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.     

Dipeptidyl peptidase IV-resistant [D-Ala(2)]glucose-dependent insulinotropic polypeptide (GIP) improves glucose tolerance in normal and obese diabetic rats

The therapeutic potential of glucose-dependent insulinotropic polypeptide (GIP) for improving glycemic control has largely gone unstudied. A series of synthetic GIP peptides modified at the NH(2)-terminus were screened in vitro for resistance to dipeptidyl peptidase IV (DP IV) degradation and potency to stimulate cyclic AMP and affinity for the transfected rat GIP receptor. In vitro experiments indicated that [D-Ala(2)]GIP possessed the greatest resistance to enzymatic degradation, combined with minimal effects on efficacy at the receptor. Thus, [D-Ala(2)]GIP(1--42) was selected for further testing in the perfused rat pancreas and bioassay in conscious Wistar and Zucker rats. When injected subcutaneously in normal Wistar, Fa/?, or fa/fa Vancouver Diabetic Fatty (VDF) Zucker rats, both GIP and [D-Ala(2)]GIP significantly reduced glycemic excursions during a concurrent oral glucose tolerance test via stimulation of insulin release. The latter peptide displayed greater in vivo effectiveness, likely because of resistance to enzymatic degradation. Hence, despite reduced bioactivity in diabetic models at physiological concentrations, GIP and analogs with improved plasma stability still improve glucose tolerance when given in supraphysiological doses, and thus may prove useful in the treatment of diabetic states.     

Matrix metalloproteinases contribute to insulin insufficiency in Zucker diabetic fatty rats

To assess the molecular changes associated with pancreatic beta-cell dysfunction occurring during the onset of type 2 diabetes, we profiled pancreatic islet mRNAs from diabetic male and high-fat-fed female Zucker diabetic fatty (ZDF) rats and their nondiabetic lean counterparts on custom islet-specific oligonucleotide arrays. The most prominent changes in both the male and female models of type 2 diabetes were increases in the mRNAs encoding proteases and extracellular matrix components that are associated with tissue remodeling and fibrosis. The mRNAs for metalloproteinase (MMP)-2, -12, and -14 were sharply increased with the onset of islet dysfunction and diabetes. Zymography of islet extracts revealed a concurrent, >10-fold increase in MMP-2 protease activity in islets from 9-week-old male ZDF rats. Treatment of female ZDF rats receiving a diabetogenic diet with PD166793, a broad-spectrum MMP inhibitor, substantially prevented diabetes. The effect of this compound was due in part to marked beta-cell expansion. These studies indicate that MMPs contribute to islet fibrosis and insulin insufficiency in ZDF rats. Class-targeted protease inhibitors should be explored for their potential therapeutic utility in preservation of beta-cell mass in type 2 diabetes.     

Glucose-dependent insulinotropic polypeptide: a bifunctional glucose-dependent regulator of glucagon and insulin secretion in humans

OBJECTIVE

To evaluate the glucose dependency of glucose-dependent insulinotropic polypeptide (GIP) effects on insulin and glucagon release in 10 healthy male subjects ([means ± SEM] aged 23 ± 1 years, BMI 23 ± 1 kg/m², and HbA_1c 5.5 ± 0.1%).

RESEARCH DESIGN AND METHODS

Saline or physiological doses of GIP were administered intravenously (randomized and double blinded) during 90 min of insulin-induced hypoglycemia, euglycemia, or hyperglycemia.

RESULTS

During hypoglycemia, GIP infusion caused greater glucagon responses during the first 30 min compared with saline (76 ± 17 vs. 28 ± 16 pmol/L per 30 min, P < 0.008), with similar peak levels of glucagon reached after 60 min. During euglycemia, GIP infusion elicited larger glucagon responses (62 ± 18 vs. −11 ± 8 pmol/L per 90 min, P < 0.005). During hyperglycemia, comparable suppression of plasma glucagon (−461 ± 81 vs. −371 ± 50 pmol/L per 90 min, P = 0.26) was observed with GIP and saline infusions. In addition, during hyperglycemia, GIP more than doubled the insulin secretion rate (P < 0.0001).

CONCLUSIONS

In healthy subjects, GIP has no effect on glucagon responses during hyperglycemia while strongly potentiating insulin secretion. In contrast, GIP increases glucagon levels during fasting and hypoglycemic conditions, where it has little or no effect on insulin secretion. Thus, GIP seems to be a physiological bifunctional blood glucose stabilizer with diverging glucose-dependent effects on the two main pancreatic glucoregulatory hormones.The regulation of pancreatic islet function is crucial in glucose homeostasis (1). Despite intensive research, the regulation and function of the pancreatic α- and β-cells in health and disease remain enigmatic (2). During the past 30 years, the involvement of several gut-derived peptides in the regulation of pancreatic islet secretion has been progressively uncovered. One such gut-derived factor is glucose-dependent insulinotropic polypeptide (GIP), a polypeptide hormone secreted from the small-intestinal K cells in response to nutrient intake (3). GIP is well founded as an incretin hormone potentiating insulin release from β-cells in healthy humans (4–6). In addition to insulinotropic effects, early studies also delineated the glucagon-releasing properties of GIP by demonstrating that GIP stimulates glucagon secretion from α-cells in the perfused rat pancreas at glucose concentrations <5.5 mmol/L (4). However, a subsequent study was not able to reproduce the glucagon-releasing properties in humans during fasting and hyperglycemic conditions (5). Thus, additional investigations of the potential glucagonotropic effect of GIP was not pursued for nearly 25 years, until Meier et al. (7) used an improved immunoassay and demonstrated that GIP has dose-dependent glucagon-releasing properties when administered as bolus injections to healthy humans during euglycemia (plasma glucose [PG] 5.7 mmol/L). In contrast, using the same immunoassay, Vilsbøll et al. (8) reported GIP to be without glucagon-releasing properties when administered as a physiological infusion (1.5 pmol/kg/min for 30 min) in healthy humans clamped at euglycemia (5.1 mmol/L) as well as slightly elevated glucose levels (6 and 7 mmol/L, respectively). During overt hyperglycemic conditions, several studies have shown that GIP infusion does not have a glucagonotropic effect in healthy subjects (8–10). Consequently, although the relevance of GIP as an insulinotropic hormone in healthy individuals seems unquestionable, controversy exists regarding the glucagonotropic effects of GIP. We hypothesized that the glucagonotropic effects of GIP, such as its insulinotropic effects, are glucose dependent. Therefore, we aimed to evaluate, in the same individuals, the effects of GIP on plasma concentrations of glucagon and insulin at three distinct glycemic levels: hypoglycemia, euglycemia, and hyperglycemia.     

Intrinsic gluconeogenesis is enhanced in renal proximal tubules of Zucker diabetic fatty rats

Recent studies indicate that renal gluconeogenesis is substantially stimulated in patients with type 2 diabetes, but the mechanism that is responsible for such stimulation remains unknown. Therefore, this study tested the hypothesis that renal gluconeogenesis is intrinsically elevated in the Zucker diabetic fatty rat, which is considered to be an excellent model of type 2 diabetes. For this, isolated renal proximal tubules from diabetic rats and from their lean nondiabetic littermates were incubated in the presence of physiologic gluconeogenic precursors. Although there was no increase in substrate removal and despite a reduced cellular ATP level, a marked stimulation of gluconeogenesis was observed in diabetic relative to nondiabetic rats, with near-physiologic concentrations of lactate (38%), glutamine (51%) and glycerol (66%). This stimulation was caused by a change in the fate of the substrate carbon skeletons resulting from an increase in the activities and mRNA levels of the key gluconeogenic enzymes that are common to lactate, glutamine, and glycerol metabolism, i.e., mainly of phosphoenolpyruvate carboxykinase and, to a lesser extent, of glucose-6-phosphatase and fructose-1,6-bisphosphatase. Experimental evidence suggests that glucocorticoids and cAMP were two factors that were responsible for the long-term stimulation of renal gluconeogenesis observed in the diabetic rats. These data provide the first demonstration in an animal model that renal gluconeogenesis is upregulated by a long-term mechanism during type 2 diabetes. Together with the increased renal mass (38%) observed, they lend support to the view so far based only on in vivo studies performed in humans that renal gluconeogenesis may be stimulated by and crucially contribute to the hyperglycemia of type 2 diabetes.     

Distinct effects of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 on insulin secretion and gut motility

Glucose-induced insulin secretion from pancreatic beta-cells depends critically on ATP-sensitive K(+) channel (K(ATP) channel) activity, but it is not known whether K(ATP) channels are involved in the potentiation of insulin secretion by glucose-dependent insulinotropic polypeptide (GIP). In mice lacking K(ATP) channels (Kir6.2(-/-) mice), we found that pretreatment with GIP in vivo failed to blunt the rise in blood glucose levels after oral glucose load. In Kir6.2(-/-) mice, potentiation of insulin secretion by GIP in vivo was markedly attenuated, indicating that K(ATP) channels are essential in the insulinotropic effect of GIP. In contrast, pretreatment with glucagon-like peptide-1 (GLP-1) in Kir6.2(-/-) mice potentiated insulin secretion and blunted the rise in blood glucose levels. We also found that GLP-1 inhibited gut motility whereas GIP did not. Perfusion experiments of Kir6.2(-/-) mice revealed severely impaired potentiation of insulin secretion by 1 nmol/l GIP and substantial potentiation by 1 nmol/l GLP-1. Although both GIP and GLP-1 increase the intracellular cAMP concentration and potentiate insulin secretion, these results demonstrate that the GLP-1 and GIP signaling pathways involve the K(ATP) channel differently.     

Glucose-dependent insulinotropic polypeptide receptor knockout mice have altered bone turnover

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone, which is secreted from endocrine cells in the small intestine after meal ingestion. GIP has been shown to affect osteoblastic function in vitro; however, the in vivo effects of GIP on bone remodeling remain unclear. In the present study, we investigated the role of GIP in modulating bone turnover, by evaluating serum markers of bone turnover, bone density, bone morphology, and changes in biomechanical bone strength over time (one to five months) in GIP receptor knockout mice (GIPR−/− mice). The GIPR−/− mice showed a decreased bone size, lower bone mass, altered bone microarchitecture and biomechanical properties, and altered parameters for bone turnover, especially in bone formation. Moreover, the effects of GIP on bone mass were site-specific and compensatory mechanism developed over time and ameliorated the impact of the loss of GIP signaling on bone mass. Further, GIPR−/− mice had earlier age-related changes than wild-type mice in body composition, including bone mass, lean body mass, and fat percentage. In summary, our results indicate that GIP has an anabolic effect on bone mass and bone quality and suggests that GIP may be a hormonal link between nutrient ingestion and utilization.     

Glucose-dependent insulinotropic polypeptide receptor knockout mice have altered bone turnover

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone, which is secreted from endocrine cells in the small intestine after meal ingestion. GIP has been shown to affect osteoblastic function in vitro; however, the in vivo effects of GIP on bone remodeling remain unclear. In the present study, we investigated the role of GIP in modulating bone turnover, by evaluating serum markers of bone turnover, bone density, bone morphology, and changes in biomechanical bone strength over time (one to five months) in GIP receptor knockout mice (GIPR−/− mice). The GIPR−/− mice showed a decreased bone size, lower bone mass, altered bone microarchitecture and biomechanical properties, and altered parameters for bone turnover, especially in bone formation. Moreover, the effects of GIP on bone mass were site-specific and compensatory mechanism developed over time and ameliorated the impact of the loss of GIP signaling on bone mass. Further, GIPR−/− mice had earlier age-related changes than wild-type mice in body composition, including bone mass, lean body mass, and fat percentage. In summary, our results indicate that GIP has an anabolic effect on bone mass and bone quality and suggests that GIP may be a hormonal link between nutrient ingestion and utilization.     

Hyperinsulinism, glucose-dependent insulinotropic polypeptide, and the enteroinsular axis in morbidly obese patients before and after gastric bypass

The role of glucose-dependent insulinotropic polypeptide (GIP) in the hyperinsulinism of morbid obesity and its correction after gastric bypass was studied in 12 morbidly obese (150 +/- 15 kg) patients. After oral glucose, significant increases in serum glucose, insulin, and GIP levels occurred both before and after gastric bypass. Compared with preoperative values, fasting concentrations and integrated incremental areas for glucose, insulin, and GIP were decreased after a 25% weight loss after gastric bypass. The hyperinsulinism of morbid obesity and its amelioration after gastric bypass may be caused by markedly elevated levels of GIP before surgery and its reduced release after bypass. Reduced release of GIP after gastric bypass may partly occur because of exclusion of ingested glucose from contact with the mucosa of the duodenum and proximal jejunum, sites with the highest concentration of GIP.     

Effects of tungstate, a new potential oral antidiabetic agent, in Zucker diabetic fatty rats

Tungstate was orally administered to 7.5-week-old male Zucker diabetic fatty (ZDF) rats that already showed moderate hyperglycemia (180 +/- 16 mg/dl). The animals became normoglycemic for approximately 10 days. Then, glycemia started to rise again, although it did not reach the initial values until day 24, when levels stabilized at approximately 200 mg/dl for the duration of the experiment. Untreated ZDF rats showed steadily increased blood glucose levels between 7.5 and 10 weeks of age, when they reached a maximum value of 450 +/- 19 mg/dl, which was maintained throughout the experiment. In addition, tolerance to intraperitoneal glucose load improved in treated diabetic rats. Serum levels of triglycerides were elevated in untreated diabetic rats compared with their lean counterparts (ZLC). In the liver of diabetic animals, glucokinase (GK), glycogen phosphorylase a (GPa), liver-pyruvate kinase (L-PK), and fatty acid synthase (FAS) activities decreased by 81, 30, 54, and 35%, respectively, whereas phosphoenolpyruvate carboxykinase (PEPCK) levels increased by 240%. Intracellular glucose-6-phosphate (G6P) decreased by 40%, whereas glycogen levels remained unaffected. Tungstate treatment of these rats induced a 42% decrease in serum levels of triglycerides and normalized hepatic G6P concentrations, GPa activity, and PEPCK levels. GK activity in treated diabetic rats increased to 50% of the values of untreated ZLC rats. L-PK and FAS activity increased to higher values than those in untreated lean rats (1.7-fold L-PK and 2.4-fold FAS). Hepatic glycogen levels were 55% higher than those in untreated diabetic and healthy rats. Tungstate treatment did not significantly change the phosphotyrosine protein profile of primary cultured hepatocytes from diabetic animals. These data suggest that tungstate administration to ZDF rats causes a considerable reduction of glycemia, mainly through a partial restoration of hepatic glucose metabolism and a decrease in lipotoxicity.     

Beta-cell mass dynamics in Zucker diabetic fatty rats. Rosiglitazone prevents the rise in net cell death

The evolution of diabetes in the male leptin receptor-deficient (fa/fa) Zucker diabetic fatty (ZDF) rat is associated with disruption of normal islet architecture, beta-cell degranulation, and increased beta-cell death. It is unknown whether these changes precede or develop as a result of the increasing plasma glucose, or whether the increased beta-cell death can be prevented. Early intervention with thiazolidinediones prevents disruption of the islet architecture. To determine the specific effects of rosiglitazone (RSG) on beta-cell mass dynamics, male fa/fa (obese) and +/fa or +/+ (lean) rats age 6 weeks were fed either chow (control group [CN]) or chow mixed with rosiglitazone (RSG group) at a dosage of 10 micromol. kg(-1) body wt.day(-1). Rats were killed after 0, 2, 4, 6, or 10 weeks of treatment (at age 6, 8, 10, 12, or 16 weeks). Plasma glucose increased from 8.9 +/- 0.4 mmol/l at 0 weeks to 34.2 +/- 1.8 mmol/l (P = 0.0001) at 6 weeks of treatment in obese CN rats and fell from 8.0 +/- 0.3 to 6.3 +/- 0.4 mmol/l in obese RSG rats (P = 0.02). beta-cell mass fell by 51% from 2 to 6 weeks of treatment (ages 8-12 weeks) in obese CN rats (6.9 +/- 0.9 to 3.4 +/- 0.5 mg; P < 0.05), whereas beta-cell mass was unchanged in obese RSG rats. At 10 weeks of treatment (age 16 weeks), beta-cell mass in obese CN rats was only 56% of that of obese RSG rats (4.4 +/- 0.4 vs. 7.8 +/- 0.3 mg, respectively; P = 0.0001). The beta-cell replication rate fell from a baseline value of 0.95 +/- 0.12% in lean rats and 0.94 +/- 0.07% in obese rats (at 0 weeks) to approximately 0.3-0.5% in all groups by 6 weeks of treatment (age 12 weeks). After 10 weeks of treatment, beta-cell replication was higher in obese RSG rats than in CN rats (0.59 +/- 0.14 vs. 0.28 +/- 0.05%, respectively; P < 0.02). Application of our mass balance model of beta-cell turnover indicated that net beta-cell death was fivefold higher in obese CN rats as compared with RSG rats after 6 weeks of treatment (age 12 weeks). The increase in beta-cell death in obese CN rats during the 6-week observation period was well correlated with the increase in plasma glucose (r2 = 0.90, P < 0.0001). These results suggest that the development of hyperglycemia in ZDF rats is concomitant with increasing net beta-cell death. beta-cell proliferation compensates for the increased beta-cell loss at a time when plasma glucose is moderately elevated, but compensation ultimately fails and the plasma glucose levels increase beyond approximately 20 mmol/l. Treatment with rosiglitazone, previously shown to reduce insulin resistance, prevents the loss of beta-cell mass in obese ZDF rats by maintaining beta-cell proliferation and preventing increased net beta-cell death.     

Hepatic glucose cycling does not contribute to the development of hyperglycemia in Zucker diabetic fatty rats.

Hepatic glucose cycling, whereby glucose is taken up by the liver, partially metabolized, then recycled to glucose, makes a substantial contribution to the development of hyperglycemia in IDDM. This stimulation of glucose cycling appears to be associated with elevated rates of fatty acid oxidation. Whether hepatic glucose cycling also contributes to the development of hyperglycemia in NIDDM is unclear. Using a model of NIDDM, the Zucker diabetic fatty (ZDF) rat, we determined whether glucose cycling was enhanced. Hepatocytes from ZDF rats exhibited higher rates of glucose phosphorylation and glycolysis, but there was no increase in the rate of cycling between glucose and glucose-6-phosphate or between glycolytically derived pyruvate and glucose. Despite the increased rates of glycolysis, the production of CO2 in liver cells from ZDF rats was no different from rates measured in cells from control animals. Instead, there was a large increase in the accumulation of lactate and pyruvate in the ZDF liver cells. The addition of 2-bromopalmitate, an inhibitor of fatty acid oxidation that inhibited glucose cycling in hepatocytes from IDDM rats, had no effect on glucose cycling in cells from ZDF rats. We therefore conclude that, unlike in IDDM, hepatic glucose cycling does not contribute to the development of hyperglycemia in the NIDDM Zucker rat.     

Intramyocellular lipid and insulin resistance: a longitudinal in vivo 1H-spectroscopic study in Zucker diabetic fatty rats

Insulin resistance plays an important role in the pathogenesis of human type 2 diabetes. In humans, a negative correlation between insulin sensitivity and intramyocellular lipid (IMCL) content has been shown; thus, IMCL becomes a marker for insulin resistance. Recently, magnetic resonance spectroscopy (MRS) has been established as a dependable method for selective detection and quantification of IMCL in humans. To validate the interrelation between insulin sensitivity and IMCL in an animal model of type 2 diabetes, we established volume selective (1)H-MRS at 7 Tesla to noninvasively assess IMCL in the rat. In male obese Zucker Diabetic Fatty rats and their lean littermates, IMCL levels were determined repeatedly over 4 months, and insulin sensitivity was measured by the euglycemic-hyperinsulinemic clamp method at 6-7 and at 22-24 weeks of age. A distinct relation between IMCL and insulin sensitivity was demonstrated as well as age dependence for both parameters. Rosiglitazone treatment caused a clear reduction of IMCL and hepatic fat despite increased body weight, and a marked improvement of insulin sensitivity. Thus, the insulin sensitizing properties of rosiglitazone were consistent with a redistribution of lipids from nonadipocytic (skeletal muscle, liver) back into fat tissue.     

Glucagon-like peptide-1 receptor activation antagonizes voltage-dependent repolarizing K(+) currents in beta-cells: a possible glucose-dependent insulinotropic mechanism

Glucagon-like peptide-1 (GLP-1) acts through its G-protein-coupled receptor to enhance glucose-stimulated insulin secretion from pancreatic beta-cells. This is believed to result from modulation of at least two ion channels: ATP-sensitive K(+) (K(ATP)) channels and voltage-dependent Ca(2+) channels. Here, we report that GLP-1 receptor signaling also regulates the activity of beta-cell voltage-dependent K(+) (K(V)) channels, themselves potent glucose-dependent regulators of insulin secretion. GLP-1 receptor activation with exendin 4 (10(-8) mol/l) in rat beta-cells antagonized K(V) currents by 43.3 +/- 6.3%, whereas the GLP-1 receptor antagonist exendin 9-39 had no effect. The effect of GLP-1 receptor activation on K(V) currents could be replicated (current reduction of 55.7 +/- 6.0%) by G-protein activation with GMP-PNP (10 nmol/l). The cAMP pathway antagonist Rp-cAMPS (100 micro mol/l) prevented current inhibition by exendin 4, implicating cAMP signaling in GLP-1 receptor modulation of beta-cell K(V) currents. Finally, exendin 4 (10(-8) mol/l) increased the amplitude (130 +/- 5.7%) and duration (285 +/- 15.9%) of the beta-cell depolarization response to current injection, independent of any effect on K(ATP) or Ca(2+) channels. The present results demonstrate that GLP-1 receptor signaling can antagonize beta-cell repolarization by reducing voltage-dependent K(+) currents, an effect likely to contribute to GLP-1's glucose-dependent insulinotropic effect.     

Renal cyclooxygenase-2 in obese Zucker (fatty) rats

BACKGROUND: Cyclooxygenase (COX) isoforms, COX-1 and COX-2, are involved in production of prostanoids in the kidney. Increases in renal COX-2 expression have been implicated in the pathophysiology of progressive renal injury, including type 1 diabetes. Thromboxane A(2) (TxA(2)) has been suggested as the key mediator of these effects resulting in up-regulation of prosclerotic cytokines and extracellular matrix proteins. Unlike type 1 diabetes, renal COX has not been studied in models of type 2 diabetes. METHODS: Renal cortical COX protein expression, and urinary excretion of stable metabolites of prostaglandin E(2) (PGE(2)) and TxA(2), in association with metabolic parameters, were determined in 4-and 12-week-old Zucker fatty rats (fa/fa rat) (ZDF4 and ZDF12), a model of type 2 diabetes, and in age-matched littermates with no metabolic defect (Zucker lean) (ZL4 and ZL12). RESULTS: Western blotting revealed increased COX-2 expression in ZDF4 as compared to ZL4 (245 +/- 130%) (P < 0.05). This increase in COX-2 was even more apparent in 12-week-old ZDF rats (650 +/- 120%) (P < 0.01). All groups of rats demonstrated COX-2-positive cells in typical cortical localizations [macula densa, thick ascending loop of Henle (TALH)]. In contrast to COX-2, COX-1 expression was 30% lower in ZDF12. These changes in COX expression were associated with enhanced urinary excretion of prostanoids, in parallel with the development of metabolic abnormalities. Moreover, increases in prostanoid excretion in ZDF12 were in part reduced by wortmannin (100 mug/kg), used as inhibitor of insulin signaling. CONCLUSION: Renal cortical COX-2 protein expression and function were increased in ZDF rats, as compared to controls, whereas COX-1 exhibited opposite regulation. The changes in COX-2 paralleled metabolic abnormalities, and were at least in part a four consequence of hyperinsulinemia. These abnormalities may play a role in renal pathophysiology in this model of type 2 diabetes.     

Inhibition of fructose 1,6-bisphosphatase reduces excessive endogenous glucose production and attenuates hyperglycemia in Zucker diabetic fatty rats

Gluconeogenesis is increased in type 2 diabetes and contributes significantly to fasting and postprandial hyperglycemia. We recently reported the discovery of the first potent and selective inhibitors of fructose 1,6-bisphosphatase (FBPase), a rate-controlling enzyme of gluconeogenesis. Herein we describe acute and chronic effects of the lead inhibitor, MB06322 (CS-917), in rodent models of type 2 diabetes. In fasting male ZDF rats with overt diabetes, a single dose of MB06322 inhibited gluconeogenesis by 70% and overall endogenous glucose production by 46%, leading to a reduction in blood glucose of >200 mg/dl. Chronic treatment of freely feeding 6-week-old male Zucker diabetic fatty (ZDF) rats delayed the development of hyperglycemia and preserved pancreatic function. Elevation of lactate ( approximately 1.5-fold) occurred after 4 weeks of treatment, as did the apparent shunting of precursors into triglycerides. Profound glucose lowering ( approximately 44%) and similar metabolic ramifications were associated with 2-week intervention therapy of 10-week-old male ZDF rats. In high-fat diet-fed female ZDF rats, MB06322 treatment for 2 weeks fully attenuated hyperglycemia without evidence of metabolic perturbation other than a modest reduction in glycogen stores ( approximately 20%). The studies confirm that excessive gluconeogenesis plays an integral role in the pathophysiology of type 2 diabetes and suggest that FBPase inhibitors may provide a future treatment option.     

PPAR-alpha agonist fenofibrate induces renal CYP enzymes and reduces blood pressure and glomerular hypertrophy in Zucker diabetic fatty rats

We have previously shown that fenofibrate, a peroxisome proliferator-activated receptor-alpha activator, increases renal cytochrome P450 (CYP)-derived eicosanoids and improves endothelial function in pre-diabetic obese rats. The present study was designed to explore the efficacy of fenofibrate on blood pressure and renal injury in the advanced stage of type-2 diabetes. 26-week-old male Zucker diabetic fatty rats (ZDF) were fed fenofibrate (100 mg/kg/day) for 6 weeks. Chronic treatment with fenofibrate normalized systolic blood pressure and reduced glomerular size by 19% in diabetic rats. Western blot and fluorescent immunostaining revealed that the over-expression of collagen type IV and alpha-smooth muscle actin was significantly attenuated in the kidney of fenofibrate-treated ZDF (F-ZDF) rats. In addition, fenofibrate administration dramatically decreased the cyclin D1 protein level in the kidney of diabetic rats. In contrast, renal CYP2C23 and CYP4A proteins were significantly increased in F-ZDF rats. These fenofibrate effects were observed in the absence of significant changes in glucose, insulin or lipid levels. Taken together, our results demonstrate that fenofibrate may lower blood pressure and attenuate glomerular hypertrophy and collagen accumulation through the downregulation of cyclin D1 and upregulation of CYP monooxygenases in the late stage of type-2 diabetes.     

Glucose-dependent insulinotropic polypeptide (GIP) receptor deletion leads to reduced bone strength and quality

Bone is permanently remodeled by a complex network of local, hormonal and neuronal factors that affect osteoclast and osteoblast biology. In this context, a role for gastro-intestinal hormones has been proposed based on evidence that bone resorption dramatically falls after a meal. Glucose-dependent insulinotropic polypeptide (GIP) is one of the candidate hormones as its receptor, glucose-dependent insulinotropic polypeptide receptor (GIPR), is expressed in bone. In the present study we investigated bone strength and quality by three-point bending, quantitative x-ray microradiography, microCT, qBEI and FTIR in a GIPR knockout (GIPR KO) mouse model and compared with control wild-type (WT) animals. Animals with a deletion of the GIPR presented with a significant reduction in ultimate load (-− 11%), stiffness (− 16%), total absorbed (− 28%) and post-yield energies (− 27%) as compared with WT animals. Furthermore, despite no change in bone outer diameter, the bone marrow diameter was significantly increased and as a result cortical thickness was significantly decreased by 20% in GIPR deficient animals. Bone resorption at the endosteal surface was significantly increased whilst bone formation was unchanged in GIPR deficient animals. Deficient animals also presented with a pronounced reduction in the degree of mineralization of bone matrix. Furthermore, the amount of mature cross-links of collagen matrix was significantly reduced in GIPR deficient animals and was associated with lowered intrinsic material properties. Taken together, these data support a positive effect of the GIPR on bone strength and quality.