Differential effects of oxyntomodulin and GLP-1 on glucose metabolism

Glucagon-like peptide-1 (GLP-1) and oxyntomodulin (OXM) are peptide hormones secreted postprandially from the gut that stimulate insulin secretion in a glucose-dependent manner. OXM activates both the GLP-1 receptor (GLP1R) and the glucagon receptor (GCGR). It has been suggested that OXM acutely modulates glucose metabolism solely through GLP1R agonism. Because OXM activates the GLP1R with lower affinity than GLP-1, we generated a peptide analog (Q→E, OXMQ3E) that does not exhibit glucagon receptor agonist activity but retains the same affinity as OXM for GLP1R. We compared the effects of OXM and OXMQ3E in a glucose tolerance test and, to better characterize the effect on glucose metabolism, we performed controlled infusions of OXM or OXMQ3E during a hyperglycemic clamp performed in wild-type, Glp1r(-/-), and Gcgr(-/-) mice. Our findings show that OXM, but not OXMQ3E, activates the GCGR in vivo. Second, OXM and OXMQ3E improve glucose tolerance following an acute glucose challenge and during a hyperglycemic clamp in mice. Finally, OXM infusion during a glucose clamp reduces the glucose infusion rate (GIR) despite a simultaneous increase in insulin levels in Glp1r(-/-) mice, whereas OXM and OXMQ3E increase GIR to a similar extent in Gcgr(-/-) mice. In conclusion, activation of the GCGR seems to partially attenuate the acute beneficial effects on glucose and contributes to the insulinotropic action of oxyntomodulin.     

The glucagon receptor is involved in mediating the body weight-lowering effects of oxyntomodulin

Oxyntomodulin (OXM) is a peptide secreted postprandially from the L-cells of the gut that has a weak affinity for both the glucagon-like peptide-1 receptor (GLP1R) and the glucagon receptor (GCGR). Peripheral administration of OXM in humans and rodents causes weight loss reducing food intake and increasing energy expenditure. It has been suggested that OXM modulates energy intake solely through GLP1R agonism. Because glucagon decreases food intake in rodents and humans, we examined whether activation of the GCGR is involved in the body weight-lowering effects of OXM. We identified an equipotent GLP1R-selective peptide agonist that differs from OXM by only one residue (Q3→E, OXMQ3E), but has no significant GCGR agonist activity in vitro and ~100-fold reduced ability to stimulate liver glycogenolysis. Chronic treatment of obese mice with OXM and OXMQ3E demonstrated that OXM exhibits superior weight loss and lipid-lowering efficacy, and antihyperglycemic activity that is comparable to the corresponding GLP1R-selective agonist. Studies in Glp1r(-/-) mice and coadministration of OXM and a GCGR antagonist revealed that the antiobesity effect of OXM requires activation of both GLP1R and GCGR. Our data provide new insight into the mechanism of action of OXM and suggest that activation of GCGR is involved in the body weight-lowering action of OXM.     

Metabolic responses to xenin-25 are altered in humans with Roux-en-Y gastric bypass surgery

Xenin-25 (Xen) is a neurotensin-related peptide secreted by a subset of enteroendocrine cells located in the proximal small intestine. Many effects of Xen are mediated by neurotensin receptor-1 on neurons. In healthy humans with normal glucose tolerance (NGT), Xen administration causes diarrhea and inhibits postprandial glucagon-like peptide-1 (GLP‐1) release but not insulin secretion. This study determines (i) if Xen has similar effects in humans with Roux-en-Y gastric bypass (RYGB) and (ii) whether neural pathways potentially mediate effects of Xen on glucose homeostasis.Eight females with RYGB and no history of type 2 diabetes received infusions with 0, 4 or 12 pmol Xen/kg/min with liquid meals on separate occasions. Plasma glucose and gastrointestinal hormone levels were measured and insulin secretion rates calculated. Pancreatic polypeptide and neuropeptide Y levels were surrogate markers for parasympathetic input to islets and sympathetic tone, respectively. Responses were compared to those in well-matched non-surgical participants with NGT from our earlier study.Xen similarly increased pancreatic polypeptide and neuropeptide Y responses in patients with and without RYGB. In contrast, the ability of Xen to inhibit GLP-1 release and cause diarrhea was severely blunted in patients with RYGB. With RYGB, Xen had no statistically significant effect on glucose, insulin secretory, GLP-1, glucose-dependent insulinotropic peptide, and glucagon responses. However, insulin and glucose-dependent insulinotropic peptide secretion preceded GLP-1 release suggesting circulating GLP-1 does not mediate exaggerated insulin release after RYGB. Thus, Xen has unmasked neural circuits to the distal gut that inhibit GLP-1 secretion, cause diarrhea, and are altered by RYGB.     

Add-on therapy with anagliptin in Japanese patients with type-2 diabetes mellitus treated with metformin and miglitol can maintain higher concentrations of biologically active GLP-1/total GIP and a lower concentration of leptin

Metformin, α-glucosidase inhibitors (α-GIs), and dipeptidyl peptidase 4 inhibitors (DPP-4Is) reduce hyperglycemia without excessive insulin secretion, and enhance postprandial plasma concentration of glucagon-like peptide-1 (GLP-1) in type-2 diabetes mellitus (T2DM) patients. We assessed add-on therapeutic effects of DPP-4I anagliptin in Japanese T2DM patients treated with metformin, an α-GI miglitol, or both drugs on postprandial responses of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), and on plasma concentration of the appetite-suppressing hormone leptin. Forty-two Japanese T2DM patients with inadequately controlled disease (HbA1c: 6.5%–8.0%) treated with metformin (n = 14), miglitol (n = 14) or a combination of the two drugs (n = 14) received additional treatment with anagliptin (100 mg, p.o., b.i.d.) for 52 weeks. We assessed glycemic control, postprandial responses of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), and on plasma concentration of leptin in those patients. Add-on therapy with anagliptin for 52 weeks improved glycemic control and increased the area under the curve of biologically active GLP-1 concentration without altering obesity indicators. Total GIP concentration at 52 weeks was reduced by add-on therapy in groups treated with miglitol compared with those treated with metformin. Add-on therapy reduced leptin concentrations. Add-on therapy with anagliptin in Japanese T2DM patients treated with metformin and miglitol for 52 weeks improved glycemic control and enhanced postprandial concentrations of active GLP-1/total GIP, and reduce the leptin concentration.     

DPP‐IV‐resistant,long‐acting oxyntomodulin derivatives

Obesity is one of the major risk factors for type 2 diabetes, and the development of agents, that can simultaneously achieve glucose control and weight loss, is being actively pursued. Therapies based on peptide mimetics of the gut hormone glucagon‐like peptide 1 (GLP‐1) are rapidly gaining favor, due to their ability to increase insulin secretion in a strictly glucose‐dependent manner, with little or no risk of hypoglycemia, and to their additional benefit of causing a modest, but durable weight loss. Oxyntomodulin (OXM), a 37‐amino acid peptide hormone of the glucagon (GCG) family with dual agonistic activity on both the GLP‐1 (GLP1R) and the GCG (GCGR) receptors, has been shown to reduce food intake and body weight in humans, with a lower incidence of treatment‐associated nausea than GLP‐1 mimetics. As for other peptide hormones, its clinical application is limited by the short circulatory half‐life, a major component of which is cleavage by the enzyme dipeptidyl peptidase IV (DPP‐IV). SAR studies on OXM, described herein, led to the identification of molecules resistant to DPP‐IV degradation, with increased potency as compared to the natural hormone. Analogs derivatized with a cholesterol moiety display increased duration of action in vivo. Moreover, we identified a single substitution which can change the OXM pharmacological profile from a dual GLP1R/GCGR agonist to a selective GLP1R agonist. The latter finding enabled studies, described in detail in a separate study (Pocai A, Carrington PE, Adams JR, Wright M, Eiermann G, Zhu L, Du X, Petrov A, Lassman ME, Jiang G, Liu F, Miller C, Tota LM, Zhou G, Zhang X, Sountis MM, Santoprete A, Capitò E, Chicchi GG, Thornberry N, Bianchi E, Pessi A, Marsh DJ, SinhaRoy R. Glucagon‐like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 2009; 58: 2258–2266), which highlight the potential of GLP1R/GCGR dual agonists as a potentially superior class of therapeutics over the pure GLP1R agonists currently in clinical use. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Tyr1 and Ile7 of glucose-dependent insulinotropic polypeptide (GIP) confer differential ligand selectivity toward GIP and glucagon-like peptide-1 receptors

Glucagon like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones released in response to food intake and potentiate insulin secretion from pancreatic β cells through their distinct yet related G protein-coupled receptors, GLP1R and GIPR. While GLP-1 and GIP exhibit similarity in their N-terminal sequence and overall α-helical structure, GLP-1 does not bind to GIPR and vice versa. To determine which amino acid residues of these peptide ligands are responsible for specific interaction with their respective receptors, we generated mutant GIP in which several GLP-1-specific amino acid residues were substituted for the original amino acids. The potency of the mutant ligands was examined using HEK293 cells transfected with GLP1R or GIPR expression plasmids together with a cAMP-responsive element-driven luciferase (CRE-luc) reporter plasmid. A mutated GIP peptide in which Tyr¹, Ile⁷, Asp¹⁵, and His¹⁸ were replaced by His, Thr, Glu, and Ala, respectively, was able to activate both GLP1R and GIPR with moderate potency. Replacing the original Tyr¹ and/or Ile⁷ in the N-terminal moiety of this mutant peptide allowed full activation of GIPR but not of GLP1R. However, reintroducing Asp¹⁵ and/or His¹⁸ in the central α-helical region did not significantly alter the ligand potency. These results suggest that Tyr/His¹ and Ile/Thr⁷ of GIP/GLP-1 peptides confer differential ligand selectivity toward GIPR and GLP1R.     

The protective and anti-inflammatory effects of glucagon-like peptide-2 in an experimental rat model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease, that affects premature infants. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone and reduces the inflammation. We suspected that GLP-2 would have protective and anti-inflammatory effects in an experimental rat model of NEC. NEC was induced in newborn rats by enteral feeding with hyperosmolar formula, asphyxial stress and enteral administration of lipopolysaccharide (LPS). Rats were randomly divided into the following four groups: dam-fed, NEC, NEC + GLP-2(L) given 80 μg/kg/day of GLP-2, and NEC + GLP-2(H) given 800 μg/kg/day of GLP-2. GLP-2 was administered subcutaneously every 6 h before stress. All animals surviving beyond 96 h or any that developed signs of distress were euthanized. The clinical sickness score in the NEC + GLP-2(H) group was significantly lower than that in the NEC group. The NEC score and the survival rate in the NEC + GLP-2(H) group was significantly improved compared with those in the NEC and the NEC + GLP-2(L) groups. Villous height and crypt depth in both the GLP-2 treatment groups were significantly increased compared with those in the NEC group. There were no significant differences in the crypt cell proliferation indices among the groups. Ileal interstitial TNF-α and IL-6 level in the NEC + GLP-2(H) group was decreased to the same levels in the dam-fed group. High dose GLP-2 administration improved the incidence and survival rate for NEC. It also decreased mucosal inflammatory cytokine production. These results support a potential therapeutic role for GLP-2 in the treatment of NEC.     

Regional specificity of the gut-incretin response to small intestinal glucose infusion in healthy older subjects

The importance of the region, as opposed to the length, of small intestine exposed to glucose in determining the secretion of the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) remains unclear. We sought to compare the glycemic, insulinemic and incretin responses to glucose administered to the proximal (12–60 cm beyond the pylorus), or more distal ( > 70 cm beyond the pylorus) small intestine, or both. 10 healthy subjects (9M,1F; aged 70.3 ± 1.4 years) underwent infusion of glucose via a catheter into the proximal (glucose proximally; GP), or distal (glucose distally; GD) small intestine, or both (GPD), on three separate days in a randomised fashion. Blood glucose, serum insulin and plasma GLP-1, GIP and CCK responses were assessed. The iAUC for blood glucose was greater in response to GPD than GP (P < 0.05), with no difference between GD and GP. GP was associated with minimal GLP-1 response (P = 0.05), but substantial increases in GIP, CCK and insulin (P < 0.001 for all). GPD and GD both stimulated GLP-1, GIP, CCK and insulin (P < 0.001 for all). Compared to GP, GPD induced greater GLP-1, GIP and CCK responses (P < 0.05 for all). Compared with GPD, GD was associated with greater GLP-1 (P < 0.05), but reduced GIP and CCK (P < 0.05 for both), responses. We conclude that exposure of glucose to the distal small intestine appears necessary for substantial GLP-1 secretion, while exposure of both the proximal and distal small intestine result in substantial secretion of GIP.     

Estradiol modulates the anorexic response to central glucagon-like peptide 1

Estrogens suppress feeding in part by enhancing the response to satiation signals. Glucagon-like peptide 1 (GLP-1) acts on receptor populations both peripherally and centrally to affect food intake. We hypothesized that modulation of the central GLP-1 system is one of the mechanisms underlying the effects of estrogens on feeding. We assessed the anorexic effect of 0, 1, and 10 μg doses of GLP-1 administered into the lateral ventricle of bilaterally ovariectomized (OVX) female rats on a cyclic regimen of either 2 μg β-estradiol-3-benzoate (EB) or oil vehicle 30 min prior to dark onset on the day following hormone treatment. Central GLP-1 treatment significantly suppressed food intake in EB-treated rats at both doses compared to vehicle, whereas only the 10 μg GLP-1 dose was effective in oil-treated rats. To follow up, we examined whether physiologic-dose cyclic estradiol treatment influences GLP-1-induced c-Fos in feeding-relevant brain areas of OVX females. GLP-1 significantly increased c-Fos expression in the area postrema (AP) and nucleus of the solitary tract (NTS), and the presence of estrogens may be required for this effect in the paraventricular nucleus of the hypothalamus (PVN). Together, these data suggest that modulation of the central GLP-1 system may be one of the mechanisms by which estrogens suppress food intake, and highlight the PVN as a region of interest for future investigation.     

Evolutionarily conserved residues at glucagon-like peptide-1 (GLP-1) receptor core confer ligand-induced receptor activation

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) play important roles in insulin secretion through their receptors, GLP1R and GIPR. Although GLP-1 and GIP are attractive candidates for treatment of type 2 diabetes and obesity, little is known regarding the molecular interaction of these peptides with the heptahelical core domain of their receptors. These core domains are important not only for specific ligand binding but also for ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R/GIPR, we determined that evolutionarily conserved amino acid residues such as Ile(196) at transmembrane helix 2, Leu(232) and Met(233) at extracellular loop 1, and Asn(302) at extracellular loop 2 of GLP1R are responsible for interaction with ligand and receptor activation. Application of chimeric GLP-1/GIP peptides together with molecular modeling suggests that His(1) of GLP-1 interacts with Asn(302) of GLP1R and that Thr(7) of GLP-1 has close contact with a binding pocket formed by Ile(196), Leu(232), and Met(233) of GLP1R. This study may provide critical clues for the development of peptide and/or nonpeptide agonists acting at GLP1R.     

Adding Rapid-Acting Insulin or Glp-1 Receptor Agonist to Basal Insulin: Outcomes in A Community Setting

ObjectiveTo evaluate real-world outcomes in patients with type 2 diabetes mellitus (T2DM) receiving basal insulin who initiate add-on therapy with a rapid-acting insulin (RAI) or a glucagon-like peptide 1 (GLP-1) receptor agonist.MethodsData were extracted retrospectively from a U.S. health claims database. Adults with T2DM on basal insulin who added an RAI (basal + RAI) or GLP-1 receptor agonist (basal + GLP-1) were included. Propensity score matching (with a 1 up to 3 ratio) was used to control for differences in baseline demographics, clinical characteristics, and health resource utilization. Endpoints included prevalence of hypoglycemia, pancreatic events, all-cause and diabetes-related resource utilization, and costs at 1-year follow-up.ResultsOverall, 6,718 matched patients were included: 5,013 basal + RAI and 1,705 basal + GLP1. Patients in both groups experienced a similar proportion of any hypoglycemic event (P = .4079). Hypoglycemic events leading to hospitalization were higher in the basal + RAI cohort (2.7% vs. 1.8%; P = .0444). The basal + GLP-1 cohort experienced fewer all-cause (13.55% vs. 18.61%; P < .0001) and diabetes-related hospitalizations (11.79% vs. 15.68%; P < .0001). The basal + GLP-1 cohort had lower total all-cause health care costs ($18,413 vs. $20,821; P = .0002) but similar diabetes-related costs ($9,134 vs. $8,985; P < .0001) compared with the basal + RAI cohort.ConclusionsAdd-on therapy with a GLP-1 receptor agonist in T2DM patients receiving basal insulin was associated with fewer hospitalizations and lower total all-cause costs compared with add-on therapy using an RAI and could be considered as an alternative to an RAI in certain patients with T2DM who do not achieve effective glycemic control with basal insulin. (Endocr Pract. 2015; 21:68-76)     

The effect of encapsulated glutamine on gut peptide secretion in human volunteers

ContextWeight loss and improved blood glucose control after bariatric surgery have been attributed in part to increased ileal nutrient delivery with enhanced release of glucagon-like peptide 1 (GLP-1). Non-surgical strategies to manage obesity are required. The aim of the current study was to assess whether encapsulated glutamine, targeted to the ileum, could increase GLP-1 secretion, improve glucose tolerance or reduce meal size.MethodsA single-center, randomised, double blind, placebo-controlled, cross-over study was performed in 24 healthy volunteers and 8 patients with type 2 diabetes. Fasting participants received a single dose of encapsulated ileal-release glutamine (3.6 or 6.0 g) or placebo per visit with blood sampling at baseline and for 4 h thereafter. Glucose tolerance and meal size were studied using a 75 g oral glucose tolerance test and ad libitum meal respectively.ResultsIn healthy volunteers, ingestion of 6.0 g glutamine was associated with increased GLP-1 concentrations after 90 min compared with placebo (mean 10.6 pg/ml vs 6.9 pg/ml, p = 0.004), increased insulin concentrations after 90 min (mean 70.9 vs 48.5, p = 0.048), and increased meal size at 120 min (mean 542 g eaten vs 481 g, p = 0.008). Ingestion of 6.0 g glutamine was not associated with significant differences in GLP-1, glucose or insulin concentrations after a glucose tolerance test in healthy or type 2 diabetic participants.ConclusionsSingle oral dosing of encapsulated glutamine did not provoke consistent increases in GLP-1 and insulin secretion and was not associated with beneficial metabolic effects in healthy volunteers or patients with type 2 diabetes.     

DA-1229, a novel and potent DPP4 inhibitor, improves insulin resistance and delays the onset of diabetes

AimTo characterize the pharmacodynamic profile of DA-1229, a novel dipeptidyl peptidase (DPP) 4 inhibitor.Main methodsEnzyme inhibition assays against DPP4, DPP8 and DPP9. Antidiabetic effects of DA-1229 in HF-DIO mice and young db/db mice.Key findingsDA-1229 was shown to potently inhibit the DPP4 enzyme in human and murine soluble forms and the human membrane-bound form with IC₅₀ values of 0.98, 3.59 and 1.26 nM, respectively. As a reversible and competitive inhibitor, DA-1229 was more selective to human DPP4 (6000-fold) than to human DPP8 and DPP9. DA-1229 (0.1–3 mg/kg) dose-dependently inhibited plasma DPP4 activity, leading to increased levels of plasma GLP-1 and insulin, and thereby lowering blood glucose levels in mice. In high fat diet-fed (HF) mice, a single oral dose of 100 mg/kg of DA-1229 reduced plasma DPP4 activity by over 80% during a 24 h period. Long-term treatment with DA-1229 for 8 weeks revealed significant improvements in glucose intolerance and insulin resistance, accompanied by significant body weight reduction. However, it remains unclear whether there is a direct causal relationship between DPP4 inhibition and body weight reduction. In young db/db mice, the DA-1229 treatment significantly reduced blood glucose excursions for the first 2 weeks, resulting in significantly lower levels of HbA1c at the end of the study. Furthermore, the pancreatic insulin content of the treatment group was significantly higher than that of the db/db control.SignificanceDA-1229 as a novel and selective DPP4 inhibitor improves the insulin sensitivity in HF mice and delays the onset of diabetes in young db/db mice.     

Comparative effects of intraduodenal fat and glucose on the gut-incretin axis in healthy males

BackgroundThe interaction of nutrients with the small intestine stimulates the secretion of numerous enteroendocrine hormones that regulate postprandial metabolism. However, differences in gastrointestinal hormonal responses between the macronutrients are incompletely understood. In the present study, we compared blood glucose and plasma hormone concentrations in response to standardised intraduodenal (ID) fat and glucose infusions in healthy humans.MethodsIn a parallel study design, 16 healthy males who received an intraduodenal fat infusion were compared with 12 healthy males who received intraduodenal glucose, both at a rate of 2 kcal/min over 120 min. Venous blood was sampled at frequent intervals for measurements of blood glucose, and plasma total and active glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), insulin and glucagon.ResultsPlasma concentrations of the incretin hormones (both total and active GLP-1 and GIP) and glucagon were higher, and plasma insulin and blood glucose concentrations lower, during intraduodenal fat, when compared with intraduodenal glucose, infusion (treatment by time interaction: P < 0.001 for each).ConclusionsCompared with glucose, intraduodenal fat elicits substantially greater GLP-1, GIP and glucagon secretion, with minimal effects on blood glucose or plasma insulin in healthy humans. These observations are consistent with the concept that fat is a more potent stimulus of the ‘gut-incretin’ axis than carbohydrate.     

Early liraglutide treatment is better in glucose control, β-cell function improvement and mass preservation in db/db mice

Glucagon-like peptide-1 (GLP-1) has been proved to have effects of anti-hyperglycemia and β-cell preservation. However, it is still unclear whether there are differences between early and late GLP-1 intervention in type 2 diabetes mellitus (T2DM). We divided the mice into 5 groups: early treated group (n = 7, 8-week old, fasting glucose > 10 mmol/l), late treated group (n = 7, 10-week old, fasting glucose > 20 mmol/l), early control group (n = 7), late control group (n = 7) and wild type group (n = 7). Treated group was injected with liraglutide (a GLP-1 analog) 300 μg/kg bid for 4 weeks, while control group was given saline at the same time. The results showed that compared with control group, food intake and body weight gain were reduced in both early and late treated group (p < 0.05), and there was no significance between the two treated groups. Early liraglutide intervention showed better improvements in glucose control, acute insulin response to glucose (AIRg) and disposition index (before vs. after treatment, AIRg 1.01 ± 0.53 vs. 2.98 ± 0.63, disposition index 10.81 ± 0.89 vs. 27.4 ± 2.15) than late intervention (AIRg 0.99 ± 0.02 vs. 1.41 ± 0.32, disposition index 3.47 ± 0.38 vs. 6.43 ± 1.62, p = 0.001). The histopathology of the pancreas showed the estimated β-cell mass (BCM) was increased more in early treated group than that in late one (0.03 vs. 0.01 g). Expressions of the proliferation related genes PDX-1, MafA and GLP-1 receptor (GLP-1R) in early treated group were 1.81, 2.57 and 1.59 times as much as that in late treated group. In conclusion, early liraglutide intervention was better in glucose control, β-cell function improvement and β-cell mass preservation.     

Metabolic effect of short term administration of Hoodia gordonii,an herbal appetite suppressant

Hoodia gordonii (family: Apocynaceae) is used traditionally by the Khoi-San tribes to control hunger. It has become extremely popular and has triggered commercial interest due to its appetite suppressant property. The present study was undertaken to investigate the appetite regulatory mechanism and associated metabolic changes induced by the herb. Effect of organic solvent extract of H. gordonii on food intake and body weight of male Sprague Dawley rats was monitored at three different doses 50, 100 and 150 mg/kg body weight, given orally for five days. Subsequently, the dose of 100 mg/kg body weight was selected for further studies on the regulatory hormones and biochemical variables. Dose-dependent reduction in food intake (12–26%) was observed at a dose of 100 and 150 mg/kg body weight (p < 0.05). Appetite suppression persisted for 6 h and food intake was restored within 24 h after stopping of the treatment. There was an increase in liver glycogen stores, activity of mitochondrial CPT-1 and thyroid hormones in treated animals. The circulating levels of NPY and IGF-1 were decreased with marginal increase in leptin and CCK, in case of treated rats. There was no change in blood glucose and insulin levels were not affected significantly. The hormonal and metabolic changes due to treatment with the H. gordonii extract may be responsible for its anorectic activity.     

Insulin-releasing and cytotoxic properties of the frog skin peptide,tigerinin-1R: a structure–activity study

The frog skin host-defense peptide tigerinin-1R (RVCSAIPLPICH.NH₂) is insulinotropic both in vitro and in vivo. This study investigates the effects on insulin release and cytotoxicity of changes in cationicity and hydrophobicity produced by selected substitutions of amino acids by l-arginine, l-lysine and l-tryptophan. The [A5W], [L8W] and [I10W] analogs produced a significant (P < 0.01) increase in the rate of insulin release from BRIN-BD11 rat clonal β cells at concentration of 0.01 nM compared with 0.1 nM for tigerinin-1R. The increase in the rate of insulin release produced by a 3 μM concentration of the [S4R], [H12K], and [I10W] analogs from both BRIN-BD11 cells and mouse islets was significantly greater (P < 0.05) than that produced by tigerinin-1R. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM indicating that plasma membrane integrity had been preserved. [A5W] tigerinin-1R was the only analog tested that showed cytotoxic activity against human erythrocytes (LC₅₀ = 265 ± 16 μM) and inhibited growth of Escherichia coli (MIC = 500 μM) and Staphylococcus aureus (MIC = 250 μM). The circular dichroism spectra of tigerinin-1R and [A5W] tigerinin-1R indicate that the peptides adopt a mixture of β-sheet, random coil and reverse β-turn conformations in 50% trifluoroethanol/water and methanol/water. Administration of [S4R] tigerinin-1R (75 nmol/kg body weight) to high-fat fed mice with insulin resistance significantly (P < 0.05) enhanced insulin release and improved glucose tolerance over a 60 min period following an intraperitoneal glucose load. The study supports the claim that tigerinin-1R shows potential for development into novel therapeutic agents for treatment of type 2 diabetes mellitus.     

Anticonvulsant properties of histamine H3 receptor ligands belonging to N-substituted carbamates of imidazopropanol

Ligands targeting central histamine H₃ receptors (H₃Rs) for epilepsy might be a promising therapeutic approach. Therefore, the previously described and structurally strongly related imidazole-based derivatives belonging to carbamate class with high H₃R in vitro affinity, in-vivo antagonist potency, and H₃R selectivity profile were investigated on their anticonvulsant activity in maximal electroshock (MES)-induced and pentylenetetrazole (PTZ)-kindled seizure models in Wistar rats. The effects of systemic injection of H₃R ligands 1–13 on MES-induced and PTZ-kindled seizures were screened and evaluated against the reference antiepileptic drug (AED) Phenytoin (PHT) and the standard histamine H₃R inverse agonist/antagonist Thioperamide (THP) to determine their potential as new antiepileptic drugs. Following administration of the H₃R ligands 1–13 (5, 10 and 15 mg/kg, ip) there was a significant dose dependent reduction in MES-induced seizure duration. The protective action observed for the pentenyl carbamate derivative 4, the most protective H₃R ligand among 1–13, was significantly higher (P <0.05) than that of standard H₃R antagonist THP, and was reversed when rats were pretreated with the selective H₃R agonist R-(α)-methyl-histamine (RAMH) (10 mg/kg), or with the CNS penetrant H₁R antagonist Pyrilamine (PYR) (10 mg/kg). In addition, subeffective dose of H₃R ligand 4 (5 mg/kg, ip) significantly potentiated the protective action in rats pretreated with PHT (5 mg/kg, ip), a dose without appreciable protective effect when given alone. In contrast, pretreatment with H₃R ligand 4 (10 mg/kg ip) failed to modify PTZ-kindled convulsion, whereas the reference drug PHT was found to fully protect PTZ-induced seizure. These results indicate that some of the investigated imidazole-based H₃R ligands 1–13 may be of future therapeutic value in epilepsy.     

Carboxymethylation of a polysaccharide extracted from Ganoderma lucidum enhances its antioxidant activities in vitro

The chemical carboxylmethylated polysaccharide (C-GLP), which derived from water-insoluble crude Ganoderma lucidum polysaccharide (GLP), was prepared. Water solubility, chemical characterization, and antioxidant activities in vitro of C-GLP were determined. The solubility of C-GLP in distilled water reached 100 mg/ml, which was much higher than the solubility of GLP. Chemical analysis indicated that C-GLP was composed of Glc:Man:Gal = 33.0:1.0:3.4 with a molecular weight of 1.8 × 10⁶ Da and a carboxymethyl content of 11.07%. The signals of carboxymethyl were found in IR and ¹³C NMR spectra. Moreover, a high antioxidant activity of C-GLP was observed, especially in scavenging of hydroxyl radical (83.7% at 5 mg/ml) and hydrogen peroxide (51.6% at 10 mg/ml). This study indicates the effects of carboxymethylation on water-insoluble polysaccharide and explores a potential antioxidant in food industry and pharmaceuticals.     

Glucagon-like peptide-1 regulates calcium homeostasis and electrophysiological activities of HL-1 cardiomyocytes

Glucagon like-peptide-1 (GLP-1) is an incretin hormone with antidiabetic effects through stimulating insulin secretion, β cell neogenesis, satiety sensation, and inhibiting glucagon secretion. Administration of GLP-1 provides cardioprotective effects through attenuating cardiac inflammation and insulin resistance. GLP-1 also modulates the heart rate and systolic pressure, which suggests that GLP-1 may have cardiac electrical effects. Therefore, the purposes of this study were to evaluate whether GLP-1 has direct cardiac effects and identify the underlying mechanisms. Patch clamp, confocal microscopy with Fluo-3 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, calcium homeostasis, and calcium regulatory proteins in HL-1 atrial myocytes with and without GLP-1 (1 and 10 nM) incubation for 24 h. GLP-1 (1 and 10 nM) and control cells had similar action potential durations. However, GLP-1 at 10 nM significantly increased calcium transients and sarcoplasmic reticular Ca²⁺ contents. Compared to the control, GLP-1 (10 nM)—treated cells significantly decreased phosphorylation of the ryanodine receptor at S2814 and total phospholamban, but there were similar protein levels of sarcoplasmic reticular Ca²⁺-ATPase and the sodium–calcium exchanger. Moreover, exendin (9–39) amide (a GLP-1 receptor antagonist, 10 nM) attenuated GLP-1-mediated effects on total SR content and phosphorylated ryanodine receptor S2814. This study demonstrates GLP-1 may regulate HL-1 cell arrhythmogenesis through modulating calcium handling proteins.