Insulin secretory and antidiabetic actions of Heritiera fomes bark together with isolation of active phytomolecules.

In folklore, Heritiera fomes (H. fomes) has been extensively used in treatment of various ailments such as diabetes, cardiac and hepatic disorders. The present study aimed to elucidate the antidiabetic actions of hot water extract of H. fomes (HWHF), including effects on insulin release from BRIN BD11 cells and isolated mouse islets as well as glucose homeostasis in high-fat-fed rats. Molecular mechanisms underlying anti-diabetic activity along with isolation of active compounds were also evaluated. Non-toxic concentrations of HWHF stimulated concentration-dependent insulin release from isolated mouse islets and clonal pancreatic ß-cells. The stimulatory effect was potentiated by glucose and isobutyl methylxanthine (IBMX), persisted in presence of tolbutamide or a depolarizing concentration of KCl but was attenuated by established inhibitors of insulin release such as diazoxide, verapamil, and Ca2+ chelation. HWHF caused depolarization of the ß-cell membrane and increased intracellular Ca2+. The extract also enhanced glucose uptake and insulin action in 3T3-L1 differentiated adipocytes cells and significantly inhibited in a dose-dependent manner starch digestion, protein glycation, DPP-IV enzyme activity, and glucose diffusion in vitro. Oral administration of HWHF (250 mg/5ml/kg b.w.) to high-fat fed rats significantly improved glucose tolerance and plasma insulin responses and it inhibited plasma DPP-IV activity. HWHF also decreased in vivo glucose absorption and intestinal disaccharidase activity while increasing gastrointestinal motility and unabsorbed sucrose transit. Compounds were isolated from HWHF with similar molecular weights to quercitrin (C21 H20 O11) ranging from 447.9 to 449.9 Da which stimulated the insulin release in vitro and improved both glucose tolerance and plasma insulin responses in mice. In conclusion, H. fomes and its water-soluble phytochemicals such as quercitrin may exert antidiabetic actions mediated through a variety of mechanisms which might be useful as dietary adjunct in the management of type 2 diabetes.

Identification of Multiple Pancreatic and Extra-Pancreatic Pathways Underlying the Glucose-Lowering Actions of Acacia arabica Bark in Type-2 Diabetes and Isolation of Active Phytoconstituents.

Acacia arabica is used traditionally to treat a variety of ailments, including diabetes. This study elucidated the antidiabetic actions of A. arabica bark together with the isolation of bioactive molecules. Insulin secretion and signal transduction were measured using clonal ß cells and mouse islets. Glucose uptake was assessed using 3T3-L1 adipocytes, and in vitro systems assessed additional glucose-lowering actions. High-fat-fed (HFF) obese rats were used for in vivo evaluation, and phytoconstituents were isolated and characterised by RP-HPLC followed by LC-MS and NMR. Hot-water extract of A. arabica (HWAA) increased insulin release from clonal ß cells and mouse islets by 1.3-6.8-fold and 1.6-3.2-fold, respectively. Diazoxide, verapamil and calcium-free conditions decreased insulin-secretory activity by 30-42%. In contrast, isobutylmethylxanthine (IBMX), tolbutamide and 30 mM KCl potentiated the insulin-secretory effects. The mechanism of actions of HWAA involved membrane depolarisation and elevation of intracellular Ca2+ together with an increase in glucose uptake by 3T3-L1 adipocytes, inhibition of starch digestion, glucose diffusion, dipeptidyl peptidase-IV (DPP-IV) enzyme activity and protein glycation. Acute HWAA administration (250 mg/5 mL/kg) enhanced glucose tolerance and plasma insulin in HFF obese rats. Administration of HWAA (250 mg/5 mL/kg) for 9 days improved glucose homeostasis and ß-cell functions, thereby improving glycaemic control, and circulating insulin. Isolated phytoconstituents, including quercetin and kaempferol, increased insulin secretion in vitro and improved glucose tolerance. The results indicate that HWAA has the potential to treat type 2 diabetes as a dietary supplement or as a source of antidiabetic agents, including quercetin and kaempferol.

Insulinotropic and antidiabetic properties of Eucalyptus citriodora leaves and isolation of bioactive phytomolecules.

OBJECTIVE: The aim of this study was to delineate the mechanisms of action of the plant Eucalyptus citriodora used traditionally for the treatment of type 2 diabetes. METHODS: Insulin secretion and signal transduction were measured using clonal pancreatic ß-cells and mouse islets. Glucose uptake was assessed using 3T3-L1 adipocytes and in vitro systems assessed additional glucose-lowering actions. High-fat-fed (HFF) obese rats were used for in vivo evaluation and phytoconstituents were identified by RP-HPLC followed by LC-MS. KEY FINDINGS: Eucalyptus citriodora stimulated 1.2-4.6-fold insulin release that was inhibited by the Ca2+-channel blocker, verapamil, KATP-channel opener, diazoxide and Ca2+ free conditions. The effect was potentiated by IBMX and preserved in presence of tolbutamide or 30 mM KCl. The action mechanism involved membrane depolarization and elevation of intracellular Ca2+. Eucalyptus citriodora also significantly increased glucose uptake by 3T3-L1 cells and inhibited digestion of starch, glucose absorption, DPP-IV enzyme and glycation of protein. Administration of E. citriodora (250 mg/5 ml/kg) for 9 days to HFF obese-diabetic rats improved glycaemic control and ß-cell function. The isolated phytoconstituents responsible for the ß-cell actions included quercitrin, isoquercitrin and rhodomyrtosone E. CONCLUSIONS: Eucalyptus citriodora improves glycaemic control via multiple mechanisms. Further studies are required to assess the utility of the plant or active constituents in the therapy of type 2 diabetes.

Anti-hyperglycaemic and insulin-releasing effects of Camellia sinensis leaves and isolation and characterisation of active compounds.

Anti-diabetic actions of Camellia sinensis leaves, used traditionally for type 2 diabetes (T2DM) treatment, have been determined. Insulin release, membrane potential and intra-cellular Ca were studied using the pancreatic ß-cell line, BRIN-BD11 and primary mouse pancreatic islets. Cellular glucose-uptake/insulin action by 3T3-L1 adipocytes, starch digestion, glucose diffusion, dipeptidyl peptidase-4 (DPP-IV) activity and glycation were determined together with in vivo studies assessing glucose homoeostasis in high-fat-fed (HFF) rats. Active phytoconstituents with insulinotropic activity were isolated using reversed-phase HPLC, LCMS and NMR. A hot water extract of C. sinensis increased insulin secretion in a concentration-dependent manner. Insulinotropic effects were significantly reduced by diazoxide, verapamil and under Ca-free conditions, being associated with membrane depolarisation and increased intra-cellular Ca2+. Insulin-releasing effects were observed in the presence of KCl, tolbutamide and isobutylmethylxanthine, indicating actions beyond K+ and Ca2+ channels. The extract also increased glucose uptake/insulin action in 3T3L1 adipocyte cells and inhibited protein glycation, DPP-IV enzyme activity, starch digestion and glucose diffusion. Oral administration of the extract enhanced glucose tolerance and insulin release in HFF rats. Extended treatment (250 mg/5 ml per kg orally) for 9 d led to improvements of body weight, energy intake, plasma and pancreatic insulin, and corrections of both islet size and ß-cell mass. These effects were accompanied by lower glycaemia and significant reduction of plasma DPP-IV activity. Compounds isolated by HPLC/LCMS, isoquercitrin and rutin (464·2 Da and 610·3 Da), stimulated insulin release and improved glucose tolerance. These data indicate that C. sinensis leaves warrant further evaluation as an effective adjunctive therapy for T2DM and source of bioactive compounds.

Evaluation of the Antidiabetic and Insulin Releasing Effects of A. squamosa, Including Isolation and Characterization of Active Phytochemicals.

Annona squamosa is generally referred to as a 'custard apple'. Antidiabetic actions of hot water extract of Annona squamosa (HWAS) leaves together with isolation of active insulinotropic compounds were studied. Insulin release, membrane potential and intracellular Ca2+ were determined using BRIN-BD11 cells and isolated mouse islets. 3T3L1 adipocytes and in vitro models were used to determine cellular glucose uptake, insulin action, starch digestion, glucose diffusion, DPP-IV activity and glycation. Glucose intolerant high-fat fed rats were used for in vivo studies. Active compounds were isolated and characterized by HPLC, LCMS and NMR. HWAS stimulated insulin release from clonal ß-cells and mouse islets. Using fluorescent indicator dyes and modulators of insulin secretion, effects could be attributed to depolarization of ß-cells and influx of Ca2+. Secretion was stimulated by isobutylmethylxanthine (IBMX), tolbutamide or 30 mM KCl, indicating additional non-KATP dependent pathways. Extract stimulated cellular glucose uptake and insulin action and inhibited starch digestion, protein glycation, DPP-IV enzyme activity and glucose diffusion. Oral HWAS improved glucose tolerance and plasma insulin in high-fat fed obese rats. Treatment for 9 days with HWAS (250 mg/5 mL/kg), partially normalised energy intake, body weight, pancreatic insulin content, and both islet size and beta cell mass. This was associated with improved oral glucose tolerance, increased plasma insulin and inhibition of plasma DPP-IV activity. Isolated insulinotropic compounds, including rutin (C27H30O16), recapitulated the positive actions of HWAS on beta cells and in vivo glucose tolerance and plasma insulin responses. Annona squamosa is attractive as a dietary adjunct in treatment of T2DM and as a source of potential antidiabetic agents including rutin.

A modified Tat peptide for selective intracellular delivery of macromolecules.

OBJECTIVES: The Tat peptide has been widely used for the intracellular delivery of macromolecules. The aim of this study was to modify the peptide to enable regulation of cellular uptake through a dependency on activation by proteases present in the local environment. METHODS: The native Tat peptide sequence was altered to inhibit the initial interaction of the peptide with the cell membrane through the addition of the consensus sequence for urokinase plasminogen activator (uPA). uPA expression was characterised and semi-quantitatively rated in three cell lines (U251mg, MDA-MB-231 and HeLa). The modified peptide was incubated with both recombinant enzyme and with cells varying in uPA activity. Cellular uptake of the modified Tat peptide line was compared with that of the native peptide and rated according to uPA activity measured in each cell line. KEY FINDINGS: uPA activity was observed to be high in U251mg and MDA-MB-231 and low in HeLa. In MDA-MB-231 and HeLa, uptake of the modified peptide correlated with the level of uPA expression detected (93 and 52%, respectively). In U251mg, however, the uptake of the modified peptide was much less (19% observed reduction) than the native peptide despite a high level of uPA activity detected. CONCLUSIONS: Proteolytic activation represents an interesting strategy for the targeted delivery of macromolecules using peptide-based carriers and holds significant potential for further exploitation.

Administration of an acylated GLP-1 and GIP preparation provides added beneficial glucose-lowering and insulinotropic actions over single incretins in mice with Type 2 diabetes and obesity.

The present study examined the glucose-lowering and insulinotropic properties of acylated GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic polypeptide) peptides in Type 2 diabetes and obesity. GLP-1, GIP, Liraglutide, N-AcGIP(Lys(37)Myr) (N-acetylGIP with myristic acid conjugated at Lys(37)), a simple combination of both peptides and a Lira-AcGIP preparation [overnight preparation of Liraglutide and N-AcGIP(Lys(37)Myr)] were incubated with DPP-IV (dipeptidyl peptidase-IV) to assess peptide stability, and BRIN-BD11 cells were used to evaluate cAMP production and insulin secretion. Acute glucose-lowering and insulinotropic actions were evaluated in Swiss TO mice. Subchronic studies on glucose homoeostasis, insulin secretion, food intake and bodyweight were evaluated in ob/ob mice. Liraglutide, N-AcGIP(Lys(37)Myr), a simple combination of both peptides and the Lira-AcGIP preparation demonstrated improved DPP-IV resistance (P<0.001), while stimulating cAMP production and insulin secretion (1.4-2-fold; P<0.001). The Lira-AcGIP preparation was more potent at lowering plasma glucose (20-51% reduction; P<0.05-P<0.001) and stimulating insulin secretion (1.5-1.8-fold; P<0.05-P<0.001) compared with Liraglutide and N-AcGIP(Lys(37)Myr) or a simple peptide combination. Daily administration of the Lira-AcGIP preparation to ob/ob mice lowered bodyweight (7-9%; P<0.05), food intake (23%; P<0.05) and plasma glucose (46% reduction; P<0.001), while increasing plasma insulin (1.5-1.6-fold; P<0.001). The Lira-AcGIP preparation enhanced glucose tolerance, insulin response to glucose and insulin content (P<0.05-P<0.001). These findings demonstrate that a combined preparation of the acylated GLP-1 and GIP peptides Liraglutide and N-AcGIP(Lys(37)Myr) markedly improved glucose-lowering and insulinotropic properties in diabetic obesity compared with either incretin mimetic given individually.

Glucagon-like peptide-1 analogues enhance synaptic plasticity in the brain: a link between diabetes and Alzheimer's disease.

Type 2 diabetes has been identified as a risk factor for patients with Alzheimer's disease. Insulin signalling is often impaired in Alzheimer's disease, contributing to the neurodegenerative process. One potential strategy to help prevent this is the normalisation of insulin signalling in the brain. Therefore, the present study was designed to test the effects of novel enzyme-resistant analogues of the insulin-releasing incretin hormone, glucagon-like peptide 1 (GLP-1). The effects of Liraglutide (Victoza) and other novel GLP-1 analogues were tested on synaptic plasticity (LTP) in area CA1 of the hippocampus. At a dose of 15nmol in 5microl i.c.v., Liraglutide (P<0.005), Asp(7)GLP-1 (P<0.001), N-glyc-GLP-1 (P<0.01), and Pro(9)GLP-1 (P<0.001). In contrast, the GLP-1 receptor antagonist exendin(9-39)amide impaired LTP (P<0.001). Co-injection of exendin(9-39) and Liraglutide showed no effect on LTP. These results clearly demonstrate that Liraglutide and other GLP-1 analogues elicit effects on neurotransmission in the brain. Furthermore, GLP-1 peptides are not only effective in modulating insulin-release and achieving glycaemic control in type 2 diabetes, but are also effective in modulating synaptic plasticity. These findings are consistent with our previous observations that the novel analogue (Val(8))GLP-1 enhances LTP and reverses the impairments of LTP induced by beta-amyoid fragments. Therefore, the drug effects seen here could potentially ameliorate the impairments in neuronal communication and cognitive processes observed in Alzheimer's disease.

Characterisation and biological activity of Glu3 amino acid substituted GIP receptor antagonists.

Glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal hormone, which regulates insulin release and glucose homeostasis, but is rapidly inactivated by enzymatic N-terminal truncation. Here we report the enzyme resistance and biological activity of several Glu(3)-substituted analogues of GIP namely; (Ala(3))GIP, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP. Only (Lys(3))GIP demonstrated moderately enhanced resistance to DPP-IV (p<0.05 to p<0.01) compared to native GIP. All analogues demonstrated a decreased potency in cAMP production (EC(50) 1.47 to 11.02 nM; p<0.01 to p<0.001) with (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated cAMP production (p<0.05). In BRIN-BD11 cells, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP did not stimulate insulin secretion with both (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated insulin secretion (p<0.05). Injection of each GIP analogue together with glucose in ob/ob mice significantly increased the glycaemic excursion compared to control (p<0.05 to p<0.001). This was associated with lack of significant insulin responses. (Ala(3))GIP, (Phe(3))GIP and (Tyr(3))GIP, when administered together with GIP, significantly reduced plasma insulin (p<0.05 to p<0.01) and impaired the glucose-lowering ability (p<0.05 to p<0.01) of the native peptide. The DPP-IV resistance and GIP antagonism observed were similar but less pronounced than (Pro(3))GIP. These data demonstrate that position 3 amino acid substitution of GIP with (Ala(3)), (Phe(3)), (Tyr(3)) or (Pro(3)) provides a new class of functional GIP receptor antagonists.

Antagonistic effects of two novel GIP analogs, (Hyp3)GIP and (Hyp3)GIPLys16PAL, on the biological actions of GIP and longer-term effects in diabetic ob/ob mice.

This study examines the actions of the novel enzyme-resistant, NH2-terminally modified GIP analog (Hyp(3))GIP and its fatty acid-derivatized analog (Hyp(3))GIPLys(16)PAL. Acute effects are compared with the established GIP receptor antagonist (Pro(3))GIP. All three peptides exhibited DPP IV resistance, and significantly inhibited GIP stimulated cAMP formation and insulin secretion in GIP receptor-transfected fibroblasts and in clonal pancreatic BRIN-BD11 cells, respectively. Likewise, in obese diabetic ob/ob mice, intraperitoneal administration of GIP analogs significantly inhibited the acute antihyperglycemic and insulin-releasing effects of native GIP. Administration of once daily injections of (Hyp(3))GIP or (Hyp(3))GIPLys(16)PAL for 14 days resulted in significantly lower plasma glucose levels (P < 0.05) after (Hyp(3))GIP on days 12 and 14 and enhanced glucose tolerance (P < 0.05) and insulin sensitivity (P < 0.05 to P < 0.001) in both groups by day 14. Both (Hyp(3))GIP and (Hyp(3))GIPLys(16)PAL treatment also reduced pancreatic insulin (P < 0.05 to P < 0.01) without affecting islet number. These data indicate that (Hyp(3))GIP and (Hyp(3))GIPLys(16)PAL function as GIP receptor antagonists with potential for ameliorating obesity-related diabetes. Acylation of (Hyp(3))GIP to extend bioactivity does not appear to be of any additional benefit.

Comparison of the anti-diabetic effects of GIP- and GLP-1-receptor activation in obese diabetic (ob/ob) mice: studies with DPP IV resistant N-AcGIP and exendin(1-39)amide.

BACKGROUND: The two major incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are being actively explored as anti-diabetic agents because they lower blood glucose through multiple mechanisms. The rapid inactivation of GIP and GLP-1 by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV) makes their biological actions short-lived, but stable agonists such as N-acetylated GIP (N-AcGIP) and exendin(1-39)amide have been advocated as stable and specific GIP and GLP-1 analogues. METHODS: The present study examined the sub-chronic (14 days) anti-diabetic actions of single daily doses of N-AcGIP and exendin(1-39)amide given alone or in combination to obese diabetic (ob/ob) mice over a 14-day period. RESULTS: Initial experiments confirmed the potent anti-hyperglycaemic and insulinotropic properties of N-AcGIP and exendin(1-39)amide. Sub-chronic administration of N-AcGIP alone or in combination with exendin(1-39)amide significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to control ob/ob mice. This was associated with a significant enhancement of the insulin response to glucose and a notable improvement of insulin sensitivity. Combined treatment with N-AcGIP and exendin(1-39)amide also significantly decreased glycated haemoglobin. Exendin(1-39)amide alone had no significant effect on any of the metabolic parameters monitored. In addition, no significant effects were observed on body weight and food intake in any of the treatment groups. CONCLUSIONS: The results illustrate significant anti-diabetic potential of N-AcGIP alone and in combination with exendin(1-39)amide.

Soluble beta-amyloid[25-35] reversibly impairs hippocampal synaptic plasticity and spatial learning.

Beta-amyloid is a peptide that appears to be responsible for cognitive impairments in patients with Alzheimer's disease. Recent research shows that soluble oligomers of beta-amyloid affect synaptic activity and learning, well before any amyloid has aggregated into plaques. Here we show that injection of 3x10 nmol amyloid [25-35] i.c.v. transiently impairs learning of a radial arm maze and the induction of hippocampal long-term potentiation. Furthermore, hippocampal field potentials had been recorded over a period of 21 days and were found to be reduced from day 9 to day 15 (P<0.001), after which the reduction had reversed to baseline. In the spatial 8-arm learning task, animals had to learn which 3 out of 8 arms had been baited. A significant impairment of working and long-term memory was observed at day 12-20 (P<0.001), but not at days 3-11 or 20-28. Long-term potentiation induction in the hippocampus area CA1 was also impaired at day 12-20 (P<0.001), but not at other days. A scrambled peptide sequence version of amyloid did not have any effect. These results emphasise that soluble amyloid fragments already have detrimental effects on brain function well before aggregation occurs. They also show that these effects are reversible, and therefore most likely do not involve neuronal death. The neurodegeneration seen in Alzheimer's disease brains is most likely a downstream effect, linked to processes such as immune response activation and free radical production. These results suggest that treatment at very early stages of Alzheimer's disease could prevent later irreversible neuronal degeneration.

Characterisation and glucoregulatory actions of a novel acylated form of the (Pro3)GIP receptor antagonist in type 2 diabetes.

In this study, we tested the biological activity of a novel acylated form of (Pro3)glucose-dependent insulinotropic polypetide [(Pro3)GIP] prepared by conjugating palmitic acid to Lys16 to enhance its efficacy in vivo by promoting binding to albumin and extending its biological actions. Like the parent molecule (Pro3)GIP, (Pro3)GIPLys16PAL was completely stable to the actions of DPP-IV and significantly (p<0.01 to p<0.001) inhibited GIP-stimulated cAMP production and cellular insulin secretion. Furthermore, acute administration of (Pro3)GIPLys16PAL also significantly (p<0.05 to p<0.001) countered the glucose-lowering and insulin-releasing actions of GIP in ob/ob mice. Daily injection of (Pro3)GIPLys16PAL (25 nmol/kg bw) in 14-18-week-old ob/ob mice over 14 days had no effect on body weight, food intake or non-fasting plasma glucose and insulin concentrations. (Pro3)GIPLys16PAL treatment also failed to significantly alter the glycaemic response to an i.p. glucose load or test meal, but insulin concentrations were significantly reduced (1.5-fold; p<0.05) after the glucose load. Insulin sensitivity was enhanced (1.3-fold; p<0.05) and pancreatic insulin was significantly reduced (p<0.05) in the (Pro3)GIPLys16PAL-treated mice. These data demonstrate that acylation of Lys16 with palmitic acid in (Pro3)GIP does not improve its biological effectiveness as a GIP receptor antagonist.

Impairments of hippocampal synaptic plasticity induced by aggregated beta-amyloid (25-35) are dependent on stimulation-protocol and genetic background.

The aggregation of beta-amyloid to plaques in the brain is one of the hallmarks of Alzheimer disease (AD). Numerous studies have tried to elucidate to what degree amyloid peptides play a role in the neurodegenerative developments seen in AD. While most studies report an effect of amyloid on neural activity and cognitive abilities of rodents, there have been many inconsistencies in the results. This study investigated to what degree the different genetic backgrounds affect the outcome of beta-amyloid fragment (25-35) on synaptic plasticity in vivo in the rat hippocampus. Two strains, Wistar and Lister hooded rats, were tested. In addition, the effects of a strong (600 stimuli) and a weak stimulation protocol (100 stimuli) on impairments of LTP were analysed. Furthermore, since the state of amyloid aggregation appears to play a role in the induction of toxic processes, it was tested by dual polarisation interferometry to what degree and at what speed beta-amyloid (25-35) can aggregate in vitro. It was found that 100 nmol beta-amyloid (25-35) injected icv did impair LTP in Wistar rats when using the weak but not the strong stimulation protocol (P < 0.001). One-hundred nano mole of the reverse sequence amyloid (35-25) had no effect. LTP in Lister Hooded rats was not impaired by amyloid at any stimulation protocol. The aggregation studies showed that amyloid (25-35) aggregated within hours, while amyloid (35-25) did not. These results show that the genetic background and the stimulation protocol are important variables that greatly influence the experimental outcome. The fact that amyloid (25-35) aggregated quickly and showed neurophysiological effects, while amyloid (35-25) did not aggregate and did not show any effects indicates that the state of aggregation plays an important role in the physiological effects.

Beneficial effects of sub-chronic activation of glucagon-like peptide-1 (GLP-1) receptors on deterioration of glucose homeostasis and insulin secretion in aging mice.

Aging is associated with an increased incidence of glucose intolerance and type 2 diabetes. Glucagon-like peptide-1 (GLP-1) is an important insulinotropic peptide secreted from the gastrointestinal tract in response to nutrient absorption. The present study was designed to assess the sub-chronic glucose regulatory effects of the potent long-acting GLP-1 receptor agonist, (Val(8))GLP-1, in aging 45-49 week old mice. Daily injection of (Val(8))GLP-1 (25 n mol/kg body weight) for 12 days had no significant effect on food intake, body weight, non-fasting plasma glucose and insulin concentrations. However, after 12 days, the glycaemic response to intraperitoneal glucose was improved (P<0.05) in (Val(8))GLP-1 treated mice. In keeping with this, glucose-mediated insulin secretion was enhanced (P<0.05) and insulin sensitivity improved (P<0.05) compared to controls. These data indicate that sub-chronic activation of the GLP-1 receptor by daily treatment with (Val(8))GLP-1 counters aspects of the age-related impairment of pancreatic beta-cell function and insulin sensitivity.

Evaluation of the antidiabetic activity of DPP IV resistant N-terminally modified versus mid-chain acylated analogues of glucose-dependent insulinotropic polypeptide.

Glucose dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with therapeutic potential for type 2 diabetes due to its insulin-releasing and antihyperglycaemic actions. However, development of GIP-based therapies is limited by N-terminal degradation by DPP IV resulting in a very short circulating half-life. Numerous GIP analogues have now been generated exhibiting DPP IV resistance and extended bioactivity profiles. In this study, we report a direct comparison of the long-term antidiabetic actions of three such GIP molecules, N-AcGIP, GIP(Lys(37)PAL) and N-AcGIP(Lys(37)PAL) in obese diabetic (ob/ob) mice. An extended duration of action of each GIP analogue was demonstrated prior to examining the effects of once daily injections (25nmolkg(-1) body weight) over a 14-day period. Administration of either N-AcGIP, GIP(Lys(37)PAL) or N-AcGIP(Lys(37)PAL) significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to saline treated controls. All three analogues significantly enhanced glucose and nutrient-induced insulin release, and improved insulin sensitivity. The metabolic and insulin secretory responses to native GIP were also enhanced in 14-day analogue treated mice, revealing no evidence of GIP-receptor desensitization. These effects were accompanied by significantly enhanced pancreatic insulin following N-AcGIP(Lys(37)PAL) and increased islet number and islet size in all three groups. Body weight, food intake and circulating glucagon were unchanged. These data demonstrate the therapeutic potential of once daily injection of enzyme resistant GIP analogues and indicate that N-AcGIP is equally as effective as related palmitate derivatised analogues of GIP.

Novel glucagon-like peptide-1 (GLP-1) analog (Val8)GLP-1 results in significant improvements of glucose tolerance and pancreatic beta-cell function after 3-week daily administration in obese diabetic (ob/ob) mice.

This study evaluates the antidiabetic potential of an enzyme-resistant analog, (Val8)GLP-1. The effects of daily administration of a novel dipeptidyl peptidase IV-resistant glucagon-like peptide-1 (GLP-1) analog, (Val8)GLP-1, on glucose tolerance and pancreatic beta-cell function were examined in obese-diabetic (ob/ob) mice. Acute intraperitoneal administration of (Val8)GLP-1 (6.25-25 nmol/kg) with glucose increased the insulin response and reduced the glycemic excursion in a dose-dependent manner. The effects of (Val8)GLP-1 were greater and longer lasting than native GLP-1. Once-daily subcutaneous administration of (Val8)GLP-1 (25 nmol/kg) for 21 days reduced plasma glucose concentrations, increased plasma insulin, and reduced body weight more than native GLP-1 without a significant change in daily food intake. Furthermore, (Val8)GLP-1 improved glucose tolerance, reduced the glycemic excursion after feeding, increased the plasma insulin response to glucose and feeding, and improved insulin sensitivity. These effects were consistently greater with (Val8)GLP-1 than with native GLP-1, and both peptides retained or increased their acute efficacy compared with initial administration. (Val8)GLP-1 treatment increased average islet area 1.2-fold without changing the number of islets, resulting in an increased number of larger islets. These data demonstrate that (Val8)GLP-1 is more effective and longer acting than native GLP-1 in obese-diabetic ob/ob mice.

GIP(Lys16PAL) and GIP(Lys37PAL): novel long-acting acylated analogues of glucose-dependent insulinotropic polypeptide with improved antidiabetic potential.

Glucose-dependent insulinotropic polypeptide (GIP) is a physiological insulin releasing peptide. We have developed two novel fatty acid derivatized GIP analogues, which bind to serum albumin and demonstrate enhanced duration of action in vivo. GIP(Lys(16)PAL) and GIP(Lys(37)PAL) were resistant to dipeptidyl peptidase IV (DPP IV) degradation. In vitro studies demonstrated that GIP analogues retained their ability to activate the GIP receptor through production of cAMP and to stimulate insulin secretion. Intraperitoneal administration of GIP analogues to obese diabetic (ob/ob) mice significantly decreased the glycemic excursion and elicited increased and prolonged insulin responses compared to native GIP. A protracted glucose-lowering effect was observed 24 h following GIP(Lys(37)PAL) administration. Once a day injection for 14 days decreased nonfasting glucose, improved glucose tolerance, and enhanced the insulin response to glucose. These data demonstrate that fatty acid derivatized GIP peptides represent a novel class of long-acting stable GIP analogues for therapy of type 2 diabetes.

Effects on glucose homeostasis and insulin secretion of long term activation of the glucose-dependent insulinotropic polypeptide (GIP) receptor by N-AcGIP(LysPAL37) in normal mice.

Glucose-dependent insulinotropic polypeptide (GIP) is a key hormone of the enteroinsular axis. The present study was designed to assess the metabolic effects in healthy mice of long term activation of the GIP receptor by N-AcGIP(LysPAL37), a potent long-acting GIP receptor agonist. Daily injection of N-AcGIP(LysPAL37) (25 nmol/kg body weight) for 14 days had no significant effect on food intake, body weight, glycated hemoglobin levels, non-fasting plasma glucose and insulin concentrations compared to saline treated controls. No significant differences in post-prandial plasma glucose and insulin concentrations were observed between the two groups following 15 min feeding. However, after 14 days, the glycemic response to intraperitoneal (i.p.) glucose was significantly improved in the N-AcGIP(LysPAL37) treated mice compared to controls (P < 0.01). In keeping with this, glucose-mediated insulin secretion was significantly enhanced in the N-AcGIP(LysPAL37) treated group (P < 0.05). No changes in insulin sensitivity or pancreatic insulin content of the N-AcGIP(LysPAL37) treated mice were detected. No adverse reactions were noted and the effects of N-AcGIP(LysPAL37) were reversed by 14 days cessation of treatment. These data indicate that long term activation of the GIP receptor by daily treatment with N-AcGIP(LysPAL37) improved glucose tolerance due to enhancement of pancreatic beta cell glucose responsiveness and insulin secretion.

Antidiabetic potential of two novel fatty acid derivatised, N-terminally modified analogues of glucose-dependent insulinotropic polypeptide (GIP): N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37).

Fatty acid derivatisation was used to develop two novel, long-acting, N-terminally modified, glucose-dependent insulinotropic polypeptide (GIP) analogues, N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37). In contrast to GIP, which was rapidly degraded by in vitro incubation with dipeptidylpeptidase IV (DPP IV) (52% intact after 2 h), the analogues remained fully intact for up to 24 h. Both fatty acid-derivatised analogues stimulated cAMP production in GIP receptor Chinese hamster lung (CHL) fibroblasts (EC50 12.1-13.0 nM) and significantly improved in vitro insulin secretion from BRIN-BD11 cells (1.1- to 2.4-fold; p < 0.05 to p < 0.001) compared to control (5.6 mM glucose). Administration of N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) together with glucose in obese diabetic (ob/ob) mice significantly reduced the glycaemic excursion (1.4- and 1.5-fold, respectively; p < 0.05 to p < 0.01) and improved the insulinotropic response (1.5- and 2.3-fold, respectively; p < 0.01 to p < 0.001) compared to native peptide. Dose-response studies with N-AcGIP(LysPAL37) revealed that even the lowest concentration (6.25 nmol/kg) induced a highly significant decrease (1.4-fold; p < 0.001) in the overall glycaemic excursion, coupled with a significant increase (2.0-fold; p < 0.01) in circulating insulin. Furthermore, basal glucose values remained significantly reduced (p < 0.05) and insulin values increased 24 h following a single injection of N-AcGIP(LysPAL37). The glucose-lowering action of the fatty acid-derivatised peptide was greater than that of N-AcGIP. These data demonstrate that novel fatty acid-derivatised analogues of N-terminally modified AcGIP function as long-acting GIP-receptor agonists, with significant antidiabetic potential.