Incretins play an important role in FFA4/GPR120 regulation of glucose metabolism by GW-9508.

AIMS: To assess the role of GPR120 in glucose metabolism and incretin regulation from enteroendocrine L- and K-cells with determination of the cellular localisation of GPR120 in intestinal tissue and clonal Glucagon-Like Peptide-1 (GLP-1)/Gastric Inhibitory Polypeptide (GIP) cell lines. MAIN METHODS: Anti-hyperglycaemic, insulinotropic and incretin secreting properties of the GPR120 agonist, GW-9508 were explored in combination with oral and intraperitoneal glucose tolerance tests (GTT) in lean, diabetic and incretin receptor knockout mice. Cellular localisation of GPR120 was assessed by double immunofluorescence. KEY FINDINGS: Compared to intraperitoneal injection, oral administration of GW-9508 (0.1 µmol/kg body weight) together with glucose reduced the glycaemic excursion by 22-31 % (p < 0.05-p < 0.01) and enhanced glucose-induced insulin release by 30 % (p < 0.01) in normal mice. In high fat fed diabetic mice, orally administered GW-9508 lowered plasma glucose by 17-27 % (p < 0.05-p < 0.01) and augmented insulin release by 22-39 % (p < 0.05-p < 0.001). GW-9508 had no effect on the responses of GLP-1 receptor knockout mice and GIP receptor knockout mice. Consistent with this, oral GW-9508 increased circulating total GLP-1 release by 39-44 % (p < 0.01) and total GIP by 37-47 % (p < 0.01-p < 0.001) after 15 and 30 min in lean NIH Swiss mice. Immunocytochemistry demonstrated GPR120 expression on mouse enteroendocrine L- and K-cells, GLUTag cells and pGIP/Neo STC-1 cells. SIGNIFICANCE: GPR120 is expressed on intestinal L- and K-cells and stimulates GLP-1/GIP secretory pathways involved in mediating enhanced insulin secretion and improved glucose tolerance, following oral GW-9508. These novel data strongly support the development of potent and selective GPR120 agonists as an effective therapeutic approach for diabetes.

Antidiabetic actions of GPR55 agonist Abn-CBD and sitagliptin in obese-diabetic high fat fed mice.

GPR55 has been recognized as a novel anti-diabetic target exerting positive effects on beta cell function and mass. This study evaluated the metabolic actions and therapeutic efficacy of GPR55 agonist abnormal cannabidiol (Abn-CBD) administered alone and in combination with sitagliptin in diet-induced obese-diabetic mice. Chronic effects of 21-day oral administration of Abn-CBD (0.1 µmol/kg BW) monotherapy and in combination with sitagliptin (50 mg/kg BW) were assessed in obese-diabetic HFF mice (n = 8). Assessments of plasma glucose, circulating insulin, DPP-IV activity, CRP, amylase, lipids, body weight and food intake were undertaken. Glucose tolerance, insulin sensitivity, DEXA scanning and islet morphology analysis were performed at 21-days. Sitagliptin, Abn-CBD alone and in combination with sitagliptin attenuated plasma glucose by 37-53 % (p < 0.01 - p < 0.001) and enhanced circulating insulin concentrations by 23-31 % (p < 0.001). Abn-CBD alone and with sitagliptin reduced bodyweight by 9-10 % (p < 0.05). After 21-days, Abn-CBD in combination with sitagliptin (44 %; p < 0.01) improved glucose tolerance, whilst enhancing insulin sensitivity by 79 % (p < 0.01). Abn-CBD increased islet area (86 %; p < 0.05), beta cell mass (p < 0.05) and beta cell proliferation (164 %; p < 0.001), whilst in combination with sitagliptin islet area was decreased (50 %; p < 0.01). Abn-CBD alone, in combination with sitagliptin or sitagliptin alone decreased triglycerides by 34-65 % (p < 0.001) and total cholesterol concentrations by 15-25 % (p < 0.001). In addition, Abn-CBD in combination with sitagliptin reduced fat mass by 19 % (p < 0.05) and reduced CRP concentrations (39 %; p < 0.05). These findings advocate Abn-CBD monotherapy and in combination with sitagliptin as a novel and effective approach for bodyweight control and the treatment of glucose intolerance and dyslipidaemia in type-2-diabetes.

Development and characterisation of novel, enzymatically stable oxytocin analogues with beneficial antidiabetic effects in high fat fed mice.

BACKGROUND: There is growing evidence to support beneficial effects of the hypothalamic synthesised hormone, oxytocin, on metabolism. However, the biological half-life of oxytocin is short and receptor activation profile unspecific. METHODS: We have characterised peptide-based oxytocin analogues with structural modifications aimed at improving half-life and receptor specificity. Following extensive in vitro and in vivo characterisation, antidiabetic efficacy of lead peptides was examined in high fat fed (HFF) mice. RESULTS: Following assessment of stability against enzymatic degradation, insulin secretory activity, receptor activation profile and in vivo bioactivity, analogues 2 N (Ac-C ˂YIQNC >PLG-NH2) and D7R ((d-C)YIQNCYLG-NH2) were selected as lead peptides. Twice daily injection of either peptide for 22 days reduced body weight, energy intake, plasma glucose and insulin and pancreatic glucagon content in HFF mice. In addition, both peptides reduced total- and LDL-cholesterol, with concomitant elevations of HDL-cholesterol, and D7R also decreased triglyceride levels. The two oxytocin analogues improved glucose tolerance and insulin responses to intraperitoneal, and particularly oral, glucose challenge on day 22. Both oxytocin analogues enhanced insulin sensitivity, reduced HOMA-IR and increased bone mineral density. In terms of pancreatic islet histology, D7R reversed high fat feeding induced elevations of islet and beta cell areas, which was associated with reductions in beta cell apoptosis. Islet insulin secretory responsiveness was improved by 2 N, and especially D7R, treatment. CONCLUSION: Novel, enzymatically stable oxytocin analogues exert beneficial antidiabetic effects in HFF mice. GENERAL SIGNIFICANCE: These observations emphasise the, yet untapped, therapeutic potential of long-acting oxytocin-based agents for obesity and type 2 diabetes.

Zinc-induced activation of GPR39 regulates glucose homeostasis through glucose-dependent insulinotropic polypeptide secretion from enteroendocrine K-cells.

The role of Zn2+-sensing receptor GPR39 on glucose homeostasis and incretin regulation was assessed in enteroendocrine L- and K-cells. Anti-hyperglycaemic, insulinotropic and incretin secreting properties of Zn2+ were explored in normal, diabetic and incretin receptor knockout mice. Compared to intraperitoneal injection, oral administration of Zn2+ (50 µmol/kg body weight) with glucose (18 mmol/kg) in lean mice reduced the glycaemic excursion by 25-34% (p < 0.05-p < 0.001) and enhanced glucose-induced insulin release by 46-48% (p < 0.05-p < 0.01). In diabetic mice, orally administered Zn2+ lowered glucose by 24-31% (p < 0.01) and augmented insulin release by 32% (p < 0.01). In glucagon like peptide-1 (GLP-1) receptor knockout mice, Zn2+ reduced glucose by 15-28% (p < 0.05-p < 0.01) and increased insulin release by 35-43% (p < 0.01). In contrast Zn2+ had no effect on responses of glucose-dependent insulinotropic polypeptide (GIP) receptor knockout mice. Consistent with this, Zn2+ had no effect on circulating total GLP-1 whereas GIP release was stimulated by 26% (p < 0.05) in lean mice. Immunocytochemistry demonstrated GPR39 expression on mouse enteroendocrine L- and K-cells, GLUTag cells and pGIP/Neo STC-1 cells. Zn2+ had a direct effect on GIP secretion from pGIPneo STC-1 cells, increasing GIP secretion by 1.3-fold. GPR39 is expressed on intestinal L- and K-cells, and stimulated GIP secretion plays an integral role in mediating enhanced insulin secretion and glucose tolerance following oral administration of Zn2+. This suggests development of potent and selective GPR39 agonists as a therapeutic approach for diabetes.

Tissue expression of DPP-IV in obesity-diabetes and modulatory effects on peptide regulation of insulin secretion.

Dipeptidyl peptidase type 4 (DPP-4) inhibitors represent an important class of glucose-lowering drug for type 2 diabetes. DPP-4 enzyme activity has been observed to be significantly altered in type 2 diabetes. Here, the role of DPP-4 was examined in a high fat fed (HFF) mouse model of insulin resistance. HFF mice had an increased bodyweight (p < .01), were hyperglycaemic (p < .01) and hyperinsulinaemic (p < .05). Compared to normal diet, HFF mice exhibited increased plasma DPP-4 activity (p < .01). Tissue distribution patterns in lean and HFF mice demonstrated highest levels of DPP-4 activity in lung (20-26 µmol/min/mg protein) and small intestine (13-14 µmol/min/mg protein), and lowest activity in the spleen (3.8 µmol/min/mg protein). Modulation of DPP-4 activity by high fat feeding was observed in several tissues with increases in the lung (p < .05), liver (p < .05), kidney (p < .05) and pancreas (p < .05). With a high fat diet, DPP-4 gene expression was upregulated in the liver (p < .001) and downregulated in the pancreas (p < 0.001) and small intestine (p < .001). Immunohistochemical analysis revealed increased DPP-4 immunostaining localised primarily in the pancreatic islets of HFF mice (p < .01) with no change in islet GLP-1 expression. Treatment of HFF mice with metformin for 21-days resulted in inhibition of circulating DPP-4 activity (p < .05), decreased blood glucose (p < .05) and increased GLP-1 gene expression (p < .001). These data indicate that DPP-4 is modulated in a tissue specific manner and is dependent on physiological conditions such as hyperglycaemia and insulin resistance, suggesting a significant role in disorders such as diabetes.

Development of novel ligands for peptide GPCRs.

Incretin based glucagon-like peptide-1 receptor (GLP-1R) agonists which target a G-protein coupled receptor (GPCR) are currently used in the treatment of type 2 diabetes. This review focuses on GPCRs from pancreatic ß-cells, including GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucagon, somatostatin, pancreatic polypeptide (PP), cholecystokinin (CCK), peptide YY (PYY), oxyntomodulin (OXM) and ghrelin receptors. In addition, fatty acids GPCRs are thought to have an increasing role in regulating peptide secretions namely short fatty acids GPCR (GPR41, GPR43), medium chain fatty acid GPCR (GPR84), long chain fatty acid GPCR (GPR40, GPR120) and cannabinoid-like GPCR (GPR55, GPR119). Several pre-clinical and clinical trials are currently ongoing in peptide GPCR based therapies, including dual and triple agonist peptides which activate two or more GPCRs simultaneously.

Metabolic effects of orally administered small-molecule agonists of GPR55 and GPR119 in multiple low-dose streptozotocin-induced diabetic and incretin-receptor-knockout mice.

AIMS/HYPOTHESIS: Abnormal cannabidiol (Abn-CBD) and AS-1269574 are potent selective agonists for GPR55 and GPR119, respectively. The present study evaluated the actions and ability of these small-molecule agonists to counteract experimental diabetes in mice. METHODS: Diabetes was induced in NIH Swiss mice by five consecutive daily intraperitoneal injections of 40 mg/(kg body weight) streptozotocin. Diabetic mice received daily oral administration of Abn-CBD or AS-1269574 (0.1 µmol/kg) or saline vehicle (0.9% wt/vol. NaCl) over 28 days. Body weight, food intake, fluid intake, plasma glucose, insulin, glucose tolerance, insulin release, lipid profile and pancreatic morphology were examined. Mechanism of action of agonists was assessed in acute studies using incretin-receptor-knockout mice. RESULTS: Abn-CBD and AS-1269574 decreased plasma glucose (20-26%, p < 0.05) and increased circulating insulin (47-48%, p < 0.05) by 10-28 days, compared with saline-treated diabetic controls. Food intake and polydipsia were reduced by both agonists (21-23%, p < 0.05 and 33-35%, p < 0.01, respectively). After 28 days of treatment, plasma glucagon concentrations were reduced (p < 0.01) and glucose tolerance was enhanced by 19-44% by Abn-CBD (p < 0.05 or p < 0.001) and AS-1269574 (p < 0.05 to p < 0.001). Plasma insulin responses were improved (p < 0.01) and insulin resistance was decreased (p < 0.05 or p < 0.01) in both Abn-CBD- and AS-1269574-treated groups. Triacylglycerols were decreased by 19% with Abn-CBD (p < 0.05) and 32% with AS-1269574 (p < 0.01) while total cholesterol was reduced by 17% (p < 0.01) and 15% (p < 0.05), respectively. Both agonists enhanced beta cell proliferation (p < 0.001) although islet area was unchanged. Acute studies in Gipr- and Glp1r-knockout mice revealed an important role for the glucagon-like peptide 1 (GLP-1) receptor in the actions of both agonists, with the glucose-lowering effects of Abn-CBD also partly mediated through the glucose-dependent insulinotropic peptide (GIP) receptor. CONCLUSIONS/INTERPRETATION: These data highlight the potential for fatty acid G-protein-coupled receptor-based therapies as novel insulinotropic and glucose-lowering agents acting partly through the activation of incretin receptors.

G protein-coupled receptors: signalling and regulation by lipid agonists for improved glucose homoeostasis.

G protein-coupled receptors (GPCRs) play a pivotal role in cell signalling, controlling many processes such as immunity, growth, cellular differentiation, neurological pathways and hormone secretions. Fatty acid agonists are increasingly recognised as having a key role in the regulation of glucose homoeostasis via stimulation of islet and gastrointestinal GPCRs. Downstream cell signalling results in modulation of the biosynthesis, secretion, proliferation and anti-apoptotic pathways of islet and enteroendocrine cells. GPR40 and GPR120 are activated by long-chain fatty acids (>C12) with both receptors coupling to the Gαq subunit that activates the Ca(2+)-dependent pathway. GPR41 and GPR43 are stimulated by short-chain fatty acids (C2-C5), and activation results in binding to Gαi that inhibits the adenylyl cyclase pathway attenuating cAMP production. In addition, GPR43 also couples to the Gαq subunit augmenting intracellular Ca(2+) and activating phospholipase C. GPR55 is specific for cannabinoid endogenous agonists (endocannabinoids) and non-cannabinoid fatty acids, which couples to Gα12/13 and Gαq proteins, leading to enhancing intracellular Ca(2+), extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and Rho kinase. GPR119 is activated by fatty acid ethanolamides and binds to Gαs utilising the adenylate cyclase pathway, which is dependent upon protein kinase A. Current research indicates that GPCR therapies may be approved for clinical use in the near future. This review focuses on the recent advances in preclinical diabetes research in the signalling and regulation of GPCRs on islet and enteroendocrine cells involved in glucose homoeostasis.

GPR39 receptors and actions of trace metals on pancreatic beta cell function and glucose homoeostasis.

AIMS: G-protein-coupled receptor 39 (GPR39) has been implicated in glucose homoeostasis, appetite control and gastrointestinal tract function. METHODS: This study used clonal BRIN-BD11 cells and mouse pancreatic islets to assess the insulin-releasing actions of trace metals believed to act via GPR39, and the second messenger pathways involved in mediating their effects. Micromolar concentrations of Zn(2+), Cu(2+), Ni(2+) and Co(2+) were examined under normoglycaemic and hyperglycaemic conditions. Mechanistic studies investigated changes of intracellular Ca(2+), cAMP generation and assessment of cytotoxicity by LDH release. Cellular localisation of GPR39 was determined by double immunohistochemical staining. RESULTS: All trace metals (7.8-500 µmol/l) stimulated insulin release with Cu(2+) being the most potent in isolated islets, with an EC50 value of 87 µmol/l. Zn(2+) was the most selective with an EC50 value of 125 µmol/l. Enhancement of insulin secretion was also observed with Ni(2+) (179 µmol/l) and Co(2+) (190 µmol/l). These insulin-releasing effects were confirmed using clonal BRIN-BD11 cells which exhibited enhanced intracellular Ca(2+) (p < 0.05-p < 0.001) and cAMP generation (p < 0.05-p < 0.001) in response to trace metals. Oral administration of Zn(2+), Ni(2+) and Cu(2+) (50 µmol/kg together with 18 mmol/kg glucose) decreased the glycaemic excursion (p < 0.05-p < 0.01) and augmented insulin secretion (p < 0.05-p < 0.01) in NIH Swiss mice. CONCLUSIONS: This study has demonstrated the presence of GPR39 and the insulinotropic actions of trace metals on BRIN-BD11 cells and pancreatic beta cells, together with their antihyperglycaemic actions in vivo. These data suggest that development of agonists capable of specifically activating GPR39 may be a useful new therapeutic approach for diabetes management.

Activation of GPR119 by fatty acid agonists augments insulin release from clonal ß-cells and isolated pancreatic islets and improves glucose tolerance in mice.

G-protein coupled receptor 119 (GPR119) is emerging as a potential target for the treatment of type 2 diabetes with beneficial effects on glucose homeostasis. This study assessed the insulin-secreting properties of various GPR119 agonists and the distribution of GPR119 in pancreatic islets. Endogenous ligands [oleoylethanolamide (OEA), palmitoylethanolamine (PEA)] and chemically synthetic analogues (AS-1269574, PSN-375963) were investigated in clonal BRIN-BD11 cells and mouse pancreatic islets. Secondary messenger assays such as intracellular Ca²âº and cAMP in response to agonists at normoglycaemic and hyperglycaemic conditions were assessed. Cytotoxicity was assessed by LDH release. AS-1269574 was the most potent and selective agonist tested in isolated islets, with an EC50 value of 9.7×10⁻7 mol/l, enhancing insulin release maximally by 63.2%. Stimulation was also observed with GPR119 ligands; OEA (3.0×10⁻6 mol/l; 37.5%), PSN-375963 (2.4×10⁻6 mol/l; 28.7%) and PEA (1.2×10⁻6 mol/l; 22.2%). Results were corroborated by studies using BRIN-BD11 cells, which revealed augmentation of intracellular Ca²âº and cAMP. Both OEA and AS-1269574 enhanced insulin release and improved glucose tolerance in vivo in NIH Swiss mice. These results demonstrate the cellular localisation of GPR119 on islet cells (ß and pancreatic polypeptide cells), its activation of the ß-cell stimulus-secretion coupling pathway and glucose lowering effects in vivo.

Emerging applications of metabolomic and genomic profiling in diabetic clinical medicine.

Clinical and epidemiological metabolomics provides a unique opportunity to look at genotype-phenotype relationships as well as the body\x{2019}s responses to environmental and lifestyle factors. Fundamentally, it provides information on the universal outcome of influencing factors on disease states and has great potential in the early diagnosis, therapy monitoring, and understanding of the pathogenesis of disease. Diseases, such as diabetes, with a complex set of interactions between genetic and environmental factors, produce changes in the body\x{2019}s biochemical profile, thereby providing potential markers for diagnosis and initiation of therapies. There is clearly a need to discover new ways to aid diagnosis and assessment of glycemic status to help reduce diabetes complications and improve the quality of life. Many factors, including peptides, proteins, metabolites, nucleic acids, and polymorphisms, have been proposed as putative biomarkers for diabetes. Metabolomics is an approach used to identify and assess metabolic characteristics, changes, and phenotypes in response to influencing factors, such as environment, diet, lifestyle, and pathophysiological states. The specificity and sensitivity using metabolomics to identify biomarkers of disease have become increasingly feasible because of advances in analytical and information technologies. Likewise, the emergence of high-throughput genotyping technologies and genome-wide association studies has prompted the search for genetic markers of diabetes predisposition or susceptibility. In this review, we consider the application of key metabolomic and genomic methodologies in diabetes and summarize the established, new, and emerging metabolomic and genomic biomarkers for the disease. We conclude by summarizing future insights into the search for improved biomarkers for diabetes research and human diagnostics.

Evaluation of the degradation and metabolic effects of the gut peptide xenin on insulin secretion, glycaemic control and satiety.

Recently, glucagon-like peptide 1 (GLP1) and glucose-dependent insulinotropic polypeptide (GIP) have received much attention regarding possible roles in aetiology and treatment of type 2 diabetes. However, peptides co-secreted from the same enteroendocrine cells are less well studied. The present investigation was designed to characterise the in vitro and in vivo effects of xenin, a peptide co-secreted with GIP from intestinal K-cells. We examined the enzymatic stability, insulin-releasing activity and associated cAMP production capability of xenin in vitro. In addition, the effects of xenin on satiety, glucose homoeostasis and insulin secretion were examined in vivo. Xenin was time dependently degraded (t(1/2)=162±6 min) in plasma in vitro. In clonal BRIN-BD11 cells, xenin stimulated insulin secretion at 5.6 mM (P<0.05) and 16.7 mM (P<0.05 to P<0.001) glucose levels compared to respective controls. Xenin also exerted an additive effect on GIP, GLP1 and neurotensin-mediated insulin secretion. In clonal ß-cells, xenin did not stimulate cellular cAMP production, alter membrane potential or elevate intra-cellular Ca(2)(+). In normal mice, xenin exhibited a short-acting (P<0.01) satiety effect at high dosage (500 nmol/kg). In overnight fasted mice, acute injection of xenin enhanced glucose-lowering and elevated insulin secretion when injected concomitantly or 30 min before glucose. These effects were not observed when xenin was administered 60 min before the glucose challenge, reflecting the short half-life of the native peptide in vivo. Overall, these data demonstrate that xenin may have significant metabolic effects on glucose control, which merit further study.

Insulinotropic actions of nateglinide in type 2 diabetic patients and effects on dipeptidyl peptidase-IV activity and glucose-dependent insulinotropic polypeptide degradation.

BACKGROUND: Nateglinide restores early-phase insulin secretion to feeding and reduces postprandial hyperglycaemia in type 2 diabetes. This study evaluated the effects of nateglinide on dipeptidyl peptidase-IV (DPP-IV) activity and glucose-dependent insulinotropic polypeptide (GIP) degradation. Research design and methods Blood samples were collected from type 2 diabetic subjects (n=10, fasting glucose 9.36+/-1.2 mmol/l) following administration of oral nateglinide (120 mg) 10 min prior to a 75 g oral glucose load in a randomised crossover design. RESULTS: Plasma glucose reached 18.2+/-1.7 and 16.7+/-1.7 mmol/l at 90 min in control and placebo groups (P<0.001). These effects were accompanied by prompt 32% inhibition of DPP-IV activity after 10 min (19.9+/-1.6 nmol/ml per min, P<0.05), reaching a minimum of 1.9+/-0.1 nmol/ml per min at 120 min (P<0.001) after nateglinide. Insulin and C-peptide levels increased significantly compared with placebo, to peak after 90 min at 637.6+/-163.9 pmol/l (P<0.05) and 11.8+/-1.4 mg/l (P<0.01) respectively. DPP-IV-mediated degradation of GIP was significantly less in patients receiving nateglinide compared with placebo. Inhibition of DPP-IV activity corresponded with a time- and concentration-dependent inhibitory effect of nateglinide on DPP-IV-mediated truncation of GIP(1-42) to GIP(3-42) in vitro. Comparison of in vitro inhibition of DPP-IV by nateglinide and vildagliptin revealed IC(50) values of 17.1 and 2.1 microM respectively. CONCLUSIONS: Although considerably less potent than specified DPP-IV inhibitors, the possibility that some of the beneficial actions of nateglinide are indirectly mediated through DPP-IV inhibition and increased bioavailability of GIP and other incretins merits consideration.

Sub-chronic administration of the 11beta-HSD1 inhibitor, carbenoxolone, improves glucose tolerance and insulin sensitivity in mice with diet-induced obesity.

We have examined the metabolic effects of daily administration of carbenoxolone (CBX), a naturally occurring 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) inhibitor, in mice with high fat diet-induced insulin resistance and obesity. Eight-week-old male Swiss TO mice placed on a synthetic high fat diet received daily intraperitoneal injections of either saline vehicle or CBX over a 16-day period. Daily administration of CBX had no effect on food intake, but significantly lowered body weight (1.1- to 1.2-fold) compared to saline-treated controls. Non-fasting plasma glucose levels were significantly decreased (1.6-fold) by CBX treatment on day 4 and remained lower throughout the treatment period. Circulating plasma corticosterone levels were not significantly altered by CBX treatment. Plasma glucose concentrations of CBX-treated mice were significantly reduced (1.4-fold) following an intraperitoneal glucose load compared with saline controls. Similarly, after 16-day treatment with CBX, exogenous insulin evoked a significantly greater reduction in glucose concentrations (1.4- to 1.8-fold). 11beta-HSD1 gene expression was significantly down-regulated in liver, whereas glucocorticoid receptor gene expression was increased in both liver and adipose tissue following CBX treatment. The reduced body weight and improved metabolic control in mice with high fat diet-induced obesity upon daily CBX administration highlights the potential value of selective 11beta-HSD1 inhibition as a new route for the treatment of type 2 diabetes and obesity.

Decreased dipeptidyl peptidase-IV activity and glucagon-like peptide-1(7-36)amide degradation in type 2 diabetic subjects.

Dipeptidyl peptidase (DPP-IV) rapidly metabolizes hormones such as glucagon-like peptide-1(7-36)amide. This study evaluated circulating DPP-IV activity in type 2 diabetic patients in relation to GLP-1 degradation and metabolic control. Blood samples were collected from type 2 diabetic patients in three main categories: good glycaemic control (HbA(1c) <7%, upper limit of non-diabetic range), moderate glycaemic control (HbA(1c) 7-9%) and poor glycaemic control (HbA(1c) >9%). Age- and sex-matched non-diabetic subjects were used as controls. Circulating DPP-IV activity of healthy control subjects was 22.5+/-0.7 nmol/ml/min (n=70). In the combined groups of type 2 diabetic subjects, circulating DPP-IV activity was significantly decreased at 18.1+/-0.7 nmol/ml/min (p<0.001, n=54). DPP-IV activity was negatively correlated with both glucose (p<0.01) and HbA(1c) (p<0.01) in this population. Furthermore, DPP-IV activity was reduced 1.2-fold (p<0.01, n=25), 1.3-fold (p<0.001, n=19) and 1.3-fold (p<0.05, n=10) in good, moderate and poorly controlled diabetic groups, 18.7+/-1.0, 17.4+/-1.4 and 18.0+/-1.5 nmol/ml/min, respectively. Degradation of GLP-1 by in vitro incubation with pooled plasma samples from healthy and type 2 diabetic subjects revealed decreased degradation to the inactive metabolite, GLP-1(9-36), in the diabetic group. These data indicate decreased DPP-IV activity and GLP-1 degradation in type 2 diabetes. DPP-IV enzyme activity appears to be depressed in response to poor glycaemic control.

Effects of antidiabetic drugs on dipeptidyl peptidase IV activity: nateglinide is an inhibitor of DPP IV and augments the antidiabetic activity of glucagon-like peptide-1.

Dipeptidyl peptidase IV (DPP IV) is the primary inactivator of glucoregulatory incretin hormones. This has lead to development of DPP IV inhibitors as a new class of agents for the treatment of type 2 diabetes. Recent reports indicate that other antidiabetic drugs, such as metformin, may also have inhibitory effects on DPP IV activity. In this investigation we show that high concentrations of several antidiabetic drug classes, namely thiazolidinediones, sulphonylureas, meglitinides and morphilinoguanides can inhibit DPP IV. The strongest inhibitor nateglinide, the insulin-releasing meglitinide was effective at low therapeutically relevant concentrations as low as 25 micromol/l. Nateglinide also prevented the degradation of glucagon-like peptide-1 (GLP-1) by DPP IV in a time and concentration-dependent manner. In vitro nateglinide and GLP-1 effects on insulin release were additive. In vivo nateglinide improved the glucose-lowering and insulin-releasing activity of GLP-1 in obese-diabetic ob/ob mice. This was accompanied by significantly enhanced circulating concentrations of active GLP-1(7-36)amide and lower levels of DPP IV activity. Nateglinide similarly benefited the glucose and insulin responses to feeding in ob/ob mice and such actions were abolished by co-administration of exendin(9-39) and (Pro(3))GIP to block incretin hormone action. These data indicate that the use of nateglinide as a prandial insulin-releasing agent may partly rely on inhibition of GLP-1 degradation as well as beta-cell K(ATP) channel inhibition.

Meal-induced 24-hour profile of circulating glycated insulin in type 2 diabetic subjects measured by a novel radioimmunoassay.

Increasing evidence supports a role for glycated insulin in the insulin-resistant state of type 2 diabetes. We measured 24-hour profiles of plasma glycated insulin, using a novel radioimmunoassay (RIA), to evaluate the effects of meal stimulation and intermittent fasting on circulating concentrations of plasma glycated insulin in type 2 diabetes. Patients (n = 6; hemoglobin A(1c) [HbA(1c)], 7.2% +/- 0.6%; fasting plasma glucose, 7.4 +/- 0.7 mmol/L; body mass index [BMI], 35.7 +/- 3.5 kg/m(2); age, 56.3 +/- 4.4 years) were admitted for 24 hours and received a standardized meal regimen. Half-hourly venous samples were taken for plasma glycated insulin, glucose, insulin, and C-peptide concentrations between 8 am and midnight and 2-hourly overnight. The mean plasma glycated insulin concentration over 24 hours was 27.8 +/- 1.2 pmol/L with a mean ratio of insulin:glycated insulin of 11:1. Circulating glucose, insulin, C-peptide, and glycated insulin followed a basal and meal-related pattern with most prominent increments following breakfast, lunch, and evening meal, respectively. The mean concentrations of glycated insulin during the morning, afternoon, evening, and night-time periods were 24.4 +/- 2.5, 28.7 +/- 2.3, 31.1 +/- 2.1, and 26.2 +/- 1.5 pmol/L, respectively, giving significantly higher molar ratios of insulin:glycated insulin of 18.0:1, 14.2:1, and 12.7:1 compared with 7.0:1 at night (P <.01 to P <.001). These data demonstrate that glycated insulin circulates at relatively high concentrations in type 2 diabetes with a diurnal pattern of basal and meal-stimulated release. A higher proportion of glycated insulin circulates at night suggestive of differences in metabolic clearance compared with native insulin.

Evaluation of the site(s) of glycation in human proinsulin by ion-trap LCQ electrospray ionization mass spectrometry.

The glycation of beta cell proteins is known to occur under hyperglycemic states. The site(s) of glycation in human proinsulin was investigated following exposure to a hyperglycemic environment under reducing conditions in vitro. Proinsulin and glycated proinsulin were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and identified using LCQ ion-trap electrospray ionization mass spectrometry. This revealed a major peak (>70% total) of monoglycated proinsulin (M(r) 9552.2 Da), a second peak (approximately 27%) of nonglycated proinsulin (M(r) 9389.8 Da), and a third minor peptide peak (approximately 3%) corresponding to diglycated proinsulin (M(r) 9717.9 Da). Following reduction of disulphide bridges with dithiothreitol, intact peptides were incubated with endoproteinase Glu-C to release nine daughter fragments for LC-MS analysis. This strategy revealed an N-terminal fragment of monoglycated proinsulin Phe(1)-Glu(13), which contained a single glucitol adduct (M(r) 1642.0 Da). A similar treatment of small amounts of purified diglycated proinsulin revealed a fragment with Phe(1)-Glu(13) linked by a disulphide bridge to Gln(70)-Glu(82) containing two glucitol adducts (M(r) 3292.7 Da). In summary, these studies indicate that the major site of glycation in proinsulin, like insulin, is the amino terminal Phe(1) residue. However, small amounts of diglycated proinsulin occur naturally, involving an additional site of glycation located between Gln(70) and Glu(82).

Demonstration of glycated insulin in human diabetic plasma and decreased biological activity assessed by euglycemic-hyperinsulinemic clamp technique in humans.

The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA(1c) 8.1 +/- 0.2%, plasma glucose 8.7 +/- 1.3 mmol/l [means +/- SE]) revealed two major insulin-like peaks with retention times of 14-16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H)(3+) species at 1,991.1 m/z, representing monoglycated insulin with an intact M(r) of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin (M(r) 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 +/- 2.3 pmol/l, corresponding to approximately 9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe(1)-glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 micro g x kg(-1) x min(-1), followed by 2 h at 83.0 micro g x kg(-1) x min(-1); corresponding to 0.4 and 2.0 mU x kg(-1) x min(-1)). At the lower dose, the exogenous glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin (P < 0.01) and approximately 70% more glycated insulin was required to induce a similar rate of insulin-mediated glucose uptake. Maximal responses at the higher rates of infusion were similar for glycated and control insulin. Inhibitory effects on endogenous glucose production, insulin secretion, and lipolysis, as indicated by measurements of C-peptide, nonesterified free fatty acids, and glycerol, were also similar. Receptor binding to CHO-T cells transfected with human insulin receptor and in vivo metabolic clearance revealed no differences between glycated and native insulin, suggesting that impaired biological activity is due to a postreceptor effect. The present demonstration of glycated insulin in human plasma and related impairment of physiological insulin-mediated glucose uptake suggests a role for glycated insulin in glucose toxicity and impaired insulin action in type 2 diabetes.

Evaluation of glycated glucagon-like peptide-1(7-36)amide in intestinal tissue of normal and diabetic animal models.

Glucagon-like peptide-1(7-36)amide (tGLP-1) is an important insulin-releasing hormone of the enteroinsular axis which is secreted by endocrine L-cells of the small intestine following nutrient ingestion. The present study has evaluated tGLP-1 in the intestines of normal and diabetic animal models and estimated the proportion present in glycated form. Total immunoreactive tGLP-1 levels in the intestines of hyperglycaemic hydrocortisone-treated rats, streptozotocin-treated mice and ob/ob mice were similar to age-matched controls. Affinity chromatographic separation of glycated and non-glycated proteins in intestinal extracts followed by radioimmunoassay using a fully cross-reacting anti-serum demonstrated the presence of glycated tGLP-1 within the intestinal extracts of all control animals (approximately 19% of total tGLP-1 content). Chemically induced and spontaneous animal models of diabetes were found to possess significantly greater levels of glycated tGLP-1 than controls, corresponding to between 24--71% of the total content. These observations suggest that glycated tGLP-1 may be of physiological significance given that such N-terminal modification confers resistance to DPP IV inactivation and degradation, extending the very short half-life (<3 min) and bioactivity of the native peptide.