Novel fatty acid chain modified GLP-1 derivatives with prolonged in vivo glucose-lowering ability and balanced glucoregulatory activity

Glucagon-like peptide-1 is a potent hypoglycemic hormone with beneficial properties for the treatment of diabetes. However, its half-life is short because the rapid metabolic degradation. This study aims to prolong the half-life of glucagon-like peptide-1 through conjugation with the fatty acid side chain which helps the conjugates to interact with the albumin. Firstly, we chose two optimized polypeptide chains which have tremendous hypoglycemic effect named Cys₁₇-Gly₈-GLP-1(7-36)-NH₂ and Cys₃₇-Gly₈-GLP-1(7-37)-NH₂, and various fatty acid chains were modified. All conjugates preserved relatively strong GLP-1R activation and I-6 behaved best in glucose-lowering ability. The prolonged antidiabetic effects of I-6 were further confirmed by hypoglycemic efficacy test in vivo. Meanwhile, once daily injection of I-6 to diabetic mice achieved long-term beneficial effects on glucose tolerance, body weight and blood chemistry. It is concluded that I-6 is a promising agent for further investigation of its potential to treat obese patients with diabetes.     

Evaluation of Insulin Glargine and Exenatide Alone and in Combination: a Randomized Clinical Trial with Continuous Glucose Monitoring and Ambulatory Glucose Profile Analysis

Objective: Characterize the effectiveness of insulin glargine alone, exenatide alone, or combined in subjects taking stable doses of metformin and evaluate their impact on hemoglobin A1C, hypoglycemia, weight, and glucose variability.Methods: Open-label, randomized, parallel-arm study of adults with type 2 diabetes naïve to both insulin and glucagon-like peptide 1 (GLP-1) agonist who were not at A1C goal despite treatment with metformin. This prospective interventional study employed blinded continuous glucose monitoring ambulatory glucose profile (AGP) reports over 32 weeks. Subjects were randomized to treatment with glargine (Iglar), exenatide (GLP-1), or combination of glargine and exenatide (Iglar + GLP-1). At midpoint, those not at A1C target had the second medication added; those on Iglar + GLP-1 continued therapy optimization.Results: Decreases in A1C were: 7.6 to 6.2% for Iglar + GLP-1, 7.5 to 6.6% for Iglar, and 7.5 to 6.4% for GLP-1. Iglar + GLP-1 achieved A1C targets faster (14 to 16 weeks) but had more hypoglycemia. Hypoglycemia rates increased slightly for all arms. Weight loss was achieved in all regimens including GLP-1. Glucose variability was not reduced to the same extent in the Iglar arm as the GLP-1 arm.Conclusion: Addition of Iglar and/or GLP-1 to metformin for patients not at treatment goal was safe and effective. The order of medication addition needs to consider individualized AGP patterns and goals. Iglar + GLP-1 resulted in rapid A1C lowering, whereas GLP-1 was noted to have less hypoglycemia. Weight loss was most pronounced in GLP-1 monotherapy, suggesting that GLP-1 may mitigate the weight gain of Iglar. Any treatment with GLP-1 showed significant decreases in glucose variability.Abbreviations: A1C = hemoglobin A1c; AGP = ambulatory glucose profile; CGM = continuous glucose monitoring; GLM = general linear model; GLP-1 = glucagon-like peptide 1 (exenatide); Iglar = insulin glargine; SGLT2 = sodium-glucose cotransporter 2; SMBG = self-monitoring blood glucose; SU = sulfonylurea; T2D = type 2 diabetes mellitus     

The 5-S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. Evolution of the eukaryotic 5-S RNA binding protein.

The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (Mr = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4--9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL18 from Escherichia coli and HL13 from Halobacterium cutirubrum: Ala1-Phe2-Gln3-Lys4-Asp5-Ala6-Lys7-Ser8-Ser9-Ala10-Tyr11-Ser12-Ser13-Arg14-Phe15-Gln16-Tyr17-Pro18-Phe19-Arg20-Arg21-Arg22-Arg23-Glu24-Gly25-Lys26-Thr27-Asp28-Tyr29-Tyr35; of particular interest was homology in the cluster of basic residues (18--23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (Mr = 24700) and CN2 (Mr = 11300) by CNBr treatment; the larger fragment originated from the N terminus. The N-terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5-S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5-S RNAs, a model for the evolution of the eukaryotic 5-S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5-S RNA . protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5-S RNA-protein interaction.     

A 24-Week,Prospective, Randomized,Open-Label,Treat-To-Target Pilot Study of Obese Type 2 Diabetes Patients with Severe Insulin Resistance to Assess the Addition of Exenatide on the Efficacy of U-500 Regular Insulin Plus Metformin

ObjectiveTo compare the efficacy of 500 U/mL (U-500) regular insulin + metformin with U-500 regular insulin + metformin + exenatide in improving glycemic control in patients with severely insulin-resistant type 2 diabetes mellitus (T2DM).MethodsThirty patients with T2DM and severe insulin resistance were screened, and 28 were randomized to regular insulin U-500 + metformin or the GLP-1 analog exenatide, U-500, and metformin. Glycated hemoglobin (HbA_1c) levels, body weight, and insulin doses were documented at baseline and at 3 and 6 months. The number and severity hypoglycemic episodes were noted.ResultsThere were 7 males and 7 females in each group (U-500 + metformin and U-500 + metformin + exenatide). Overall, U-500 insulin + metformin, either alone or with the addition of exenatide, resulted in a significant improvement in HbA_1c in both groups, with no significant difference between the 2 groups. There was no meaningful weight change in those utilizing exenatide. Those on U-500 insulin and metformin alone had a tendency toward some weight gain. No severe hypoglycemia occurred during the study period. Symptomatic hypoglycemia was more common in the group on exenatide, but this occurred in only 5 patients, and the clinical significance of this is uncertain. Insulin dosage changes on U-500 regular insulin were variable but tended to be lower in those subjects on exenatide.ConclusionsU-500 regular insulin + metformin is effective for the treatment of T2DM patients with severe insulin resistance. The addition of exenatide may ameliorate potential weight gain but provides no additional improvement in glycemia. (Endocr Pract. 2014;20:1143-1150)     

Use of a combination of the RDC method and NOESY NMR spectroscopy to determine the structure of Alzheimer’s amyloid Aβ10–35 peptide in solution and in SDS micelles

The spatial structure of Alzheimer’s amyloid Aβ_10–35-NH₂ peptide in aqueous solution at pH 7.3 and in SDS micelles was investigated by use of a combination of the residual dipolar coupling method and two-dimensional NMR spectroscopy (TOCSY, NOESY). At pH 7.3 Aβ_10–35-NH₂ adopts a compact random-coil conformation whereas in SDS micellar solutions two helical regions (residues 13–23 and 30–35) of Aβ_10–35-NH₂ were observed. By use of experimental data, the structure of “peptide–micelle” complex was determined; it was found that Aβ_10–35-NH₂ peptide binds to the micelle surface at two regions (residues 17–20 and 29–35).     

Endothelial dysfunction induced by triglycerides is not restored by exenatide in rat conduit arteries ex vivo

Exenatide (synthetic exendin-4) is a stable analogue of glucagon-like peptide 1 (GLP-1) and has recently been approved for clinical use against type 2 diabetes. Exenatide is believed to exert its effects via the GLP-1 receptor with almost the same potency as GLP-1 in terms of lowering blood glucose. Short term exenatide treatment normalizes the altered vascular tone in type 2 diabetic rats, probably due to the reduction in glycemia. The aim of this study was to investigate whether exenatide directly protects against triglyceride-induced endothelial dysfunction in rat femoral arterial rings ex vivo. Short term pre-incubation with Intralipid^® (0.5 and 2%) was found to dose-dependently induce endothelial dysfunction, in that it elicited a significant reduction in ACh-induced vasorelaxation by 29% and 35%, respectively. Paradoxically, this occurred with a concomitant increase in endothelial nitric oxide synthase (eNOS) activity. No such reduction in vasorelaxation by Intralipid^® was seen in response to the NO donor sodium nitroprusside (SNP), revealing an endothelium-dependent vascular dysfunction by Intralipid^®. However, exenatide did not protect against Intralipid^®-induced endothelial dysfunction. More surprisingly, the maximum vasorelaxation induced by exenatide (without Intralipid^®) was only 3 ± 2%, compared to the 23 ± 4%, 38 ± 4%, 79 ± 3% and 97 ± 4% relaxations induced by GLP-1, GLP-1 (9-36), ACh and SNP, respectively. This unexpected finding prompted us to ascertain that the exenatide preparation was biologically active, and both exenatide (10^− 11 mol/l) and GLP-1 (10^− 9 mol/l) significantly increased insulin secretion in pancreatic β-cells from ob/ob mice in vitro. In conclusion, exenatide could neither confer any acute protective effects against triglyceride-induced endothelial dysfunction nor exert any significant vasorelaxant actions in this model of rat conduit arteries ex vivo.     

Greater Combined Reductions in Hba1C ≥1.0% and Weight ≥5.0% with Semaglutide Versus Comparators in type 2 Diabetes

Objective: Semaglutide is a glucagon-like peptide 1 (GLP-1) analog for the once-weekly treatment of type 2 diabetes (T2D). In the global SUSTAIN clinical trial program, semaglutide demonstrated superior glycated hemoglobin (HbA_1c) and body weight reductions versus comparators. This post hoc analysis compared the proportion of patients achieving combined reductions in glycemia and body weight versus comparators.Methods: A total of 5,119 subjects with T2D in the phase 3 SUSTAIN 1 through 5 and 7 trials, from 33 countries, were included in this post hoc analysis. Subjects received subcutaneous semaglutide 0.5 or 1.0 mg, placebo or active comparator (sitagliptin 100 mg, exenatide extended release 2.0 mg, insulin glargine, dulaglutide 0.75 or 1.5 mg). The main endpoint was a composite of ≥1.0% HbA_1c reduction and ≥5.0% weight loss at end of treatment.Results: Significantly greater proportions of subjects achieved the composite endpoint with semaglutide 0.5 (25 to 38%) and 1.0 mg (38 to 59%) versus comparators (2 to 23%). More subjects treated with semaglutide versus comparators achieved ≥1.0% HbA_1c reductions (58 to 77% and 75 to 83% for semaglutide 0.5 and 1.0 mg versus 12 to 68%) and ≥5.0% weight loss (37 to 46%, 45 to 66% versus 4 to 30%). Proportions of subjects achieving targets were significantly higher with semaglutide 1.0 versus 0.5 mg in four of five trials. Semaglutide was well tolerated, with a safety profile similar to other GLP-1 receptor agonists.Conclusion: Significantly more subjects achieved both ≥1.0% HbA_1c reduction and ≥5.0% weight loss with once-weekly subcutaneous semaglutide treatment versus comparators in the SUSTAIN trials. A dose-dependent effect was observed with semaglutide.Abbreviations: AE = adverse event; CV = cardiovascular; ER = extended release; GLP-1 = glucagon-like peptide 1; GLP-1 RA = glucagon-like peptide 1 receptor agonist; HbA_1c = glycated hemoglobin; OAD = oral antidiabetic drug; sc = subcutaneous; T2D = type 2 diabetes     

Effects of exenatide on glycemia and renal water and ion excretion differ in frogs and rats

The aim of this study was to compare the effects of exenatide, a glucagon-like peptide-1 (GLP-1) mimetic, on glucose and water–salt homeostasis in animals with different levels of renal proximal tubular reabsorption, rats (Rattus norvegicus) and frogs (Rana temporaria). Following the glucose tolerance test, in rats exenatide promoted fast recovery of normoglycemia, whereas in frogs it delayed this process. In water-loaded rats, exenatide augmented solute-free water clearance and enhanced natriuresis in furosemide-treated animals. In frogs, exenatide did not alter the urinary flow rate, urinary sodium excretion and solute-free water clearance under water diuresis and furosemide treatment. It is suggested that the involvement of GLP-1 in regulation of water–salt homeostasis in mammals was preceded by a key evolutionary transformation, the increase in the glomerular filtration rate and proximal tubular reabsorption.     

Clinical Outcomes Using Long-Term Combination Therapy with Insulin Glargine and Exenatide in Patients with Type 2 Diabetes Mellitus

ObjectiveTo examine the long-term effects of combination insulin glargine/exenatide treatment on glycemic control.MethodsWe conducted a 24-month retrospective US chart review of patients with inadequately controlled type 2 diabetes (T2DM) and hemoglobin A_1c (A1C) levels > 7.0% for whom glargine and exenatide were coprescribed in differing order (glargine added after exenatide [exenatide/glargine]; exenatide added after glargine [glargine/exenatide]). Treatment order groups were combined to form a pooled treatment group. Changes from baseline in A1C, patients with A1C ≤ 7.0%, body weight, glargine/exenatide daily dose, oral antidiabetic drug (OAD) use, and hypoglycemia were evaluated.ResultsTreatment groups were similar at baseline; however, patients in the glargine/exenatide group (n = 121) (vs exenatide/glargine group [n = 44]) had longer disease duration (11.8 vs 8.0 years) and took fewer OADs (1.7 vs 2.3). Overall, baseline A1C was 8.8 ± 1.3% and weight was 109.5 ± 25.3 kg. Significant A1C reductions emerged at month 6 and persisted throughout 24 months (vs baseline) in both treatment groups (pooled: –0.7 ± 1.6; P < .001), and 33.0% of patients achieved an A1C level ≤ 7.0%. After 24 months of exenatide/glargine, body weight remained unchanged (0.7 ± 8.3 kg; P = .640). With glargine/exenatide, body weight decreased (–2.5 ± 6.7 kg; P = .001). At month 24, daily glargine dose was 0.40 ± 0.23 units/kg for the exenatide/glargine group and 0.47 ± 0.30 units/kg for the glargine/exenatide group. Hypoglycemia frequency was similar in both treatment groups.ConclusionsRegardless of treatment order, long-term combined therapy with glargine and exenatide for up to 24 months in patients with inadequately controlled T2DM suggests reduction of A1C without significant weight gain or increased hypoglycemia risk. (Endocr Pract. 2012;18:17-25)     

A Proteinase from Germinated Barley : II. Hydrolytic Specificity of a 30 Kilodalton Cysteine Proteinase From Green Malt

The hydrolytic specificity of a 30 kilodalton cysteine proteinase purified from germinated barley (Hordeum vulgare L. cv Morex) was investigated using high performance liquid chromatography to characterize its hydrolysis of two small barley seed proteins, the α- and β-hordothionins. The reduced and pyridylethylated thionins were rapidly cleaved, resulting in the production of a limited number of peptides. Peptide bonds Gly9-Arg10, Cys 16-Arg17, Cys25-Ala26, and Thr34-Ser35 were most susceptible to hydrolysis, the peptide bonds Arg5-Ser6, Arg19-Gly20 in both thionins and Lys38-Cys39 in β-hordothionin and Cys29-Arg30 of α-hordothionin being broken at much slower rates. The hydrolysis patterns were highly reproducible from assay to assay and with various enzyme preparations. The specificity was apparently defined by the amino acids in the P₂ position, not those immediately adjacent to the susceptible bonds. The P₂ amino acid residues of the released peptides were always either leucine, valine, tyrosine, or pyridylethylcysteine. From these observations and from the rates of release of the various peptides, it appears that the barley 30 kilodalton endoproteinase has an S2 subsite that preferentially binds the leucine side chain: i.e. for hydrolyzing the peptide bond P₁-P₁′ in the general sequence NH₂—P₂-P₁-P₁′—COOH, the enzyme is selective for leucine and, to a lesser extent, valine and tyrosine at position P₂. The barley proteinase thus resembles two other cysteine proteinases, papain and Streptococcal proteinase, in its specificity.     

The determination of low-molecular-mass thiols with 4-(hydroxymercuric)benzoic acid as a tag using HPLC coupled online with UV/HCOOH-induced cold vapor generation AFS

An alternative analytical method was established for simultaneous determination of main urinary low-molecular-mass (LMM) thiols including cysteine (Cys), cysteinylglycine (Cys–Gly), homocysteine (HCys), γ-glutamyl cysteine (γ-Glu–Cys) and glutathione (GSH) as well as N-acetylcysteine (NAC) using RPLC coupled on line with UV/HCOOH-induced cold vapor generation atomic fluorescence spectrometry (UV/HCOOH–CVG–AFS) with 4-(hydroxymercuric)benzoic acid (PHMB) as a tag. The LMM thiols were stabilized and labeled by PHMB allowing the determination of reduced form thiols (R-thiols) and total thiols (T-thiols) without and with Tris-(2-carboxyethyl)-phosphine reduction. UV/HCOOH-induced Hg cold vapor generation was used instead of K₂SO₈–KBH₄/NaOH–HCl and/or KBrO₃/KBr–KBH₄/NaOH–HCl systems as an effective interface between RPLC and CVG–AFS. The limits of detection (3σ) of RPLC–(UV/HCOOH)–CVG–AFS with PHMB labeling for Cys, HCys, Cys–Gly, γ-Glu–Cys and GSH as well as NAC were 4.6, 5.9, 5.9, 8.1, 7.3 and 5.9 nM with the RSD of 4.4, 5.1, 3.6, 7.5 4.2 and 3.7% (n = 6 at 2 μM), respectively, satisfying the simultaneous determination of the main urinary LMM thiols. This developed method was applied successfully to determine the LMM R-thiols and T-thiols in 10 urine samples contributed by 10 healthy volunteers.     

Exenatide can reduce glucose independent of islet hormones or gastric emptying

Exenatide is a long-acting glucagon-like peptide-1 (GLP-1) mimetic used in the treatment of type 2 diabetes. There is increasing evidence that GLP-1 can influence glycemia not only via pancreatic (insulinotropic and glucagon suppression) and gastric-emptying effects, but also via an independent mechanism mediated by portal vein receptors. The aim of our study was to investigate whether exenatide has an islet- and gastric-independent glycemia-reducing effect, similar to GLP-1. First, we administered mixed meals, with or without exenatide (20 microg sc) to dogs. Second, to determine whether exenatide-induced reduction in glycemia is independent of slower gastric emptying, in the same animals we infused glucose intraportally (to simulate meal test glucose appearance) with exenatide, exenatide + the intraportal GLP-1 receptor antagonist exendin-(9-39), or saline. Exenatide markedly decreased postprandial glucose: net 0- to 135-min area under the curve = +526 +/- 315 and -536 +/- 197 mg.dl(-1).min(-1) with saline and exenatide, respectively (P < 0.05). Importantly, the decrease in plasma glucose occurred without a corresponding increase in postprandial insulin but was accompanied by delayed gastric emptying and lower glucagon. Significantly lower glycemia was induced by intraportal glucose infusion with exenatide than with saline (92 +/- 1 vs. 97 +/- 1 mg/dl, P < 0.001) in the absence of hyperinsulinemia or glucagon suppression. The exenatide-induced lower glycemia was partly reversed by intraportal exendin-(9-39): 95 +/- 3 and 92 +/- 3 mg/dl with exenatide + antagonist and exenatide, respectively (P < 0.01). Our results suggest that, similar to GLP-1, exenatide lowers glycemia via a novel mechanism independent of islet hormones and slowing of gastric emptying. We hypothesize that receptors in the portal vein, via a neural mechanism, increase glucose clearance independent of islet hormones.     

Biological activity of AC3174, a peptide analog of exendin-4

Exenatide, the active ingredient of BYETTA (exenatide injection), is an incretin mimetic that has been developed for the treatment of patients with type 2 diabetes. Exenatide binds to and activates the known GLP-1 receptor with a potency comparable to that of the mammalian incretin GLP-1(7-36), thereby acting as a glucoregulatory agent. AC3174 is an analog of exenatide with leucine substituted for methionine at position 14, [Leu(14)]exendin-4. The purpose of these studies was to evaluate the glucoregulatory activity and pharmacokinetics of AC3174. In RINm5f cell membranes, the potency of AC3174 for the displacement of [(125)I]GLP-1 and activation of adenylate cyclase was similar to that of exenatide and GLP-1. In vivo, AC3174, administered as a single IP injection, significantly decreased plasma glucose concentration and glucose excursion following the administration of an oral glucose challenge in both non-diabetic (C57BL/6) and diabetic db/db mice (P<0.05 vs. vehicle-treated). The magnitude of glucose lowering of AC3174 was comparable to exenatide. The ED(50) values of AC3174 for glucose lowering (60 minute post-dose) were 1.2 microg/kg in db/db mice and 1.3 microg/kg in C57BL/6 mice. AC3174 has insulinotropic activity in vivo. Administration of AC3174 resulted in a 4-fold increase in insulin concentrations in normal mice following an IP glucose challenge. AC3174 was also shown to inhibit food intake and decrease gastric emptying in rodent models. AC3174 was stable in human plasma (>90% of parent peptide was present after 5 h of incubation). In rats, the in vivo half-life of AC3174 was 42-43 min following SC administration. In summary, AC3174 is an analog of exenatide that binds to the GLP-1 receptor in vitro and shares many of the biological and glucoregulatory activities of exenatide and GLP-1 in vivo.     

Patient Factors Associated with Glucagonlike Peptide 1 Receptor Agonist use with and without Insulin

ObjectiveTo evaluate both the patient factors associated with the initiation of exenatide and the real-world treatment patterns of exenatide use with and without insulin.MethodsUsing retrospective electronic medical records from the General Electric Centricity database, we performed analyses of 2 cohorts to separately evaluate factors associated with initiation of exenatide among patients with type 2 diabetes mellitus and differences between those who initiated exenatide with and without concurrent insulin use. Cohort 1 was used to assess predictors of exenatide initiation and included adults with type 2 diabetes who were active in the database when exenatide became available (October 1, 2005). Cohort 2 was used to identify characteristics of patients who initiated exenatide with and without insulin.ResultsCohort 1 included 190444 adults, and cohort 2 included 9810 adults. In cohort 1, 7383 patients initiated exenatide therapy; factors associated with exenatide initiation were female sex, younger age, body weight of 102.3 kg or greater, body mass index of 35 kg/m² or greater, residence in the southern United States, a lower Charlson Comorbidity Index, previous or existing therapy with triple oral antidiabetic drugs, and insulin plus oral antidiabetic drugs. In cohort 2, 2470 exenatide-treated patients initiated exenatide with insulin (25%) (with or without oral antidiabetic drugs). They were more likely to weigh more than 113.6 kg, have a body mass index greater than 40 kg/m², have a Charlson Comorbidity Index of 2 or greater, and have a baseline hemoglobin A_1c level greater than 9%.ConclusionsExenatide concomitant with insulin use (with or without oral antidiabetic drugs) was common, and was more likely to be prescribed in patients with morbid obesity, comorbid conditions, and poor glycemic control. Randomized controlled trials are needed to confirm the safe and effective use of this combination. (Endocr Pract. 2011;17:707-716)     

A novel neurotensin/xenin fusion peptide enhances β-cell function and exhibits antidiabetic efficacy in high-fat fed mice

Neurotensin and xenin possess antidiabetic potential, mediated in part through augmentation of incretin hormone, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), action. In the present study, fragment peptides of neurotensin and xenin, acetyl-neurotensin and xenin-8-Gln, were fused together to create Ac-NT/XN-8-Gln. Following assessment of enzymatic stability, effects of Ac-NT/XN-8-Gln on in vitro β-cell function were studied. Subchronic antidiabetic efficacy of Ac-NT/XN-8-Gln alone, and in combination with the clinically approved GLP-1 receptor agonist exendin-4, was assessed in high-fat fed (HFF) mice. Ac-NT/XN-8-Gln was highly resistant to plasma enzyme degradation and induced dose-dependent insulin-releasing actions (P<0.05 to P<0.01) in BRIN-BD11 β-cells and isolated mouse islets. Ac-NT/XN-8-Gln augmented (P<0.001) the insulinotropic actions of GIP, while possessing independent β-cell proliferative (P<0.001) and anti-apoptotic (P<0.01) actions. Twice daily treatment of HFF mice with Ac-NT/XN-8-Gln for 32 days improved glycaemic control and circulating insulin, with benefits significantly enhanced by combined exendin-4 treatment. This was reflected by reduced body fat mass (P<0.001), improved circulating lipid profile (P<0.01) and reduced HbA1c concentrations (P<0.01) in the combined treatment group. Following an oral glucose challenge, glucose levels were markedly decreased (P<0.05) only in combination treatment group and superior to exendin-4 alone, with similar observations made in response to glucose plus GIP injection. The combined treatment group also presented with improved insulin sensitivity, decreased pancreatic insulin content as well as increased islet and β-cell areas. These data reveal that Ac-NT/XN-8-Gln is a biologically active neurotensin/xenin fusion peptide that displays prominent antidiabetic efficacy when administered together with exendin-4.     

NMR insights into the core of GED assembly by H/D exchange coupled with DMSO dissociation and analysis of the denatured state

GTPase effector domain (GED) of dynamin forms megadalton-sized assembly in vitro, rendering its structural characterization highly challenging. To probe the core of the GED assembly, we performed H/D exchange in native state and analyzed the residual amides following dissociation by dimethyl sulfoxide (DMSO). The data indicated a hierarchy in solvent exposure: Ser2-Glu13, Glu23-Phe32, Asp37-Gln43, Val51-Met55, and Lys60-Asp64 followed by the remaining segments. This reflects the chain packing in the core of the assembly. The segment Leu65-Pro138 in the C-terminal half is largely in the interior of the core, while the N-terminal segment Asp37-Asp64 traverses into and out of the core. Next, we characterized the structural and motional behavior of the DMSO-denatured state. The stretches Gly9-Lys18, Asp37-Arg42, Lys68-Met74, and Ser136-Thr137 were seen to display alternate conformations in slow exchange. In the major population, both α and β propensities were seen along the polypeptide chain. Spectral density analysis of ¹⁵N R₁, R₂, and ¹H-¹⁵N nuclear Overhauser effect collected at 600 and 800 MHz suggested the presence of four domains of slow motions, namely, A (Leu40-Tyr91), A′ (Leu124-Ile130); B (Asn97-Gln107), B′ (Tyr117-Leu120), two of which flank the region Arg109-Met116, for which no peaks are seen in the heteronuclear single quantum coherence spectrum. These domains would identify folding and association initiation sites of GED. Interestingly, they also coincide with the helical domain in the native state, suggesting that helix formation leads to self-association of GED.     

Amphipathic short helix-stabilized peptides with cell-membrane penetrating ability

We synthesized four types of arginine-based amphipathic nonapeptides, including two homochiral peptides, R-(l-Arg-l-Arg-Aib)₃-NH₂ (R = 6-FAM-β-Ala: FAM-1; R = Ac: Ac-1) and R-(d-Arg-d-Arg-Aib)₃-NH₂ (R = 6-FAM-β-Ala: ent-FAM-1; R = Ac: ent-Ac-1); a heterochiral peptide, R-(l-Arg-d-Arg-Aib)₃-NH₂ (R = 6-FAM-β-Ala: FAM-2; R = Ac: Ac-2); and a racemic mixture of diastereomeric peptides, R-(rac-Arg-rac-Arg-Aib)₃-NH₂ (R = 6-FAM-β-Ala: FAM-3; R = Ac: Ac-3), and then investigated the relationship between their secondary structures and their ability to pass through cell membranes. Peptides 1 and ent-1 formed stable one-handed α-helical structures and were more effective at penetrating HeLa cells than the non-helical peptides 2 and 3.     

Theoretical description of halogen bonding – an insight based on the natural orbitals for chemical valence combined with the extended-transition-state method (ETS-NOCV)

In the present study we have characterized the halogen bonding in selected molecules H₃N–ICF₃ (1-NH ₃ ), (PH₃)₂C–ICF₃ (1-CPH ₃ ), C₃H₇Br–(IN₂H₂C₃)₂C₆H₄ (2-Br), H₂–(IN₂H₂C₃)₂C₆H₄ (2-H ₂ ) and Cl–(IC₆F₅)₂C₇H₁₀N₂O₅ (3-Cl), containing from one halogen bond (1-NH ₃ , 1-CPH ₃ ) up to four connections in 3-Cl (the two Cl–HN and two Cl–I), based on recently proposed ETS-NOCV analysis. It was found based on the NOCV-deformation density components that the halogen bonding C–X ^… B (X-halogen atom, B-Lewis base), contains a large degree of covalent contribution (the charge transfer to X ^… B inter-atomic region) supported further by the electron donation from base atom B to the empty σ*(C–X) orbital. Such charge transfers can be of similar importance compared to the electrostatic stabilization. Further, the covalent part of halogen bonding is due to the presence of σ-hole at outer part of halogen atom (X). ETS-NOCV approach allowed to visualize formation of the σ-hole at iodine atom of CF₃I molecule. It has also been demonstrated that strongly electrophilic halogen bond donor, [C₆H₄(C₃H₂N₂I)₂][OTf]₂, can activate chemically inert isopropyl bromide (2-Br) moiety via formation of Br–I bonding and bind the hydrogen molecule (2-H ₂ ). Finally, ETS-NOCV analysis performed for 3-Cl leads to the conclusion that, in terms of the orbital-interaction component, the strength of halogen (Cl–I) bond is roughly three times more important than the hydrogen bonding (Cl–HN).

Exenatide prevents high-glucose-induced damage of retinal ganglion cells through a mitochondrial mechanism

Exenatide (exendin-4 analogue) is widely used in clinics and shows a neuroprotective effect. The main objectives of the present study were to prove that retinal ganglion cells (RGC-5) express GLP-1R, to ascertain whether exenatide prevents a high-glucose-induced RGC-5 impairment, to determine the appropriate concentration of exenatide to protect RGC-5 cells, and to explore the neuroprotective mechanisms of exenatide. Immunofluorescence and Western blot analyses demonstrated that RGC-5 cells express GLP-1R. We incubated RGC-5 cells with 25 mM glucose prior to incubation with either 25 mM glucose, 55 mM glucose (high), high glucose plus exenatide or high glucose plus a GLP-1R antagonist. The survival rates of the cells were measured by CCK-8, and cellular injury was detected by electron microscopy. There were statistical differences between the high-glucose group and the control group (P<0.05). Exenatide improved the survival rate of the cells and suppressed changes in the mitochondrial morphology. The optimum concentration of exenatide to protect the RGC-5 cells from high-glucose-induced RGC injury was 0.5 μg/ml, and this protective effect could be inhibited by exendin (9-39). To further study the mechanism underlying the beneficial effects of exenatide, the expression levels of cytochrome c, Bcl-2, Bax and caspase-3 were analysed by Western blot. The present study showed that treatment with exenatide significantly inhibited cytochrome c release and decreased the intracellular expression levels of Bax and caspase-3, whereas Bcl-2 was increased (P<0.05). These results suggested that GLP-1R activation can inhibit the cellular damage that is induced by high glucose. A mitochondrial mechanism might play a key role in the protective effect of exenatide on the RGC-5 cells, and exenatide might be beneficial for patients with diabetic retinopathy.     

A new GLP-1 analogue with prolonged glucose-lowering activity in vivo via backbone-based modification at the N-terminus

Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic hormone with wonderful glucose-lowering activity. However, its clinical use in type II diabetes is limited due to its rapid degradation at the N-terminus by dipeptidyl peptidase IV (DPP-IV). Among the N-terminal modifications of GLP-1, backbone-based modification was rarely reported. Herein, we employed two backbone-based strategies to modify the N-terminus of tGLP-1. Firstly, the amide N-methylated analogues 2–6 were designed and synthesized to make a full screening of the N-terminal amide bonds, and the loss of GLP-1 receptor (GLP-1R) activation indicated the importance of amide H-bonds. Secondly, with retaining the N-terminal amide H-bonds, the β-peptide replacement strategy was used and analogues 7–13 were synthesized. By two rounds of screening, analogue 10 was identified. Analogue 10 greatly improved the DPP-IV resistance with maintaining good GLP-1R activation in vitro, and showed approximately a 4-fold prolonged blood glucose-lowering activity in vivo in comparison with tGLP-1. This modification strategy will benefit the development of GLP-1-based anti-diabetic drugs.