Glucose Tolerance Test In Mice Research Articles

55P0110, a Novel Synthetic Compound Developed from a Plant Derived Backbone Structure, Shows Promising Anti-Hyperglycaemic Activity in Mice.

Starting off with a structure derived from the natural compound multiflorine, a derivatisation program aimed at the discovery and initial characterisation of novel compounds with antidiabetic potential. Design and discovery of the structures was guided by oral bioactivities obtained in oral glucose tolerance tests in mice. 55P0110, one among several new compounds with distinct anti-hyperglycaemic activity, was further examined to characterise its pharmacology and mode of action. Whereas a single oral dose of 55P0110 did not affect basal glycaemia, it markedly improved the glucose tolerance of healthy and diabetic mice (peak blood glucose in glucose tolerance test, mmol/l: healthy mice with 90 mg/kg 55P0110, 17.0±1.2 vs. 10.1±1.1; diabetic mice with 180 mg/kg 55P0110, 23.1±0.9 vs. 11.1±1.4; p<0.001 each). Closer examination argued against retarded glucose resorption from the gut, increased glucose excretion in urine, acute insulin-like or insulin sensitising properties, and direct inhibition of dipeptidyl peptidase-4 as the cause of glucose lowering. Hence, 55P0110 seems to act via a target not exploited by any drug presently approved for the treatment of diabetes mellitus. Whereas the insulinotropic sulfonylurea gliclazide (16 mg/kg) distinctly increased the circulating insulin-per-glucose ratio under basal conditions, 55P0110 (90 mg/kg) lacked such an effect (30 min. after dosing, nmol/mol: vehicle, 2.49±0.27; 55P0110, 2.99±0.35; gliclazide, 8.97±0.49; p<0.001 each vs. gliclazide). Under an exogenous glucose challenge, however, 55P0110 increased this ratio to the same extent as gliclazide (20 min. after glucose feeding: vehicle, 2.53±0.41; 55P0110, 3.80±0.46; gliclazide, 3.99±0.26; p<0.05 each vs. vehicle). By augmenting the glucose stimulated increase in plasma insulin, 55P0110 thus shows distinct anti-hyperglycaemic action in combination with low risk for fasting hypoglycaemia in mice. In summary, we have discovered a novel class of fully synthetic substituted quinazolidines with an attractive pharmacological profile that recommends the structures for further evaluation as candidates for the treatment of diabetes mellitus.

Activity of dietary fatty acids on FFA1 and FFA4 and characterisation of pinolenic acid as a dual FFA1/FFA4 agonist with potential effect against metabolic diseases.

Various foods are associated with effects against metabolic diseases such as insulin resistance and type 2 diabetes; however, their mechanisms of action are mostly unclear. Fatty acids may contribute by acting as precursors of signalling molecules or by direct activity on receptors. The medium- and long-chain NEFA receptor FFA1 (free fatty acid receptor 1, previously known as GPR40) has been linked to enhancement of glucose-stimulated insulin secretion, whereas FFA4 (free fatty acid receptor 4, previously known as GPR120) has been associated with insulin-sensitising and anti-inflammatory effects, and both receptors are reported to protect pancreatic islets and promote secretion of appetite and glucose-regulating hormones. Hypothesising that FFA1 and FFA4 mediate therapeutic effects of dietary components, we screened a broad selection of NEFA on FFA1 and FFA4 and characterised active compounds in concentration-response curves. Of the screened compounds, pinolenic acid, a constituent of pine nut oil, was identified as a relatively potent and efficacious dual FFA1/FFA4 agonist, and its suitability for further studies was confirmed by additional in vitro characterisation. Pine nut oil and free and esterified pure pinolenic acid were tested in an acute glucose tolerance test in mice. Pine nut oil showed a moderately but significantly improved glucose tolerance compared with maize oil. Pure pinolenic acid or ethyl ester gave robust and highly significant improvements of glucose tolerance. In conclusion, the present results indicate that pinolenic acid is a comparatively potent and efficacious dual FFA1/FFA4 agonist that exerts antidiabetic effects in an acute mouse model. The compound thus deserves attention as a potential active dietary ingredient to prevent or counteract metabolic diseases.

Glucose tolerance tests for systematic screening of glucose homeostasis in mice.

This article presents a detailed description of intraperitoneal and oral glucose tolerance tests in mice. The former is widely used in initial high-throughput phenotyping of mutant mice to assess a diabetic phenotype and alterations in glucose homeostasis. Each protocol provides a comprehensive description of each step in the workflow, including variation of the standard protocol under particular circumstances (e.g., sensitivity to food deprivation, excessive deviations in body composition, or need for extra blood samples for additional analyses). We also describe how reduction of body mass and body temperature can be used as additional readouts to monitor metabolic function in response to food deprivation.

Split Ssp DnaB mini-intein-mediated production of recombinant human glucagon-like peptide-1/7-36.

Glucagon-like peptide-1 (GLP-1) plays an important role in the regulation of postprandial insulin release. Here, we used the split DnaB mini-intein system to produce recombinant human GLP-1/7-36 (rhGLP-1) in Escherichia coli. The C-terminal domain of DnaB mini-intein (IntC) was genetically fused at the N-terminus of rhGLP-1 to produce IntC-GLP-1. IntC-GLP-1 and N-terminal domain of DnaB mini-intein (IntN) protein were prepared in a denatured buffer of pH 8.0. IntC-GLP-1 was diluted 1:8 into the phosphate buffer of pH 6.6. IntN was added into the diluted solution of IntC-GLP-1 at the molar ratio of 1:2. Then, rhGLP-1 was released from IntC-GLP-1 via inducible C-terminal peptide-bond cleavage by shifting pH from 8.0 to 6.6 at 25 °C for 24-H incubation. Then, the supernatant was applied to a Ni-Sepharose column, and the pass through fraction was collected. About 5.34 mg of rhGLP-1 with the purity of 97% was obtained from 1 L of culture medium. Mass spectrometry showed the molecular weight of 3,300.45 Da, which was equal to the theoretical value of GLP-1/7-36. The glucose-lowering activity of rhGLP-1 was confirmed by the glucose tolerance test in mice. In conclusion, the reported method was an efficient strategy to produce rhGLP-1 without using enzyme or chemical reagents, which could also be used for other similar peptides.

Improving the Pharmacokinetics of GPR40/FFA1 Full Agonists.

We recently reported the discovery of a potent GPR40 full agonist AM-1638 (1). Herein, we describe our efforts in improving the drug-like properties of the full agonists through the systematic introduction of polar groups in the C-, D-, and A-rings. This led to the discovery of new GPR40 full agonists with significantly improved pharmacokinetic propeties. Compound 8 and 20 also showed potent in vivo efficacy in oral glucose tolerance tests in mice in addition to the improvement in properties.

A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes

Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2'-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a Kd of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC50 of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.

Potent and Orally Bioavailable GPR142 Agonists as Novel Insulin Secretagogues for the Treatment of Type 2 Diabetes.

GPR142 is a G protein-coupled receptor that is predominantly expressed in pancreatic β-cells. GPR142 agonists stimulate insulin secretion in the presence of high glucose concentration, so that they could be novel insulin secretagogues with reduced or no risk of hypoglycemia. We report here the optimization of HTS hit compound 1 toward a proof of concept compound 33, which showed potent glucose lowering effects during an oral glucose tolerance test in mice and monkeys.

Recombinant production of mGLP-1 by coupling of refolding and intein-mediated self-cleavage (CRIS)

Glucagon-like peptide-1 as an endogenous glucose-lowering peptide is a promising candidate for anti-diabetic drug development. Here, we developed a convenient method by coupling of refolding and intein-mediated self-cleavage (CRIS) to improve the recombinant production of a mutated glucagon-like peptide-1 (mGLP-1). Bacterial cell culture employing auto-induction was performed at 37°C to avoid the intracellular self-cleavage of the intein fusion protein. The impacts of urea, pH, and temperature on the efficiency of CRIS were tested, and then, the optimized CRIS was established. Using the optimized method, we obtained the purified mGLP-1 with a yield of 3.41mg peptide/g bacterial cells which was 5.6-fold higher than before. After that, using chromatography, peptide electrophoresis, and mass spectrometry, we determined the purity and molecular weight of the purified peptide and then confirmed its glucose-lowering activity by performing glucose tolerance test in mice. These results suggest that CRIS is a relatively simple and efficacious method for the recombinant production of mGLP-1, and as a general method, it can also be used for the recombinant preparation of some other proteins and peptides.

Platycodon grandiflorum modifies adipokines and the glucose uptake in high-fat diet in mice and L6 muscle cells

Obesity and diabetes have become the most common human health problems worldwide. Obesity's contribution to type 2 diabetes might be due to dysregulation of adipokines and glucose uptake. In this study, we performed in-vivo and in-vitro studies to evaluate the effects of Platycodon grandiflorum extract (PGE) on adipokines and glucose uptake. Before study, platycodin D concentrations were analysed by HPLC in PGE prepared in water, in 50% ethanol and in 80% ethanol, and we selected the 80% ethanol extract as the PGE for this study based on the HPLC results. We found that inclusion of PGE in the high-fat diet (HFD) markedly attenuated food intake, body weight, epididymal fat weight, adipocyte size and blood glucose levels by the oral glucose tolerance test in mice, and maintained serum levels of adiponectin, resistin, leptin, fructosamine and triglycerides. Gene expression analysis revealed that PGE up-regulated adiponectin, and down-regulated TNF-α and leptin in fat tissue. In L6 muscle cells in vitro, PGE increased insulin-stimulated glucose uptake. We conclude that PGE may improve obesity in mice fed an HFD and glucose uptake in L6 muscle cells by modifying adipokines, and could offer clinical benefits as a supplement to treat obesity and diabetes.

Quinolone derivatives containing strained spirocycle as orally active glycogen synthase kinase 3β (GSK-3β) inhibitors for type 2 diabetics

The design, synthesis, and evaluation of 6-6-7 tricyclic quinolones containing the strained spirocycle moiety aiming at the GSK-3β inhibitor were described. Among the synthesized compounds, 44, having a cyclobutane ring on a spirocycle, showed excellent GSK-3β inhibitory activity in both cell-free and cell-based assays (IC50 = 36nM, EC50 = 3.2μM, respectively). Additionally, 44 decreased the plasma glucose concentration dose-dependently after an oral glucose tolerance test in mice.

Comparative Study for α-Glucosidase Inhibitory Effects of Total Iridoid Glycosides in the Crude Products and the Wine-processed Products from &lt;i&gt;Cornus officinalis&lt;/i&gt;

To compare the difference of α-glucosidase inhibitory effect of total iridoid glycosides from crude products and processed products (Paozhi in Chinese) of Cornus officinalis Sieb. et Zucc. by α-glucosidase and glucose tolerance test in mice. Alcohol 40% was employed to extract the total iridoid glycosides from the crude products and the processed products then the total iridoid glycosides were enriched through macroporous resins to obtain SZY-1 and SZY-2 from the crude products and the processed products, respectively. The inhibitiory activity of these extracts was investigated on α-glucosidase inhibition in vitro and glucose tolerance test in vivo. The results demonstrated that both SZY-1 and SZY-2 were successfully enriched by macroporous resins. The contents of the total iridoid glycoside in SZY-1 and SZY-2 were 75.3% and 42.8%, respectively, and exerted inhibitory effects at various concentrations on α-glucosidase. The maximal inhibition ratio (86%) of SZY-1 was markedly higher than that of SZY-2. Meanwhile, SZY-1 was more potent than SZY-2 in decreasing blood sugar in diabetic mice induced by streptozotocin. Their effects were dependent on the dosage. In conclusion, the iridoid total glycoside from crude products and processed products of C. officinalis had α-glucosidase inhibitory effects in vitro and in vivo, and the effects of the crude products ware superior to those of processed products. It was also suggested that the hypoglycemic effects of C. officinalis may be attributable to its total iridoid glycoside.

Anti-obesity effects of highly polymeric proanthocyanidins from seed shells of Japanese horse chestnut (Aesculus turbinata Blume)

Recently, we have shown that seed shells contain a large amount of highly polymeric proanthocyanidins having a series of heteropolyflavan-3-ols with doubly linked A-type linkages as well as single B-type bonds without gallic acid esterified to them. Here, we attempted to evaluate in vivo anti-obesity effects of the polymerized proanthocyanidins in mice. An oral starch or glucose tolerance test in mice revealed that the isolated two fractions of highly polymerized proanthocyanidins with the different degree of polymerization suppressed effectively the elevation of blood glucose from oral starch, but not from oral glucose, suggesting the preferential inhibition of the digestive enzymes of carbohydrates. Moreover, in vivo anti-obesity effects of the total fraction containing the proanthocyanidins as a drink were investigated in mice fed a high-fat diet. Their anti-obesity effects became more evident after 9weeks as determined by the attenuation of the elevation in body weight, the mass of peritoneal adipose tissues, and the plasma levels of total cholesterol and leptin. Furthermore, the increased size of hepatocytes and the generation of steatosis with micro- and macrovesicles in liver were normalized by the dietary supplementation of the total proanthocyanidin fraction.The findings suggest the usefulness of highly polymeric proanthocyanidins from seed shells in the application to food as a dietary supplement with anti-obesity effects in vivo through the inhibition of digestive enzymes of carbohydrates and fats.

Adipocyte Apoptosis, a Link between Obesity, Insulin Resistance, and Hepatic Steatosis

Adipocyte death has been reported in both obese humans and rodents. However, its role in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity has not been studied. We now show using real-time reverse transcription-PCR arrays that adipose tissue of obese mice display a pro-apoptotic phenotype. Moreover, caspase activation and adipocyte apoptosis were markedly increased in adipose tissue from both mice with diet-induced obesity and obese humans. These changes were associated with activation of both the extrinsic, death receptor-mediated, and intrinsic, mitochondrial-mediated pathways of apoptosis. Genetic inactivation of Bid, a key pro-apoptotic molecule that serves as a link between these two cell death pathways, significantly reduced caspase activation, adipocyte apoptosis, prevented adipose tissue macrophage infiltration, and protected against the development of systemic insulin resistance and hepatic steatosis independent of body weight. These data strongly suggest that adipocyte apoptosis is a key initial event that contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis associated with obesity in both mice and humans. Inhibition of adipocyte apoptosis may be a new therapeutic strategy for the treatment of obesity-associated metabolic complications.

Nuclear Factor of Activated T Cells Regulates Osteopontin Expression in Arterial Smooth Muscle in Response to Diabetes-Induced Hyperglycemia

Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3(-/-) mice. These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

Screening of the hypoglycemic effect of eight Vietnamese herbal drugs

The prevalence of type 2 diabetes in developing countries is increasing. In Vietnam, several plants are thought to be useful for the treatment of type 2 diabetes. This study has been performed to screen the hypoglycemic effects of eight Vietnamese herbs used in traditional medicine for the treatment of diabetes. Blood glucose levels were measured before and several times after oral or i.p. administration of the ethanol-based plant extracts in normal mice. The extracts that reduced blood glucose both orally and i.p. were also studied in glucose tolerance tests in mice. Gynostemma pentaphyllum Makino (Cucurbitaceae) at doses of 200 and 300 mg/kg i.p. or 1500 mg/kg orally reduced blood glucose in mice (P < 0.001 for all compared to control group using NaCl 0.9%). Similarly, Anemarrhena asphodeloides Bunge (Liliaceae) at 200 and 300 mg/kg i.p. (P < 0.001 vs. control group) and 1500 mg/kg orally reduced blood glucose 4 h after administration (P < 0.001). Angiopteris evecta Forst. Hoffn. (Marattiaceae) 300 mg/kg i.p. and 1500 mg/kg orally strongly reduced blood glucose levels (P < 0.001 vs. control groups). All three extracts when dosed at 1000 mg/kg orally suppressed the rise in blood glucose in normal mice during a glucose tolerance test. We have found three herbs that reduced blood glucose and inhibited increases in blood glucose after a glucose challenge in normal mice.

Discovery of the First Potent and Orally Efficacious Agonist of the Orphan G-Protein Coupled Receptor 119

GPR119 is a rhodopsin-like GPCR expressed in pancreatic beta-cells and incretin releasing cells in the GI tract. As with incretins, GPR119 increases cAMP levels in these cell types, thus making it a highly attractive potential target for the treatment of diabetes. The discovery of the first reported potent agonist of GPR119, 2-fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine (8g, AR231453), is described starting from an initial inverse agonist screening hit. Compound 8g showed in vivo activity in rodents and was active in an oral glucose tolerance test in mice following oral administration.

Engineered Enteroendocrine Cells Secrete Insulin in Response to Glucose and Reverse Hyperglycemia in Diabetic Mice

Type 1 diabetes is a metabolic disorder caused by loss of insulin-producing pancreatic beta-cells. Expression of insulin in non-beta-cells to create beta-cell surrogates has been tried to treat type 1 diabetes. Enteroendocrine K cells have characteristics similar to pancreatic beta-cells, such as a glucose-sensing system and insulin-processing proteases. In this study, we genetically engineered an enteroendocrine cell line (STC-1) to express insulin under the control of the glucose-dependent insulinotropic polypeptide promoter. We screened clones and chose one, Gi-INS-7, based on its high production of insulin. Gi-INS-7 cells expressed glucose transporter 2 (GLUT2) and glucokinase (GK) and secreted insulin in response to elevated glucose levels in vitro. To determine whether Gi-INS-7 cells can control blood glucose levels in diabetic mice, we transplanted these cells under the kidney capsule of streptozotocin (STZ)-induced diabetic mice and found that blood glucose levels became normal within 2 weeks of transplantation. In addition, glucose tolerance tests in mice that became normoglycemic after transplantation with Gi-INS-7 cells showed that exogenous glucose was cleared appropriately. These results suggest that engineered K cells may be promising surrogate beta-cells for possible therapeutic use for the treatment of type 1 diabetes.

Prolonged Remission of Diabetes by Regeneration of β Cells in Diabetic Mice Treated with Recombinant Adenoviral Vector Expressing Glucagon-like Peptide-1

Type 1 diabetes results from insulin deficiency caused by destruction of pancreatic β cells. Glucagon-like peptide (GLP)-1 stimulates β cell growth and differentiation. To determine whether continuous expression of GLP-1 in vivo can regenerate β cells and remit type 1 diabetes in mice for a prolonged time, we constructed an adenoviral vector containing the cytomegalovirus promoter/enhancer and albumin leader sequence followed by GLP-1 cDNA (rAd-GLP-1). A single administration of rAd-GLP-1 via the tail vein into streptozotocin (STZ)-induced diabetic non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in remission of diabetes within 10 days; normoglycemia remained until the experiment was terminated. The number of insulin-positive cells in the pancreas and insulin secretion significantly increased in rAd-GLP-1-treated mice compared with STZ-induced diabetic mice treated with rAd-β-galactosidase. Glucose tolerance tests in mice that achieved normoglycemia after treatment with rAd-GLP-1 showed that the kinetics of glucose clearance was similar to normal NOD/SCID mice. Treatment of autoimmune diabetic mice with rAd-GLP-1 restored normoglycemia, which was maintained for 1 year when mice were also treated with an immunoregulator to halt the autoimmune response to β cells. We suggest that regeneration of insulin-producing cells by GLP-1 gene therapy may be a potential method for prolonged control of type 1 diabetes in humans.

(3 R)-4-[(3 R)-3-Amino-4-(2,4,5-trifluorophenyl)butanoyl]-3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one, a selective dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Replacement of the triazolopiperazine ring of sitagliptin (DPP-4 IC 50 = 18 nM) with 3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one gave dipeptidyl peptidase IV (DPP-4) inhibitor 1 which is potent (DPP-4 IC 50 = 2.6 nM), selective, and efficacious in an oral glucose tolerance test in mice. It was selected for extensive preclinical development as a potential back-up candidate to sitagliptin.

Identification of novel saponins from edible seeds of Japanese horse chestnut ( Aesculus turbinata B lume) after treatment with wooden ashes and their nutraceutical activity

Natural seeds of Japanese horse chestnut ( Aesculus turbinata B lume) contain large amounts of mixed triterpenoidal saponins called escins. Recent studies have shown that escins have several biological activities including anti-inflammatory action and inhibitory effects on the absorption of ethanol and glucose. For the edible utilization of the seeds, natural seeds are usually treated with wooden ashes to remove harshness. Here, we found the novel compounds derived from escins in the edible seeds after the food processing with wooden ashes. The instrumental analyses revealed the chemical structures of escins and the derivatives. These compounds are identified as four types of deacetylescins Ia, IIa, Ib, and IIb as well as two types of desacylescins I and II. To determine their biological activity, the purified compounds were tested for their potential nutraceutical activity. The oral glucose tolerance test in mice revealed that a single oral administration of the isolated components of deacetylescins at a dose of 100 mg/kg was clearly effective in attenuating the elevation of blood glucose levels. The inhibitory effects of escins and their derivatives were in the order of escins > deacetylescins > desacylescins. Moreover, we found the inhibitory activity of those compounds on pancreatic lipase. Escins were the most potent in inhibiting the enzyme activity, and followed by desacylescins and then deacetylescins. Taken together, our results suggest the potential usefulness of novel saponins including deacetylescins and desacylescins from edible seeds as novel sources for nutraceutical foods with anti-obese effects.