3, and −12 ± 2 pg/ml (−3.7 ± 0.9, −2.6 ± 0.9, and −3.4 ± 0.6 pmol/l), respectively, (all P < 0.01) during zinc-free hyperinsulinemic euglycemia over the first 60 min. Glucagon levels remained suppressed following a decrease in zinc-free insulin with euglycemia (−14 ± 3 pg/ml [−4.0 ± 0.9 pmol/l]) and during sustained hyperinsulinemia with hypoglycemia (−14 ± 2 pg/ml [−4.0 ± 0.6 pmol/l]) but increased to −3 ± 3 pg/ml (−0.9 ± 0.9 pmol/l) (P < 0.01) following a decrease in zinc-free insulin with hypoglycemia over the next 120 min.

CONCLUSIONS

These data indicate that an increase in insulin per se suppresses glucagon secretion and a decrease in insulin per se, in concert with a low glucose concentration, stimulates glucagon secretion. Thus, they document that insulin is a β-cell secretory product that, in concert with glucose and among other signals, reciprocally regulates α-cell glucagon secretion in humans.The regulation of pancreatic islet α-cell glucagon secretion by nutrients, hormones, neurotransmitters, and drugs is complex and incompletely understood (1–8). It involves direct signaling of α-cells (1) and indirect signaling of α-cells by β-cell (2–4) and δ-cell (5) secretory products, the autonomic nervous system (6,7), and gut incretins (8). Among the intraislet mechanisms, there is evidence that indirect reciprocal β-cell–mediated signaling of α-cells normally predominates over direct α-cell signaling in the regulation of glucagon secretion in humans (9–13). The physiological concept is as follows: 1) A β-cell secretory product, or products, tonically restrains α-cell glucagon secretion during postabsorptive euglycemia. 2) A decrease in β-cell secretion, in concert with a low α-cell glucose concentration, signals an increase in α-cell glucagon secretion during hypoglycemia (9–12). 3) An increase in β-cell secretion negates direct α-cell stimulation and thus results in no change or even suppression of α-cell glucagon secretion following a mixed meal (13).Among the various candidate signaling molecules—insulin, zinc, γ-aminobutyric acid, and amylin among others (4)—there is evidence that insulin is a β-cell secretory product that normally restrains basal α-cell glucagon secretion (14). First, administered insulin suppresses glucagon secretion in several species (4,14,15) including humans (16). However, most available insulin preparations contain zinc and zinc is cosecreted with insulin from β-cells and it has been reported that a decrease in pancreatic arterial zinc, but not in insulin depleted of zinc, increases glucagon secretion during hypoglycemia in streptozotocin diabetic rats, leading the authors to conclude that the inhibitory β-cell secretory product is zinc—not insulin (17). Second, perfusion of the rat (3) and the human (18) pancreas (and incubation of rat islets [15]) with an antibody to insulin increases glucagon release. Because insulin circulates as a zinc-free monomer (19), that finding seemingly implicates insulin directly as an α-cell inhibitory factor and suggests that zinc is not the only β-cell product that reciprocally regulates α-cell glucagon secretion. However, it could be reasoned that the antibody binds insulin-zinc complexes before they dissociate at physiological pH and with dilution. Third, findings—that siRNA-mediated knockdown of insulin receptors prevented the effect of low glucose concentrations to increase glucagon release from isolated mouse islets (20) and that blockade of insulin signaling with the phosphatidylinositol 3-kinase inhibitor wortmannin prevented the effect of high glucose concentrations to decrease glucagon release from isolated rat and human islets (21)—also appear to implicate insulin as the relevant β-cell secretory product.We tested the hypothesis that an increase in insulin per se, i.e., in the absence of an increase in zinc, suppresses glucagon secretion during euglycemia and that a decrease in insulin per se, in concert with low glucose concentrations, stimulates glucagon secretion in humans. To do so, we studied patients with type 1 diabetes, individuals with essentially no endogenous insulin secretion and no α-cell glucagon secretory response to hyperinsulinemic hypoglycemia (22–29) but viable α-cells as evidenced by a glucagon secretory response to administered amino acids (22,30–32), on three separate occasions. Plasma glucagon concentrations were measured during infusion of the zinc-free insulin glulisine with clamped euglycemia over 60 min on all three occasions. Then, glucagon concentrations were measured following discontinuation of glulisine, to produce a sharp decrease in systemic and therefore α-cell zinc-free insulin levels, over 120 min with clamped euglycemia on one occasion or clamped hypoglycemia on another occasion. On a third occasion, glucagon levels were measured during sustained glulisine infusion with clamped hypoglycemia. The data document 1) a decrease in plasma glucagon during zinc-free hyperinsulinemic euglycemia and 2) an increase in plasma glucagon following a decrease in zinc-free insulin during hypoglycemia but not following a decrease in insulin without hypoglycemia or during hypoglycemia without a decrease in insulin.     

Cyclic GMP kinase I modulates glucagon release from pancreatic α-cells

OBJECTIVE

The physiologic significance of the nitric oxide (NO)/cGMP signaling pathway in islets is unclear. We hypothesized that cGMP-dependent protein kinase type I (cGKI) is directly involved in the secretion of islet hormones and glucose homeostasis.

RESEARCH DESIGN AND METHODS

Gene-targeted mice that lack cGKI in islets (conventional cGKI mutants and cGKIα and Iβ rescue mice [α/βRM] that express cGKI only in smooth muscle) were studied in comparison to control (CTR) mice. cGKI expression was mapped in the endocrine pancreas by Western blot, immuno-histochemistry, and islet-specific recombination analysis. Insulin, glucagon secretion, and cytosolic Ca²⁺ ([Ca²⁺]_i) were assayed by radioimmunoassay and FURA-2 measurements, respectively. Serum levels of islet hormones were analyzed at fasting and upon glucose challenge (2 g/kg) in vivo.

RESULTS

Immunohistochemistry showed that cGKI is present in α- but not in β-cells in islets of Langerhans. Mice that lack α-cell cGKI had significantly elevated fasting glucose and glucagon levels, whereas serum insulin levels were unchanged. High glucose concentrations strongly suppressed the glucagon release in CTR mice, but had only a moderate effect on islets that lacked cGKI. 8-Br-cGMP reduced stimulated [Ca²⁺]_i levels and glucagon release rates of CTR islets at 0.5 mmol/l glucose, but was without effect on [Ca²⁺]_i or hormone release in cGKI-deficient islets.

CONCLUSIONS

We propose that cGKI modulates glucagon release by suppression of [Ca²⁺]_i in α-cells.The complex and tightly controlled process of glucagon secretion from pancreatic α-cells is important for the maintenance of blood glucose homeostasis (1). Glucagon release is physiologically regulated by multiple signaling pathways that include neuronal control of α-cell function, paracrine factors such as insulin (2,3), and/or γ-aminobutyric acid (GABA) (4) released from β-cells, somatostatin (SST) secreted from adjacent δ-cells (5), and the inhibitory role of high blood glucose itself that directly acts on α-cells to suppresses glucagon release (3,6).Controversial data have been reported for the physiologic significance of the nitric oxide (NO) pathway for islet functions. Both types of constitutive NOS (eNOS, nNOS) isozymes have been identified in islets (7–11). It was suggested that NO stimulates glucose-induced insulin release (7,10), was a negative modulator of insulin release (8,12,13) or had no effect (14). These discrepant results are probably caused by the analysis of various β-cell–derived cell lines compared with intact islets, the use of different types and concentrations of NO-donors and NOS-inhibitors. Additional data suggested that iNOS-derived NO is involved in autoimmune reactions that cause β-cell–dysfunction leading to insulin-dependent diabetes (15,16).It has been difficult to discriminate between a direct action of NO on α-cells and an indirect effect of NO via β-cells since β-cell factors are potent inhibitors of α-cell activity (12,17,18). An important target of NO is the soluble guanylyl cyclase (sGC) that generates the second messenger cyclic guanosine-3′-5′-monophosphate (cGMP) (19). Some studies detected increased islet cGMP levels upon treatment with cytokines and l-arginine (12,15,20). cGMP analogues were reported to potentiate insulin release directly (21), suggesting that cGMP-dependent effectors are involved in the control of islet activity. The cGMP-dependent protein kinase type I (cGKI) is an important intracellular mediator of NO/cGMP signaling in many cells (22). The analysis of cGKI-deficient mice revealed that cGKI mediates the inhibitory effects of NO on platelet aggregation, the negative inotropic effect of NO/cGMP in the murine heart, and the NO-induced relaxation of blood vessels (22). However, cGKI knockout mice could not be analyzed reliably for a distorted islet function because they display abnormalities of various organ systems and die within the first 6 weeks (23). Recently, we generated an improved mouse model to study the specific roles of cGKI in vivo (24,25). These mice express either the cGKIα or cGKIβ isozyme selectively in smooth muscle cells (SMCs) on a cGKI-deficient genetic background. Since the animals show a prolonged life expectancy and normal SMC functions they were termed cGKIα and cGKIβ rescue mice (αRM and βRM, respectively).We examined the role of cGKI for the regulation of glucose homeostasis using cGKI-KO mice (23) and rescue mice (RM) (24,25) models. We show that cGKI is expressed in the α-cells of the endocrine pancreas, whereas in different gene-targeted animals the cGKI protein was not detectable. Furthermore, we demonstrate that islet cGKI regulates glucagon release by modulation of the glucose-dependent changes of [Ca²⁺]_i that trigger exocytosis. These ex-vivo findings were supported by elevated serum levels of basal glucose and glucagon in intact RM animals.     

Glucose- and hormone-induced cAMP oscillations in α- and β-cells within intact pancreatic islets

OBJECTIVE

cAMP is a critical messenger for insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Dispersed β-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown.

RESEARCH DESIGN AND METHODS

The subplasma-membrane cAMP concentration ([cAMP]_pm) was recorded in α- and β-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in α- and β-cells.

RESULTS

In islets exposed to 3 mmol/L glucose, [cAMP]_pm was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP]_pm, often with oscillations synchronized among β-cells. Whereas glucagon also induced [cAMP]_pm oscillations in most α-cells, <20% of the α-cells responded to GLP-1. Elevation of the glucose concentration to 11–30 mmol/L in the absence of hormones induced slow [cAMP]_pm oscillations in both α- and β-cells. These cAMP oscillations were coordinated with those of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in the β-cells but not caused by the changes in [Ca²⁺]_i. The transmembrane adenylyl cyclase (AC) inhibitor 2′5′-dideoxyadenosine suppressed the glucose- and hormone-induced [cAMP]_pm elevations, whereas the preferential inhibitors of soluble AC, KH7, and 1,3,5(10)-estratrien-2,3,17-β-triol perturbed cell metabolism and lacked effect, respectively.

CONCLUSIONS

Oscillatory [cAMP]_pm signaling in secretagogue-stimulated β-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP]_pm oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.Cyclic AMP and Ca²⁺ are key messengers in the regulation of insulin and glucagon secretion from pancreatic β- and α-cells, respectively, by nutrients, hormones, and neural factors. Glucose stimulation of insulin secretion involves uptake and metabolism of the sugar in the β-cells, closure of ATP-sensitive K⁺ channels, and depolarization-induced Ca²⁺ entry generating oscillations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) that trigger periodic exocytosis of secretory granules (1,2). This process is amplified by mechanism(s) acting distal to the elevation of Ca²⁺ (3). cAMP promotes exocytosis by facilitating the generation of Ca²⁺ signals (4,5), by sensitizing the secretory machinery to Ca²⁺ (4,6), and by stimulating mobilization and priming of granules via protein kinase A– and Epac-dependent pathways (7,8).Measurements of the cAMP concentration beneath the plasma membrane ([cAMP]_pm) in individual INS-1 β-cells showed that glucagon-like peptide-1(7-36)-amide (GLP-1) induces [cAMP]_pm elevation, often manifested as oscillations (9). Glucose has been regarded to only modestly raise islet cAMP, supposedly by amplifying the effect of glucagon (10), but single-cell cAMP recordings have recently shown that glucose alone induces marked elevation of cAMP in MIN6 β-cells (11,12) and primary mouse β-cells (12,13). Although one study reported that the glucose-induced cAMP response depends on elevation of [Ca²⁺]_i (11), other studies show only partial or no Ca²⁺-dependence of the cAMP signal (12,13). Like hormone stimulation, glucose induces oscillations of [cAMP]_pm, and coordination of the [cAMP]_pm and [Ca²⁺]_i elevations generates pulsatile insulin release (12,14).There are 10 isoforms of cAMP-generating adenylyl cyclases (ACs) with different regulatory properties, nine of which are transmembrane (tm) proteins stimulated by Gα_s and the plant diterpene forskolin. Such tmACs mediate the cAMP-elevating action of glucagon and incretin hormones (15). β-cells express multiple tmACs (16), and the Ca²⁺-stimulated AC8 has been proposed to be particularly important for integrating hormone- and depolarization-evoked signals (17). Soluble AC (sAC) is the only isoform that lacks transmembrane domains. It is insensitive to forskolin and G-proteins but stimulated by bicarbonate (18) and Ca²⁺ (19). Although sAC was first found in the testis, it also seems to be expressed in other tissues and was recently proposed to be involved in glucose-induced cAMP production in insulin-secreting cells (20).Like insulin secretion, exocytosis of glucagon from the α-cells is triggered by an increase of [Ca²⁺]_i (21). Glucagon release is stimulated by absence of glucose and is maximally inhibited when the sugar concentration approaches the threshold for stimulation of insulin secretion (22). Under hypoglycemic conditions, glucagon secretion is also stimulated by adrenaline, which raises [Ca²⁺]_i and [cAMP]_pm via α₁- and β-adrenergic mechanisms (23,24). There are fundamentally different ideas about the mechanisms underlying glucose inhibition of glucagon secretion, including paracrine influences from β- and δ-cells (25–29) and direct actions of glucose on the α-cells, resulting in depolarization- (30) or hyperpolarization-mediated (22) inhibition of exocytosis. Apart from the inhibitory effect of glucose, we observed that very high glucose concentrations unexpectedly stimulate glucagon secretion (31). The stimulatory component may be important under physiological conditions because the hormone is released in pulses from rat (32) and human (33) islets. Glucose thus causes alternating periods of stimulation and inhibition resulting in time-average reduction of glucagon secretion. Ca²⁺ is probably not the only messenger in glucose-regulated glucagon release (29). Like for insulin secretion, cAMP is believed to promote glucagon release by enhancing intracellular Ca²⁺ mobilization, Ca²⁺ influx through the plasma membrane, and mobilization of secretory granules (23,24,34,35). However, it has also been suggested that cAMP-mediated reduction of N-type Ca²⁺ currents can explain the inhibitory effect of GLP-1 on glucagon secretion (36).Until now, nothing was known about cAMP kinetics in α-cells and all information on primary β-cells was based on studies of dispersed islet cells. However, as a result of gap junctional coupling and paracrine influences, the electrophysiological characteristics and [Ca²⁺]_i signaling in intact islets differ considerably from those in dispersed β-cells (2). Therefore, the aim of the current study was to clarify how glucose, glucagon, and GLP-1 affect cAMP signaling in α- and β-cells within intact islets of Langerhans.     

Brain GLP-1 signaling regulates femoral artery blood flow and insulin sensitivity through hypothalamic PKC-δ

OBJECTIVE

Glucagon-like peptide 1 (GLP-1) is a gut-brain hormone that regulates food intake, energy metabolism, and cardiovascular functions. In the brain, through a currently unknown molecular mechanism, it simultaneously reduces femoral artery blood flow and muscle glucose uptake. By analogy to pancreatic β-cells where GLP-1 activates protein kinase C (PKC) to stimulate insulin secretion, we postulated that PKC enzymes would be molecular targets of brain GLP-1 signaling that regulate metabolic and vascular function.

RESEARCH DESIGN AND METHODS

We used both genetic and pharmacological approaches to investigate the role of PKC isoforms in brain GLP-1 signaling in the conscious, free-moving mouse simultaneous with metabolic and vascular measurements.

RESULTS

In normal wild-type (WT) mouse brain, the GLP-1 receptor (GLP-1R) agonist exendin-4 selectively promotes translocation of PKC-δ (but not -βII, -α, or -ε) to the plasma membrane. This translocation is blocked in Glp1r^−/− mice and in WT mice infused in the brain with exendin-9, an antagonist of the GLP-1R. This mechanism coordinates both blood flow in the femoral artery and whole-body insulin sensitivity. Consequently, in hyperglycemic, high-fat diet–fed diabetic mice, hypothalamic PKC-δ activity was increased and its pharmacological inhibition improved both insulin-sensitive metabolic and vascular phenotypes.

CONCLUSIONS

Our studies show that brain GLP-1 signaling activates hypothalamic glucose-dependent PKC-δ to regulate femoral artery blood flow and insulin sensitivity. This mechanism is attenuated during the development of experimental hyperglycemia and may contribute to the pathophysiology of type 2 diabetes.Glucagon-like peptide 1 (GLP-1) is secreted during a meal by intestinal L cells and directly enhances glucose-induced insulin secretion (1). Furthermore, GLP-1 is an enteric neuropeptide that triggers vagal signals from the intestine to the brain to activate the gut-brain-pancreatic axis (2–7). Through interaction between neural and direct receptor-dependent mechanisms, GLP-1 regulates multiple metabolic and cardiovascular functions (5,8). Moreover, GLP-1 is produced by neurons in the brainstem (9,10) and transported to several nuclei in the hypothalamus where its receptor has been localized (11–13). To unravel the metabolic importance of the central nervous system GLP-1 receptor (GLP-1R), we previously showed that activation of brain GLP-1 signaling simultaneously reduces femoral artery blood flow and muscle glucose utilization to favor hepatic glucose storage (5,8). This mechanism is impaired in high-fat diet (HFD)-induced diabetic mice (14). Although the corresponding molecular mechanisms regulating brain GLP-1 action remain unknown, a molecular hypothesis can be postulated by analogy with pancreatic β-cells. An obvious target is protein kinase A, a dominant molecular signaling mechanism of the GLP-1R (1). However, changes in protein kinase A activity have not been consistently associated with diabetes. GLP-1 also activates both the α and ε isoforms of protein kinase C (PKC) to stimulate insulin secretion (15). These proteins are translocated from a cytoplasmic location to the plasma membrane, where proteins such as ATP-sensitive K⁺ channels are phosphorylated on conserved threonine residues (T180) in the pore-forming subunit Kir6.2 by a calcium-dependent mechanism (16,17). It is important that brain PKCs have been implicated in the control of glucose metabolism in mice (18). The central injection of the specific PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester, enhanced the hypoglycemic response to coadministered insulin (18). In healthy conscious rats, the coadministration of the hypothalamic PKC-δ inhibitor rottlerin with 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic diacylglycerol (DAG) analogue, prevented the ability of OAG to activate hypothalamic PKC-δ and to lower hepatic glucose production (19). It is noteworthy that hyperglycemia also triggers PKC activation, and in models of diabetes, these enzymes are excessively activated (20–23). Therefore, we postulated that brain GLP-1 could control metabolism or blood flow by a mechanism involving the activation of hypothalamic PKCs that might also be regulated by hyperglycemia and diabetes.     

Glucagon receptor knockout prevents insulin-deficient type 1 diabetes in mice

OBJECTIVE

To determine the role of glucagon action in the metabolic phenotype of untreated insulin deficiency.

RESEARCH DESIGN AND METHODS

We compared pertinent clinical and metabolic parameters in glucagon receptor-null (Gcgr^−/−) mice and wild-type (Gcgr^+/+) controls after equivalent destruction of β-cells. We used a double dose of streptozotocin to maximize β-cell destruction.

RESULTS

Gcgr^+/+ mice became hyperglycemic (>500 mg/dL), hyperketonemic, polyuric, and cachectic and had to be killed after 6 weeks. Despite comparable β-cell destruction in Gcgr^−/− mice, none of the foregoing clinical or laboratory manifestations of diabetes appeared. There was marked α-cell hyperplasia and hyperglucagonemia (∼1,200 pg/mL), but hepatic phosphorylated cAMP response element binding protein and phosphoenolpyruvate carboxykinase mRNA were profoundly reduced compared with Gcgr^+/+ mice with diabetes—evidence that glucagon action had been effectively blocked. Fasting glucose levels and oral and intraperitoneal glucose tolerance tests were normal. Both fasting and nonfasting free fatty acid levels and nonfasting β-hydroxy butyrate levels were lower.

CONCLUSIONS

We conclude that blocking glucagon action prevents the deadly metabolic and clinical derangements of type 1 diabetic mice.The development of a radioimmunoassay for glucagon (1) established the hormonal status of this peptide (2), originally considered to be a contaminant of the insulin extraction process. Glucagon was immunocytochemically localized to pancreatic α-cells (3) and shown to be secreted in response to increased glucose need, as in starvation and exercise (4). By 1973, it was recognized that α-cell function was grossly abnormal in diabetes, particularly in type 1 diabetes (5). Here, β-cells are largely replaced by α-cells, and, without the inhibitory action of insulin, their secretion of glucagon is unrestrained, and glucagon action on the liver is unopposed. The result is a lethal hypercatabolic state. In 1973, the discovery of somatostatin (6), a potent inhibitor of glucagon secretion, made it possible to demonstrate the essential role of glucagon in the metabolic phenotype of type 1 diabetes (7–10).Those studies led to a search for a therapeutic suppressor of diabetic hyperglucagonemia or blocker of its action on the liver. In the 37 years since the discovery of somatostatin, only one other potent glucagon-suppressing substance, leptin, has been identified (11,12). By contrast, inactivators of glucagon have been less elusive. High affinity antiglucagon antibodies have benefited diabetic animals (13), as have a variety of molecules that block binding of glucagon to the glucagon receptor and/or block its signaling (14–18). Diabetic mice with glucagon receptor knockout (19) or mice treated with Gcgr antisense oligonucleotide similarly benefit from the elimination of glucagon action (20,21). Although all of the foregoing reports demonstrate that abrogation of glucagon action reduces hepatic overproduction of glucose, a potential therapeutic asset in diabetes management, none of the foregoing diabetic models studied thus far have been totally insulin-deficient, as in type 1 diabetes. In type 1 diabetes, islets are virtually devoid of β-cells, and are largely made up of hyperplastic α-cells. In contrast to normal α-cells, which are restrained by the high local concentrations of intraislet insulin (22), type 1 diabetic α-cells are unregulated, which results in inappropriate hyperglucagonemia (5). Moreover, with no insulin to oppose the hepatic actions of the hyperglucagonemia, unrestrained glycogenolysis, gluconeogenesis, ketogenesis, and hypercatabolism lead rapidly to ketoacidosis, cachexia, coma, and death.An essential role of hyperglucagonemia in the pathogenesis of this lethal syndrome has long been suspected but never fully proven. Recent studies of adenovirally induced hyperleptinemia in type 1 diabetic mice (12) indicate that glucagon suppression normalizes all metabolic parameters for more than a month despite a total absence of insulin. More recently, the same antidiabetic efficacy has been demonstrated with recombinant leptin (11,23). However, leptin-induced actions other than suppression of glucagon, such as increased insulin-like growth factor-1 (IGF-1) (12) and insulin-like growth factor-binding protein-2 (IGFBP-2) (23), may have contributed. To obtain unassailable proof that glucagon action by itself causes the lethal consequences of insulin deficiency, we induced insulin deficiency in glucagon receptor-null (Gcgr^−/−) mice. Gcgr^−/− mice were treated with streptozotocin (STZ), the most commonly used β-cytotoxins in rodents, in an effort to achieve complete insulin deficiency in the complete absence of glucagon activity. We compared the metabolic phenotype of complete β-cell destruction in mice in which glucagon action had been transgenically abrogated by knockout of the glucagon receptor (24). Because Gcgr^−/− mice are resistant to STZ-induced β-cell destruction (24), it was necessary to use a double dose of the β-cell poison STZ. Despite β-cell destruction equivalent to the wild-type (Gcgr^+/+) mice, Gcgr^−/− remained free of all manifestations of insulin deficiency.     

The expression and function of glucose-dependent insulinotropic polypeptide in the embryonic mouse pancreas

OBJECTIVE

Glucose-dependent insulinotropic polypeptide (GIP) is a member of a structurally related group of hormones that also includes glucagon, glucagon-like peptides, and secretin. GIP is an incretin, known to modulate glucose-induced insulin secretion. Recent studies have shown that glucagon is necessary for early insulin-positive differentiation, and a similar role for incretins in regulating embryonic insulin-positive differentiation seems probable. Here we studied the role of GIP signaling in insulin-positive differentiation in the embryonic mouse pancreas.

RESEARCH DESIGN AND METHODS

The ontogeny of the GIP ligand and GIP receptor in the embryonic pancreas was investigated by immunohistochemistry and RT-PCR. GIP signaling was inhibited in cultured embryonic pancreata using morpholine-ring antisense against GIP ligand and receptor, or small interfering RNA (siRNA) for GIP ligand and receptor. Markers of endocrine cells and their progenitors were studied by immunohistochemistry and RT-PCR.

RESULTS

GIP and GIP receptor mRNA were both detected in the embryonic pancreas by embryonic day 9.5 and then persisted throughout gestation. GIP was generally coexpressed with glucagon by immunostaining. The GIP receptor was typically coexpressed with insulin. Morpholine-ring antisense or siRNA against either GIP ligand or GIP receptor both inhibited the differentiation of insulin-positive cells. Inhibition of GIP or its receptor also led to a decrease in the number of Pdx-1–positive and sox9-positive cells in the cultured embryonic pancreas. The number of Pax6- and Nkx2.2-positive cells, representative of developing pancreatic endocrine cells and β-cells, respectively, was also decreased.

CONCLUSIONS

GIP signaling may play a role in early embryonic pancreas differentiation to form insulin-positive cells or β-cells.Glucose-dependent insulinotropic polypeptide (GIP) is an incretin. Incretins are hormones released from the gut in response to nutrient ingestion that potentiate glucose-stimulated insulin secretion (1). GIP and glucagon-like peptide 1 (GLP-1) are the two main incretins, identified as mediators of the process by which administration of oral glucose provokes a greater stimulation of insulin release than does an intravenous glucose challenge (2). This connection between gut and the pancreatic islets has been termed the “enteroinsular axis” (3) and appears to be responsible for 50% of postprandial insulin release (2,4). GIP is released from enteroendocrine K-cells in the duodenum, primarily in response to the ingestion of glucose or fat, and potentiates insulin secretion in a glucose-dependent manner (5). A recent study now reports the presence of a bioactive form of GIP in the α-cells that promotes insulin secretion in the adult islet β-cells (6). GLP-1 is a proglucagon-derived peptide hormone that is synthesized and secreted by the enteroendocrine L-cells in the distal ileum and the colon. Preproglucagon can be alternatively processed to produce glicentin or oxyntomodulin and GLP-1 (7).The incretin GLP-1 can enhance β-cell growth and differentiation. GLP-1 receptor null mice, however, do not show a loss of β-cell development. Interestingly, these mice were found to have upregulated GIP release and GIP-induced insulin release (8). On the other hand, the GIP receptor (GIPR) null mice, which also did not show any obvious β-cell defect, showed enhanced sensitivity to GLP-1, suggesting that there may be important cooperation between these two incretin signaling pathways (9).We and others previously demonstrated that glucagon signaling to the glucagon receptor is necessary for early insulin-positive differentiation (10,11). This glucagon dependency also was suggested by the observation that Pax6 or prohormone convertase 2 null mice, which both lack glucagon-positive cells, also lack early insulin-positive differentiation (12,13). Although it was reported that GIP is produced in enteroendocrine K-cells in the duodenum of the adult, GIP was also found to be produced in the human fetal pancreas by 18 weeks’ gestation (14). The function of this GIP in the developing pancreas remains to be elucidated. Here we show that GIP is located in the embryonic mouse pancreas, and loss-of-function studies in vitro suggest that insulin differentiation in the embryonic pancreas depends on GIP signaling.     

Membrane Potential-Dependent Inactivation of Voltage-Gated Ion Channels in ��-Cells Inhibits Glucagon Secretion From Human Islets

OBJECTIVE

To document the properties of the voltage-gated ion channels in human pancreatic α-cells and their role in glucagon release.

RESEARCH DESIGN AND METHODS

Glucagon release was measured from intact islets. [Ca²⁺]_i was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated α-cells identified by immunocytochemistry.

RESULTS

Glucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl₂. Glucose remained inhibitory in the presence of ZnCl₂ and after blockade of type-2 somatostatin receptors. Human α-cells are electrically active at 1 mmol/l glucose. Inhibition of K_ATP-channels with tolbutamide depolarized α-cells by 10 mV and reduced the action potential amplitude. Human α-cells contain heteropodatoxin-sensitive A-type K⁺-channels, stromatoxin-sensitive delayed rectifying K⁺-channels, tetrodotoxin-sensitive Na⁺-currents, and low-threshold T-type, isradipine-sensitive L-type, and ω-agatoxin-sensitive P/Q-type Ca²⁺-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40–70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, ω-agatoxin, and isradipine. The [Ca²⁺]_i oscillations depend principally on Ca²⁺-influx via L-type Ca²⁺-channels. Capacitance measurements revealed a rapid (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below −20 mV and peaked at 0 mV. Blocking P/Q-type Ca²⁺-currents abolished depolarization-evoked exocytosis.

CONCLUSIONS

Human α-cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.Glucagon is the principal hyperglycemic hormone (1,2). It is secreted from the pancreatic α-cells in response to a fall in plasma glucose levels, β-adrenergic stimulation, lipids, and amino acids (3–5). Glucagon secretion from α-cells is regulated by paracrine (3), neuronal (6), and intrinsic mechanisms (7). Diabetes involves both impaired insulin and glucagon secretion (8). Thus, hyperglucagonemia is thought to contribute to elevated blood glucose levels, and the impaired glucagon response to hypoglycemia represents a limiting factor for insulin treatment in both type 1 and type 2 diabetes (9,10).Ion channels and electrical activity play a key role in the regulation of glucagon secretion. The properties of rodent α-cells have been characterized in some detail (5,11–13). Rodent α-cells are electrically excitable and electrically active in the absence of glucose. Action potential firing depends on the opening of voltage-activated L- and N-type Ca²⁺-channels, tetrodotoxin (TTX)-sensitive Na⁺-channels, and A-type K⁺-channels (14).The α-cells make up ∼35% of the cell population in human islets (15,16). Here, we have characterized the electrophysiological properties of isolated human α-cells and correlated our findings to changes in glucagon secretion from intact human islets. Our data indicate that glucagon secretion depends on a complex interplay among a number of depolarizing and repolarizing membrane currents.     

Influence of Insulin in the Ventromedial Hypothalamus on Pancreatic Glucagon Secretion In Vivo

OBJECTIVE

Insulin released by the β-cell is thought to act locally to regulate glucagon secretion. The possibility that insulin might also act centrally to modulate islet glucagon secretion has received little attention.

RESEARCH DESIGN AND METHODS

Initially the counterregulatory response to identical hypoglycemia was compared during intravenous insulin and phloridzin infusion in awake chronically catheterized nondiabetic rats. To explore whether the disparate glucagon responses seen were in part due to changes in ventromedial hypothalamus (VMH) exposure to insulin, bilateral guide cannulas were inserted to the level of the VMH and 8 days later rats received a VMH microinjection of either 1) anti-insulin affibody, 2) control affibody, 3) artificial extracellular fluid, 4) insulin (50 μU), 5) insulin receptor antagonist (S961), or 6) anti-insulin affibody plus a γ-aminobutyric acid A (GABA_A) receptor agonist muscimol, prior to a hypoglycemic clamp or under baseline conditions.

RESULTS

As expected, insulin-induced hypoglycemia produced a threefold increase in plasma glucagon. However, the glucagon response was fourfold to fivefold greater when circulating insulin did not increase, despite equivalent hypoglycemia and C-peptide suppression. In contrast, epinephrine responses were not altered. The phloridzin-hypoglycemia induced glucagon increase was attenuated (40%) by VMH insulin microinjection. Conversely, local VMH blockade of insulin amplified glucagon twofold to threefold during insulin-induced hypoglycemia. Furthermore, local blockade of basal insulin levels or insulin receptors within the VMH caused an immediate twofold increase in fasting glucagon levels that was prevented by coinjection to the VMH of a GABA_A receptor agonist.

CONCLUSIONS

Together, these data suggest that insulin''s inhibitory effect on α-cell glucagon release is in part mediated at the level of the VMH under both normoglycemic and hypoglycemic conditions.The pancreatic β-cell is the primary regulator of glucose homeostasis and glucagon secretion (1,2). In the presence of changes in blood glucose concentration, local changes in insulin together with zinc and/or γ-aminobutyric acid (GABA) release from the β-cell are thought to act in concert to regulate α-cell glucagon secretion (3,4). However, during hypoglycemia the central nervous system (5,6), and the VMH in particular, also plays a critical role in stimulating the release of glucagon and other counterregulatory hormones, via changes in the activity of specialized glucose-sensing neurons (7–10). Moreover, mice with selective loss of glutamate release in steroidogenic factor 1 neurons, which are expressed exclusively in the VMH, show a markedly impaired glucagon secretory response to acute hypoglycemia (11). These and other studies suggest that central regulation of pancreatic α-cell glucagon secretion may play a greater role than previously anticipated, at least during insulin-induced hypoglycemia (2,6–8).The fact that the VMH may directly influence α-cell glucagon release, and that glucose-sensing neurons in the VMH appear to use similar mechanisms for detecting fluctuations in glucose to the pancreatic α- and β-cells (7,8), suggested that insulin might play a similar regulatory role through a central mechanism. The possibility that insulin might exert a central effect is consistent with evidence that brain insulin levels mirror those in the circulation (12), that glucose-sensing neurons express insulin receptors, and that insulin alters the firing rate of these neurons (8,13–15). Moreover, insulin has been reported to act on the hypothalamus to regulate hepatic glucose production (16). However, studies in humans evaluating whether the level of circulating insulin during exogenous insulin administration influences counterregulatory hormone responses have yielded conflicting results. It has been reported that higher levels of plasma insulin may amplify (17,18) or inhibit (19–21) epinephrine responses during hypoglycemia. One study noted a significant effect of circulating insulin on accompanying glucagon responses, specifically an inhibitory action (20). Whether insulin acts centrally to influence glucose counterregulation is also uncertain. In animal studies, insulin delivered directly into the brain in very large doses has been reported to amplify counterregulatory hormone secretion (22–24) or to have no direct effect (25).This study was undertaken to determine whether insulin acts within the ventromedial hypothalamus (VMH) to modulate the secretion of glucoregulatory hormones. For this purpose, we either blocked insulin action or administered insulin to the VMH, during hypoglycemia produced in the presence or absence of elevated circulating insulin levels, respectively. In addition, we examined whether insulin acts locally in the VMH under basal conditions to regulate glucoregulatory hormone secretion in the absence of elevated levels of insulin. Our findings provide in vivo evidence for the first time that insulin acts within the VMH to influence the release of glucagon from α-cells both during hypoglycemia and under basal conditions.     

Effect of Endogenous GLP-1 on Insulin Secretion in Type 2 Diabetes

OBJECTIVE

The incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) account for up to 60% of postprandial insulin release in healthy people. Previous studies showed a reduced incretin effect in patients with type 2 diabetes but a robust response to exogenous GLP-1. The primary goal of this study was to determine whether endogenous GLP-1 regulates insulin secretion in type 2 diabetes.

METHODS

Twelve patients with well-controlled type 2 diabetes and eight matched nondiabetic subjects consumed a breakfast meal containing d-xylose during fixed hyperglycemia at 5 mmol/l above fasting levels. Studies were repeated, once with infusion of the GLP-1 receptor antagonist, exendin-(9–39) (Ex-9), and once with saline.

RESULTS

The relative increase in insulin secretion after meal ingestion was comparable in diabetic and nondiabetic groups (44 ± 4% vs. 47 ± 7%). Blocking the action of GLP-1 suppressed postprandial insulin secretion similarly in the diabetic and nondiabetic subjects (25 ± 4% vs. 27 ± 8%). However, Ex-9 also reduced the insulin response to intravenous glucose (25 ± 5% vs. 26 ± 7%; diabetic vs. nondiabetic subjects), when plasma GLP-1 levels were undetectable. The appearance of postprandial ingested d-xylose in the blood was not affected by Ex-9.

CONCLUSIONS

These findings indicate that in patients with well-controlled diabetes, the relative effects of enteral stimuli and endogenous GLP-1 to enhance insulin release are retained and comparable with those in nondiabetic subjects. Surprisingly, GLP-1 receptor signaling promotes glucose-stimulated insulin secretion independent of the mode of glucose entry. Based on rates of d-xylose absorption, GLP-1 receptor blockade did not affect gastric emptying of a solid meal.Glucagon-like peptide 1 (GLP-1) is a gut-brain peptide that is a major component of the incretin effect and is essential for normal glucose tolerance (1). Based on studies in which synthetic GLP-1, or GLP-1 receptor (GLP-1r) agonists, is administered to humans, GLP-1 has a broad range of actions that promote glucose homeostasis, including stimulating insulin secretion (2), suppressing glucagon release (3–4), delaying gastric emptying (5), and increasing hepatic glucose balance (6–7). Importantly, and unlike other insulinotropic gut peptides, the effects of GLP-1 on glucose metabolism are retained in people with diabetes (8–10). This has led to the development of novel therapeutic compounds for use in diabetic patients that are based on GLP-1r signaling (11).The physiologic role of GLP-1 in individuals with diabetes has not been determined. However, there are several reasons to question whether the GLP-1 system is fully functional in this patient group. First, there is some evidence that GLP-1 secretion in response to meal ingestion in type 2 diabetes is impaired (12–15), although this finding has not been uniform (16–17). Second, the sensitivity of insulin secretion to exogenous GLP-1 is reduced in diabetic individuals (18). Finally, it has long been believed that the augmentation of glucose-stimulated insulin secretion during enteral glucose absorption, the incretin effect, is severely attenuated in type 2 diabetes, implying that incretins such as GLP-1 are not normally active in this group of subjects.In this study, we tested the hypothesis that the effect of endogenous GLP-1 to promote insulin secretion after meal ingestion is reduced in people with diabetes. Diabetic subjects and age- and weight-matched nondiabetic subjects were studied with and without infusion of the specific GLP-1r antagonist, exendin-(9–39) (Ex-9), during fixed hyperglycemia before and after a breakfast meal.     

Exogenous Glucose�CDependent Insulinotropic Polypeptide Worsens Post prandial Hyperglycemia in T ype 2 Diabetes

OBJECTIVE

Glucose-dependent insulinotropic polypeptide (GIP), unlike glucagon-like peptide (GLP)-1, lacks glucose-lowering properties in patients with type 2 diabetes. We designed this study to elucidate the underlying pathophysiology.

RESEARCH DESIGN AND METHODS

Twenty-two insulin-naïve subjects with type 2 diabetes were given either synthetic human GIP (20 ng · kg⁻¹ · min⁻¹) or placebo (normal saline) over 180 min, starting with the first bite of a mixed meal (plus 1 g of acetaminophen) on two separate occasions. Frequent blood samples were obtained over 6 h to determine plasma GIP, GLP-1, glucose, insulin, glucagon, resistin, and acetaminophen levels.

RESULTS

Compared with placebo, GIP induced an early postprandial increase in insulin levels. Intriguingly, GIP also induced an early postprandial augmentation in glucagon, a significant elevation in late postprandial glucose, and a decrease in late postprandial GLP-1 levels. Resistin and acetaminophen levels were comparable in both interventions. By immunocytochemistry, GIP receptors were present on human and mouse α-cells. In αTC1 cell line, GIP induced an increase in intracellular cAMP and glucagon secretion.

CONCLUSIONS

GIP, given to achieve supraphysiological plasma levels, still had an early, short-lived insulinotropic effect in type 2 diabetes. However, with a concomitant increase in glucagon, the glucose-lowering effect was lost. GIP infusion further worsened hyperglycemia postprandially, most likely through its suppressive effect on GLP-1. These findings make it unlikely that GIP or GIP receptor agonists will be useful in treating the hyperglycemia of patients with type 2 diabetes.In response to glucose and fat in digested food, two enteroendocrine hormones, glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP), are secreted froml- andk-cells, respectively, in the gut. GLP-1 and GIP play important roles in postprandial glucose homeostasis. In healthy individuals, the potent insulinotropic effects of GLP-1 and GIP account for up to 60% of the insulin secreted postprandially (1). Exogenous GLP-1 acts to improve glycemic control in patients with type 2 diabetes by 1) stimulating insulin secretion in a glucose concentration–dependent manner during the fasted state, 2) suppressing glucagon secretion in the presence of hyperglycemia and euglycemia but not hypoglycemia, and 3) decelerating gastric emptying, leading to a delay in the absorption of ingested nutrients and a dampening of postprandial glucose excursion (2–4). However, it is still unclear which of these properties of exogenous GLP-1 plays a more prominent role in lowering postprandial glucose (5). GIP has not been studied as extensively as GLP-1. Similar to GLP-1, the insulinotropic effect of GIP in healthy humans is glucose concentration dependent under glucose clamp conditions (6). Unlike GLP-1, the administration of GIP in healthy humans was reported to have a dose-dependent glucagonotropic effect during euglycemia and no effect during hyperglycemic clamp conditions (6–8). Also, unlike GLP-1, GIP has no effect on gastric emptying (9).In patients with type 2 diabetes compared with healthy subjects, the ability of GLP-1 to stimulate insulin was noted to be 71%, while that of GIP was 46%; however, the glucose-lowering effect of GLP-1 was relatively preserved while that of GIP was absent (7,10–12). The underlying pathophysiology associated with this loss of glucose-lowering effect of GIP in humans is not known. Some hypotheses include defective expression of GIP receptors (13), accelerated degradation of GIP receptors (14), and downregulation of GIP signaling (15). It is argued that genetic components or GIP receptor defects do not play a role in the reduced insulinotropic response to GIP because patients with different types of diabetes, such as from chronic pancreatitis, latent autoimmune diabetes in adults, maturity-onset diabetes in the young, and newly diagnosed type 1 diabetes, also have impaired insulin response to GIP, thus suggesting that metabolic abnormalities may be the cause (16).The underlying pathophysiology associated with this loss of glucose-lowering effect of GIP despite still having some insulinotropic effect in humans is not known. The aims of this study were 1) to ascertain if a dose of GIP designed to elevate plasma GIP levels to fivefold of that observed postmeal might lower blood glucose in patients with type 2 diabetes and 2) to gain insight into the pathophysiology underlying the seeming lack of effects of GIP on glucose homeostasis in patients with type 2 diabetes.     

Beta-cell uncoupling protein 2 regulates reactive oxygen species production, which influences both insulin and glucagon secretion

OBJECTIVE

The role of uncoupling protein 2 (UCP2) in pancreatic β-cells is highly debated, partly because of the broad tissue distribution of UCP2 and thus limitations of whole-body UCP2 knockout mouse models. To investigate the function of UCP2 in the β-cell, β-cell–specific UCP2 knockout mice (UCP2BKO) were generated and characterized.

RESEARCH DESIGN AND METHODS

UCP2BKO mice were generated by crossing loxUCP2 mice with mice expressing rat insulin promoter-driven Cre recombinase. Several in vitro and in vivo parameters were measured, including respiration rate, mitochondrial membrane potential, islet ATP content, reactive oxygen species (ROS) levels, glucose-stimulated insulin secretion (GSIS), glucagon secretion, glucose and insulin tolerance, and plasma hormone levels.

RESULTS

UCP2BKO β-cells displayed mildly increased glucose-induced mitochondrial membrane hyperpolarization but unchanged rates of uncoupled respiration and islet ATP content. UCP2BKO islets had elevated intracellular ROS levels that associated with enhanced GSIS. Surprisingly, UCP2BKO mice were glucose-intolerant, showing greater α-cell area, higher islet glucagon content, and aberrant ROS-dependent glucagon secretion under high glucose conditions.

CONCLUSIONS

Using a novel β-cell–specific UCP2KO mouse model, we have shed light on UCP2 function in primary β-cells. UCP2 does not behave as a classical metabolic uncoupler in the β-cell, but has a more prominent role in the regulation of intracellular ROS levels that contribute to GSIS amplification. In addition, β-cell UCP2 contributes to the regulation of intraislet ROS signals that mediate changes in α-cell morphology and glucagon secretion.Uncoupling protein 2 (UCP2) was discovered based on sequence homology to UCP1 (1), a well-studied UCP involved in thermogenesis. UCP1 induces a strong proton leak in the inner mitochondrial membrane, which dramatically dissipates the proton motive force (PMF), consequently halting the driving force for ATP production and dissipating energy as heat (2). Despite homology to UCP1, the precise physiological function of UCP2 remains unclear (3). A mild metabolic uncoupling function whereby UCP2 facilitates a proton leak, particularly when activated by superoxide or lipid peroxidation products, has been demonstrated (4–6); however, evidence exists that disputes this classical metabolic uncoupling function (7–9).A growing body of evidence now suggests that UCP2 contributes to the control of mitochondrial-derived reactive oxygen species (ROS) production (3,4,10,11). This may provide an important mechanism to fine-tune mitochondria-generated ROS signals that regulate cell function and/or to prevent oxidative stress, a condition that results from chronic ROS accumulation and ultimately leads to oxidative damage and cytotoxicity (12,13).To combat oxidative stress, β-cells express relatively high amounts of the superoxide dismutase (SOD) family of antioxidants (∼50% of that found in liver), which convert superoxide into hydrogen peroxide (H₂O₂), yet β-cells have relatively low expression of H₂O₂-scavenging enzymes (1% of that found in liver) (14). Some argue that this makes β-cells particularly susceptible to oxidative stress and cytotoxicity, whereas others argue that this creates an environment highly sensitive to ROS-related signaling. Since ROS production is directly coupled to the metabolic rate in most tissues (15), ROS could provide a vital regulatory link between glucose metabolism and insulin secretion (16–18), and UCP2 may be an important regulator of such ROS-related signals.Since its discovery, numerous studies have demonstrated a negative link between UCP2 and β-cell function (1). UCP2 expression is upregulated in response to chronic high glucose (19,20) and fatty acid exposure (19,21–23) and is thus associated with obesity, hyperglycemia, and type 2 diabetes. More recently, mutations in the gene expressing UCP2 have been directly associated with congenital hyperinsulinemia in humans, further demonstrating this link between UCP2 and insulin secretion (24). Approximately a decade ago, whole-body UCP2 knockout (UCP2KO) mice were created on a mixed 129/SVJxC57BL/6 background (25) to explore UCP2 function in the β-cell. UCP2KO mice have reduced blood glucose levels, improved glucose tolerance, higher islet ATP content, enhanced glucose-stimulated insulin secretion (GSIS) (25), and increased intracellular ROS levels in islet cells (26,27) compared to control mice. Similar results have been demonstrated in rat insulinoma β-like cells (INS-1E), where acute knockdown of UCP2 also increased intracellular ROS and enhanced GSIS (18). However, this view of UCP2 as a negative regulator of GSIS has not been consistently supported. Backcrossing UCP2KO mice for several generations onto highly congenic background strains resulted in increased oxidative stress and impaired GSIS (28). Although the precise contribution of genetic background to these disparate effects of UCP2 on GSIS is currently unknown and is an issue that requires cautious interpretation of results, it seems that UCP2 commonly regulates ROS in all strains, further highlighting the importance of UCP2 in ROS regulation.Until now, whole-body UCP2KO mouse models have been widely used to study the role of UCP2 in β-cell function (23,25–28). However, these models can be problematic because UCP2 deletion in other tissues and cell types, including brain and other islet cells (i.e., α-cells) (25,29,30), can affect glucose-sensing and glucose homeostasis (31,32). To elucidate the function of UCP2 in the β-cell, we have created and characterized a novel β-cell–specific UCP2KO mouse (UCP2BKO). Here, we show that UCP2 does not behave as a true uncoupler in the β-cell, but rather contributes to the regulation of β-cell ROS, which in turn regulates GSIS. In addition, we suggest that β-cell UCP2 regulates intraislet ROS signals that can target and regulate the function of neighboring glucagon-secreting α-cells.     

GPR119 Is Essential for Oleoylethanolamide-Induced Glucagon-Like Peptide-1 Secretion From the Intestinal Enteroendocrine L-Cell

OBJECTIVE

Intestinal L-cells secrete the incretin glucagon-like peptide-1 (GLP-1) in response to ingestion of nutrients, especially long-chain fatty acids. The Gαs-coupled receptor GPR119 binds the long-chain fatty acid derivate oleoylethanolamide (OEA), and GPR119 agonists enhance GLP-1 secretion. We therefore hypothesized that OEA stimulates GLP-1 release through a GPR119-dependent mechanism.

RESEARCH DESIGN AND METHODS

Murine (m) GLUTag, human (h) NCI-H716, and primary fetal rat intestinal L-cell models were used for RT-PCR and for cAMP and GLP-1 radioimmunoassay. Anesthetized rats received intravenous or intraileal OEA, and plasma bioactive GLP-1, insulin, and glucose levels were determined by enzyme-linked immunosorbent assay or glucose analyzer.

RESULTS

GPR119 messenger RNA was detected in all L-cell models. OEA treatment (10 μmol/l) of mGLUTag cells increased cAMP levels (P < 0.05) and GLP-1 secretion (P < 0.001) in all models, with desensitization of the secretory response at higher concentrations. GLP-1 secretion was further enhanced by prevention of OEA degradation using the fatty acid amide hydrolase inhibitor, URB597 (P < 0.05–0.001 vs. OEA alone), and was abolished by H89-induced inhibition of protein kinase A. OEA-induced cAMP levels and GLP-1 secretion were significantly reduced in mGLUTag cells transfected with GPR119-specific small interfering RNA (P < 0.05). Application of OEA (10 μmol/l) directly into the rat ileum, but not intravenously, increased plasma bioactive GLP-1 levels in euglycemic animals by 1.5-fold (P < 0.05) and insulin levels by 3.9-fold (P < 0.01) but only in the presence of hyperglycemia.

CONCLUSIONS

The results of these studies demonstrate, for the first time, that OEA increases GLP-1 secretion from intestinal L-cells through activation of the novel GPR119 fatty acid derivate receptor in vitro and in vivo.Glucagon-like peptide-1 (GLP-1) is an intestinal hormone with potent insulinotropic effects that are essential to the maintenance of normal glucose homeostasis (1). In addition to glucose-dependent stimulation of insulin secretion, GLP-1 shows other favorable effects, increasing β-cell proliferation in rodents, as well as enhancing β-cell survival in both rodent and in human islets (2,3). Additionally, GLP-1 has been shown to protect cardiomyocytes from ischemia, and GLP-1 infusion improves cardiac function in patients with heart failure (4,5). Finally, the central nervous system effects of GLP-1 include inhibition of gastric emptying, reduction of appetite, and promotion of satiety in humans (6,7) and rodents (8,9). As a result of its potent antidiabetes and anorexic effects, GLP-1 analogs and GLP-1 degradation inhibitors have been successfully introduced to the clinic for pharmacologic treatment of patients with type 2 diabetes (10).While the biological effects of GLP-1 have been well established, the mechanisms underlying GLP-1 secretion are less well understood. GLP-1 is secreted from intestinal endocrine L-cells, localized predominantly in the distal ileum and colon (11). Rapid GLP-1 release after food intake (12,13) may be regulated by afferent innervation by the vagus nerve (14,15) as well as, in rodents, by proximal gut hormones, such as glucose-dependent insulinotropic peptide (GIP) from the duodenal K-cells (16). However, L-cells also release GLP-1 in response to direct stimulation by nutrients (16), such as carbohydrates and, most notably, fat (17,18). Monounsaturated long-chain fatty acids, such as oleic acid, are strong stimulators of GLP-1 secretion from the L-cells, both in vitro and in vivo, through a signaling pathway that requires protein kinase C (PKC) ζ (17,18). Additional studies have indicated roles for the orphan G-protein–coupled receptors, GPR40 and GPR120, in the response of the L-cell to saturated fat and α-linolenic acid, respectively (19,20). Very recently, the fatty acid derivate receptor, GPR119, was also found to be expressed in a highly tissue-specific fashion by the intestinal L-cell and the pancreatic β-cell (21,22). Furthermore, a GPR119-specific pharmacological agonist was demonstrated to increase the plasma levels of both GLP-1 and insulin in mice. However, the relevance of physiological ligands of GPR119 to GLP-1 secretion by the L-cell currently remains unknown.Oleoylethanolamide (OEA) and lysophosphatidylcholine (LPC) are endogenously occurring fatty acid derivates that are specific ligands of GPR119 (23,24). While LPC is often associated with pathophysiological processes such as atherosclerosis (25), OEA is found in a variety of tissues, including the intestinal epithelium, under physiological conditions (26). OEA is synthesized in vivo from membrane phospholipids through an N-acylphosphatidylethanolamine (NAPE)-phospholipase D (PLD)-dependent pathway (27); OEA can also be degraded into oleic acid and ethanolamide by the naturally occurring enzyme fatty acid amide hydrolase (FAAH), which is also expressed by the intestinal epithelium (28,29). Intestinal OEA levels are known to decrease during fasting and increase upon refeeding, and OEA administration to rats reduces food intake, suggesting a role for OEA in the regulation of satiety (26,30–32). As OEA was first identified as a ligand for the intranuclear peroxisome proliferator–activated receptor (PPAR) α, it has been generally assumed that the appetite reduction is dependent on PPARα activation (33). However, as GLP-1 is known to induce satiety, we hypothesized that OEA may also stimulate GLP-1 secretion from the intestinal L-cells in a GPR119-dependent fashion.     

Loss of inverse relationship between pulsatile insulin and glucagon secretion in patients with type 2 diabetes

OBJECTIVE

In patients with type 2 diabetes, glucagon levels are often increased. Furthermore, pulsatile secretion of insulin is disturbed in such patients. Whether pulsatile glucagon secretion is altered in type 2 diabetes is not known.

RESEARCH DESIGN AND METHODS

Twelve patients with type 2 diabetes and 13 nondiabetic individuals were examined in the fasting state and after mixed meal ingestion. Deconvolution analyses were performed on insulin and glucagon concentration time series sampled at 1-min intervals.

RESULTS

Both insulin and glucagon were secreted in distinct pulses, occurring at ∼5-min intervals. In patients with diabetes, postprandial insulin pulse mass was reduced by 74% (P < 0.001). Glucagon concentrations were increased in the patients during fasting and after meal ingestion (P < 0.05), specifically through an increased glucagon pulse mass (P < 0.01). In healthy subjects, the increase in postprandial insulin levels was inversely related to respective glucagon levels (P < 0.05). This relationship was absent in the fasting state and in patients with diabetes.

CONCLUSIONS

Glucagon and insulin are secreted in a coordinated, pulsatile manner. A plausible model is that the postprandial increase in insulin burst mass represses the corresponding glucagon pulses. Disruption of the insulin–glucagon interaction in patients with type 2 diabetes could potentially contribute to hyperglucagonemia.The pathogenesis of type 2 diabetes involves multiple metabolic defects, the most important ones likely being β-cell dysfunction and insulin resistance (1,2). In addition, abnormal regulation of glucagon secretion contributes to the hyperglycemia in diabetic patients (3–5), and a number of studies have reported elevated fasting glucagon concentrations in patients with type 2 diabetes as well as in individuals with impaired glucose tolerance (3,6,7). Furthermore, whereas glucagon levels typically decline after oral or intravenous glucose administration in healthy individuals, the glucose-induced suppression of glucagon secretion is markedly impaired in patients with type 2 diabetes, and mixed meal–induced glucagon excursions are typically exaggerated in such patients (3,6). The inappropriately elevated glucagon levels may contribute to the exaggerated hepatic glucose production that characterizes patients with type 2 diabetes (8).The mechanistic reasons underlying increased glucagon secretion in such patients are less well understood. Thus, some studies have reported increased numbers of α-cells in the diabetic pancreas (9,10). An alternative hypothesis is the lack of α-cell inhibition by insulin in diabetic patients (11,12). Indeed, suppression of glucagon secretion by insulin has been well established in various in vitro and in vivo models (13), and a selective loss of β-cells has been associated with the development of hyperglucagonemia (14). It has also been demonstrated that the glucagon response to hypoglycemia is lost in the absence of insulin (11,15).Secretion of insulin from pancreatic islets in nondiabetic individuals is regulated in a pulsatile manner, with distinct bursts of insulin release occurring approximately every 5 min (16–18). In contrast, the amplitude and the orderliness of insulin secretion are markedly reduced in patients with type 2 diabetes (19–24). Impaired insulin pulsatility has been suggested to contribute to the development of insulin resistance in such patients (18,20,25).For glucagon, a pulsatile secretion pattern has been reported in different large animal models (26,27). Based on these studies, a close interaction between insulin and glucagon secretion has been suggested. To examine this relationship in more detail, previous studies have examined insulin and glucagon levels in pigs before and after a selective β-cell reduction induced by the β-cytotoxin alloxan (14). It is noteworthy that there was a significant inverse relationship between postprandial insulin and glucagon secretion in healthy animals, but this pulsatile intra-islet inhibition of glucagon secretion by insulin was lost after reduction of β-cell mass, leading to overt hyperglucagonemia. Such studies have prompted speculation that reduction of intra-islet insulin secretion might also cause insufficient suppression of glucagon in patients with type 2 diabetes (14). However, to date, a pulsatile pattern of glucagon secretion has not been established in humans.Therefore, in the present studies we addressed the following questions. (1) Is there evidence of a pulsatile pattern of glucagon secretion in humans? (2) Is there an inverse relationship between insulin and glucagon secretion? (3) Are the time patterns of glucagon secretion and its interactions with insulin secretion different in normal subjects and patients with type 2 diabetes?     

Coadministration of Glucagon-Like Peptide-1 During Glucagon Infusion in Humans Results in Increased Energy Expenditure and Amelioration of Hyperglycemia

Glucagon and glucagon-like peptide (GLP)-1 are the primary products of proglucagon processing from the pancreas and gut, respectively. Giving dual agonists with glucagon and GLP-1 activity to diabetic, obese mice causes enhanced weight loss and improves glucose tolerance by reduction of food intake and by increase in energy expenditure (EE). We aimed to observe the effect of a combination of glucagon and GLP-1 on resting EE and glycemia in healthy human volunteers. In a randomized, double-blinded crossover study, 10 overweight or obese volunteers without diabetes received placebo infusion, GLP-1 alone, glucagon alone, and GLP-1 plus glucagon simultaneously. Resting EE—measured using indirect calorimetry—was not affected by GLP-1 infusion but rose significantly with glucagon alone and to a similar degree with glucagon and GLP-1 together. Glucagon infusion was accompanied by a rise in plasma glucose levels, but addition of GLP-1 to glucagon rapidly reduced this excursion, due to a synergistic insulinotropic effect. The data indicate that drugs with glucagon and GLP-1 agonist activity may represent a useful treatment for type 2 diabetes and obesity. Long-term studies are required to demonstrate that this combination will reduce weight and improve glycemia in patients.Glucagon and glucagon-like peptide (GLP)-1 are, respectively, pancreatic and intestinal hormones derived from the same proglucagon peptide but with divergent roles in metabolism. Glucagon has primarily been characterized as a counterregulatory hormone that responds to hypoglycemia and fasting by stimulating glycogenolysis and gluconeogenesis, as well as hepatic fatty acid β-oxidation and ketogenesis (1). Glucagon is an integral part of the body’s neurohormonal response to stress together with cortisol and catecholamines (2). On the other hand, GLP-1 is released postprandially and has primary roles in enhancing the β-cell insulin response to eating, enhancing β-cell survival, inhibiting gastric emptying, and suppressing appetite (3). A third proglucagon derivative, oxyntomodulin, possesses both glucagon receptor (GcgR) and GLP-1 receptor (GLP-1R) agonist activity (4,5) and is also released from the gut postprandially.GLP-1 and its analogs are used for their insulinotropic actions as therapies for type 2 diabetes. As GLP-1 suppresses appetite (6), patients generally experience weight loss with GLP-1 analog therapy (3). However, the magnitude of weight loss is restricted by dose-limiting nausea and vomiting (7), as well as by the fact that GLP-1 tends to reduce energy expenditure (EE) (8). In this connection, glucagon has emerged as a suitable therapeutic partner for GLP-1. Glucagon is also known to reduce appetite (9) but in addition increases EE (10). The combination of GLP-1 and glucagon therefore makes an attractive proposition for obesity therapy. Consistent with this hypothesis, the weak GcgR/GLP-1R coagonist oxyntomodulin is known to suppress appetite (11) and to increase EE, causing considerable weight loss in obese volunteers (12,13).The hyperglycemic effect of glucagon has deterred investigation of its potential as an obesity treatment. However, oxyntomodulin and other GcgR/GLP-1R coagonists have been shown to have neutral or beneficial glycemic effects in rodents with diet-induced obesity (14–16). This has been hypothesized by others to be due to one or more mechanisms: 1) intrinsic GLP-1R agonism having an effect opposing and neutralizing that mediated by GcgR stimulation, 2) the metabolic benefits of body weight loss outweighing any diabetogenic effect of GcgR stimulation, and 3) an unexpectedly beneficial metabolic effect of sustained GcgR stimulation (15).These observations suggest that combined administration of GcgR and GLP-1R agonists, or of a single coagonist, could be useful to treat type 2 diabetes and obesity. However, the separate and combined effects of GLP-1 and glucagon on EE and glycemia have not previously been demonstrated in humans. We therefore decided to investigate the effects of the GLP-1 and glucagon combination in healthy human volunteers. Specifically, we wished to confirm that glucagon increases resting EE and that this effect is retained when GLP-1 is combined with glucagon. Secondly, we wished to confirm that GLP-1 is able to ameliorate the hyperglycemia induced by glucagon.     

Chronic Administration of the Glucagon-Like Peptide-1 Analog, Liraglutide, Delays the Onset of Diabetes and Lowers Triglycerides in UCD-T2DM Rats

OBJECTIVE

The efficacy of liraglutide, a human glucagon-like peptide-1 (GLP-1) analog, to prevent or delay diabetes in UCD-T2DM rats, a model of polygenic obese type 2 diabetes, was investigated.

RESEARCH DESIGN AND METHODS

At 2 months of age, male rats were divided into three groups: control, food-restricted, and liraglutide. Animals received liraglutide (0.2 mg/kg s.c.) or vehicle injections twice daily. Restricted rats were food restricted to equalize body weights to liraglutide-treated rats. Half of the animals were followed until diabetes onset, whereas the other half of the animals were killed at 6.5 months of age for tissue collection.

RESULTS

Before diabetes onset energy intake, body weight, adiposity, and liver triglyceride content were higher in control animals compared with restricted and liraglutide-treated rats. Energy-restricted animals had lower food intake than liraglutide-treated animals to maintain the same body weights, suggesting that liraglutide increases energy expenditure. Liraglutide treatment delayed diabetes onset by 4.1 ± 0.8 months compared with control (P < 0.0001) and by 1.3 ± 0.8 months compared with restricted animals (P < 0.05). Up to 6 months of age, energy restriction and liraglutide treatment lowered fasting plasma glucose and A1C concentrations compared with control animals. In contrast, liraglutide-treated animals exhibited lower fasting plasma insulin, glucagon, and triglycerides compared with both control and restricted animals. Furthermore, energy-restricted and liraglutide-treated animals exhibited more normal islet morphology.

CONCLUSIONS

Liraglutide treatment delays the development of diabetes in UCD-T2DM rats by reducing energy intake and body weight, and by improving insulin sensitivity, improving lipid profiles, and maintaining islet morphology.Targeting of glucagon-like peptide-1 (GLP-1) for the pharmaceutical treatment of type 2 diabetes has shown much promise, as demonstrated by the clinical success of GLP-1 agonists and dipeptidyl peptidase IV (DPP-IV) inhibitors (1,2). As an incretin hormone, GLP-1 potentiates glucose-induced insulin secretion, avoiding hypoglycemia observed with other pharmaceutical activators of insulin secretion such as sulfonylureas (3,4). GLP-1 has also been shown to reduce excess glucagon secretion, contributing to a reduction in hyperglycemia (1), and has been suggested to reduce inflammation (5). GLP-1 signaling increases satiety (6,7) and slows gastric motility and secretion, further contributing to a reduction in food intake (8). Furthermore, GLP-1 has been shown to increase β-cell differentiation, proliferation, and insulin synthesis and decrease β-cell apoptosis (9–11). Finally, administration of GLP-1 can improve insulin sensitivity by promoting peripheral glucose uptake and decreasing hepatic gluconeogenesis, independent of changes of pancreatic hormone secretion (12–16).Early attempts to harness these antidiabetic effects through the production of GLP-1 analogs were complicated by the short half-life of endogenous GLP-1 (<2 min) because of rapid degradation by the ubiquitously expressed protease, DPP-IV (17). Thus, one method currently being pursued for the therapeutic targeting of GLP-1 is production of GLP-1 analogs that are resistant to degradation by DPP-IV. Liraglutide is a GLP-1 analog with an additional 16-carbon fatty acid and a small amino acid–based spacer that confers reversible binding of the agonist to albumin and increases resistance to DPP-IV activity, providing liraglutide with a half-life of approximately 13 h (18,19). These properties allow once-daily subcutaneous administration of liraglutide for the treatment of type 2 diabetes.The efficacy of liraglutide for the treatment of type 2 diabetes has been demonstrated in a number of clinical studies, with patients showing significant decreases in body weight, glucose, A1C, and blood pressure (20–25). However, preclinical studies of the long-term use of chronic liraglutide administration for the prevention of type 2 diabetes have not been conducted. With the increasing prevalence of type 2 diabetes, preventive measures are urgently needed. Thus, this study investigated the metabolic effects of chronic liraglutide administration and the potential of liraglutide to prevent or delay the development of type 2 diabetes in pre-diabetic UCD-T2DM rats. The UCD-T2DM rat model develops polygenic adult-onset obesity and insulin resistance, without a monogenic deficit in leptin signaling, followed by inadequate β-cell compensation and diabetes in both male and female animals (26). Thus, the UCD-T2DM rat more closely models the pathogenesis of type 2 diabetes in humans than other currently available models. Furthermore, UCD-T2DM rats demonstrate a later age of diabetes onset than other rodent models of type 2 diabetes, such as the ZDF rat, making them highly suitable for diabetes prevention studies (26,27).