A Conserved Hydrophobic Moiety and Helix-Helix Interactions Drive the Self-Assembly of the Incretin Analog Exendin-4.

Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix-helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers.

Type 2 Diabetes Mellitus: A Review of Multi-Target Drugs.

Diabetes Mellitus (DM) is a multi-factorial chronic health condition that affects a large part of population and according to the World Health Organization (WHO) the number of adults living with diabetes is expected to increase. Since type 2 diabetes mellitus (T2DM) is suffered by the majority of diabetic patients (around 90-95%) and often the mono-target therapy fails in managing blood glucose levels and the other comorbidities, this review focuses on the potential drugs acting on multi-targets involved in the treatment of this type of diabetes. In particular, the review considers the main systems directly involved in T2DM or involved in diabetes comorbidities. Agonists acting on incretin, glucagon systems, as well as on peroxisome proliferation activated receptors are considered. Inhibitors which target either aldose reductase and tyrosine phosphatase 1B or sodium glucose transporters 1 and 2 are taken into account. Moreover, with a view at the multi-target approaches for T2DM some phytocomplexes are also discussed.

A long-acting, dual-agonist analogue of lamprey GLP-1 shows potent insulinotropic, ß-cell protective, and anorexic activities and improves glucose homeostasis in high fat-fed mice.

Peptidase-resistant analogues of GLP-1 peptides from sea lamprey and paddlefish ([D-Ala2]palmitoyl-lamprey GLP-1 and [D-Ala2]palmitoyl-paddlefish GLP-1) produced significant (P ≤ 0.05) and concentration-dependent increases in insulin release from BRIN-BD11 clonal ß-cells and from isolated mouse islets. Both analogues retained the ability of the native peptides to activate both the GLP-1 receptor (GLP1R) and the glucagon receptor (GCGR). [D-Ala2]palmitoyl-lamprey GLP-1 significantly (P < 0.001) stimulated proliferation of BRIN-BD11 cells and protected against cytokine-induced apoptosis. Administration of the lamprey analogue (25 nmol/kg body weight) to lean mice up to 4 h before a glucose load improved glucose tolerance and increased plasma insulin concentrations. Twice daily administration of the lamprey GLP-1 analogue to high fat-fed mice for 21 days decreased body weight, food intake, and circulating glucose and insulin concentrations. The analogue significantly improved glucose tolerance and insulin sensitivity with beneficial effects on islet ß-cell area and insulin secretory responsiveness. Islet gene expression of Glp1r, Gcgr and Gipr significantly increased. The lamprey GLP-1 analogue shows therapeutic promise for treatment of patients with obesity-related Type 2 diabetes.

Higher-Order Structure Characterization of Pharmaceutical Proteins by 2D Nuclear Magnetic Resonance Methyl Fingerprinting.

A key challenge in the analytical assessment of therapeutic proteins is the comprehensive characterization of their higher-order structure (HOS). To directly assess HOS, a new type of assay is warranted. The most sensitive and detailed method for characterizing HOS is unquestionably nuclear magnetic resonance (NMR) spectroscopy. NMR spectroscopy provides direct information about the HOS at an atomic level, and with modern NMR spectrometers and improved pulse sequences, this has become feasible even on unlabeled proteins. Hence, NMR spectroscopy could be a very powerful tool for control of HOS following, for example, process changes resulting in structural changes, oxidation, degradation, or chemical modifications. We present a method for characterizing the HOS of therapeutic proteins by monitoring their methyl groups using 2D H, C-correlated NMR. We use a statistical model that compares the NMR spectrum of a given sample to a reference and results in one output value describing how similar the HOS of the samples are. This makes the overall result easy to interpret even for non-NMR experts. We show that the method is applicable to proteins of varying size and complexity (here up to ∼30 kDa) and that it is sufficiently sensitive for the detection of small changes in both primary and HOS.

Role of ß Cell Precursors in the Regeneration of Insulin-Producing Pancreatic ß Cells under the Influence of Glucagon-Like Peptide 1.

The effects of the pegylated form of glucagon-like peptide 1 (pegGLP-1) on oligopotent ß cell precursors (CD45-TER119-CD133+CD49flow) in the pancreas were studied in C57Bl/6 mice. Under conditions of streptozotocin-induced type 1 diabetes mellitus, intraperitoneal injection of pegGLP1 increased the content of ß cell precursors and dithizone-stained cells in the pancreas. ß Cell precursors of mice with diabetes demonstrated high self-maintenance potential. In contrast to pegGLP-1, native GLP-1 did not affect ß cell precursors in diabetic animals. Treatment of a culture of ß cell precursors from mice with diabetes induced the yield of dithizone-stained mononuclears. In conditioned mediums of dithizone-positive cells obtained as a result of differentiation of ß cell precursors from mice with diabetes, insulin was detected after administration of pegGLP-1 (10-7 M) and glucose (3 mmol/liter); the level of insulin increased with increasing glucose concentration (to 20 mmol/liter). The in vitro effect of pegGLP-1 did not differ from the effect of GLP-1 (10-7 M).

Targeted intestinal delivery of incretin secretagogues-towards new diabetes and obesity therapies.

A new strategy under development for the treatment of type 2 diabetes and obesity is to mimic some of the effects of bariatric surgery by delivering food-related stimuli to the distal gastrointestinal tract where they should enhance the release of gut hormones such as glucagon-like peptide-1 (GLP-1) and peptideYY (PYY). Methods include inhibition of food digestion and absorption in the upper GI tract, or oral delivery of stimuli in capsules or pelleted form to protect them against gastric degradation. A variety of agents have been tested in humans using capsules, microcapsules or pellets, delivering nutrients, bile acids, fatty acids and bitter compounds. This review examines the outcomes of these different approaches and supporting evidence from intestinal perfusion studies.

Rebirth of the Incretin Concept: Its conception and early development.

This paper describes the resurrection of the Incretin Concept in the early 1960s. It began with the more or less simultaneous discovery by three groups working independently in London. Dupre demonstrated that secretin given intravenously with glucose increased its rate of disappearance from the blood, McIntyre and co-workers established that hyperglycaemia evoked by oral glucose stimulated more insulin secretion than comparable hyperglycaemia produced by intravenous glucose and Marks and Samols established the insulinotropic properties of glucagon. The concept evolved with the discovery by Samols and co-workers that oral glucose stimulated the release of immunoreactive glucagon-like substances from the gut mucosa and the subsequent isolation of glucagon immunoreactive compounds, most notably oxyntomodulin and glicentin, and of gastic inhibitory polypetide (GIP). It concluded with the isolation and characterisation of glucagon-like peptide 1 (7-36) amide.

Incretin hormones receptor signaling plays the key role in antidiabetic potential of PTY-2 against STZ-induced pancreatitis.

AIMS: Incretin therapy is one of the most potential approaches in the treatment of diabetes. In contrast to markedly available drugs, the herbal incretin modulators have lesser side effects with low economic cost. The main aim of this work was to analyze the potential of previously reported DPPIV inhibitor, aqueous extract of Pueraria tuberosa tubers (PTY-2) as incretin hormones receptor agonist against streptozotocin (STZ)-induced diabetes. METHODS: Chronic diabetes was induced with STZ (65mg/kg bw) in rats for 60days and grouped into diabetic control and PTY-2. Expression of genes was assessed by PCR, IHC, and ELISA. Morphological analysis of tissue was observed using H & E stain. In silico molecular docking approach has been used to see the interaction of active phytochemicals of PTY-2 on the basis of their binding energy [kcal/mol] and dissociation constant [pM] using YASARA software. Interactive visualization was done using Discovery studio 3.0. RESULTS: In comparison to diabetic control, the size and number of islet cells along with the plasma level of GLP-1, GIP, and pancreatic expressions of GLP-1R, GIP-R, Bcl2, and insulin were enhanced significantly after PTY-2 treatment. Through in silico molecular docking, tuberostan showed the best interaction for GLP-1R with binding energy at 8.15kcal/mol and dissociation constant at 1061624.125 pM. Puererone showed the best interaction for GIP-R with binding energy at 8.31kcal/mol and dissociation constant at 810381 pM. CONCLUSIONS: In addition to previously studied DPPIV inhibitor, PTY-2 also acts as incretin receptors agonist and protects against STZ-induced diabetes by down regulating ß cells apoptosis.

Molecular Integration of Incretin and Glucocorticoid Action Reverses Immunometabolic Dysfunction and Obesity.

Chronic inflammation has been proposed to contribute to the pathogenesis of diet-induced obesity. However, scarce therapeutic options are available to treat obesity and the associated immunometabolic complications. Glucocorticoids are routinely employed for the management of inflammatory diseases, but their pleiotropic nature leads to detrimental metabolic side effects. We developed a glucagon-like peptide-1 (GLP-1)-dexamethasone co-agonist in which GLP-1 selectively delivers dexamethasone to GLP-1 receptor-expressing cells. GLP-1-dexamethasone lowers body weight up to 25% in obese mice by targeting the hypothalamic control of feeding and by increasing energy expenditure. This strategy reverses hypothalamic and systemic inflammation while improving glucose tolerance and insulin sensitivity. The selective preference for GLP-1 receptor bypasses deleterious effects of dexamethasone on glucose handling, bone integrity, and hypothalamus-pituitary-adrenal axis activity. Thus, GLP-1-directed glucocorticoid pharmacology represents a safe and efficacious therapy option for diet-induced immunometabolic derangements and the resulting obesity.

Gold nanoparticles as an efficient drug delivery system for GLP-1 peptides.

In this work, the potential application of gold nanoparticles for GLP-1 analogues delivery was studied. For this purpose, the original sequence of the incretin GLP-1 was slightly modified in the C-terminal region by adding a cysteine residue to facilitate conjugation to the gold surface. The interaction between peptides and gold nanoparticles and also the colloid stability of the conjugates were studied by UV-vis spectrophotometry, TEM, IR and XPS spectroscopy. Moreover, the permeability of these conjugates was assayed using a Caco-2/goblet monolayer model. On the basis of the stability and permeability results, one of the conjugates was chosen to be administered intraperitoneally to normoglycemic rats. The intraperitoneal delivery of the GLP-1 analogue using gold nanoparticles led to decrease levels of blood glucose in the same way as native GLP-1, thereby demonstrating that the formulation of the analogue is stable in physiological conditions and maintains the activity of this incretin.

Promising Diabetes Therapy Based on the Molecular Mechanism for Glucose Toxicity: Usefulness of SGLT2 Inhibitors as well as Incretin-Related Drugs.

Pancreatic ß-cell dysfunction and insulin resistance are the main characteristics of type 2 diabetes. Chronic exposure of ß-cells to hyperglycemia leads to the deterioration of ß-cell function. Such phenomena are well known as pancreatic ß-cell glucose toxicity. MafA, a strong transactivator of insulin gene, is particularly important for the maintenance of mature ß-cell function, but its expression level is significantly reduced under diabetic conditions which is likely associated with ß-cell failure. Reduction of incretin receptor expression level in ß-cells in diabetes is also likely associated with ß-cell failure. On the other hand, incretin-related drugs and sodium-glucose co-transporter 2 (SGLT2) inhibitors are promising diabetes therapy based on the mechanism for pancreatic ß-cell glucose toxicity. Indeed, it was shown that incretin-related drugs exerted protective effects on ß-cells through the augmentation of IRS-2 expression especially in the presence of pioglitazone. It was also shown that incretin-related drug and/or pioglitazone exerted more protective effects on ß-cells at the early stage of diabetes compared to the advanced stage. SGLT2 inhibitors, new hypoglycemic agents, also exert beneficial effects for the protection of pancreatic ß-cells as well as for the reduction of insulin resistance in various insulin target tissues. Taken together, it is important to select appropriate therapy based on the molecular mechanism for glucose toxicity.

Novel incretin analogues improve autophagy and protect from mitochondrial stress induced by rotenone in SH-SY5Y cells.

Currently, there is no viable treatment available for Parkinson's disease (PD) that stops or reverses disease progression. Interestingly, studies testing the glucagon-like-peptide-1 (GLP-1) mimetic Exendin-4 have shown neuroprotective/neurorestorative properties in pre-clinical tests and in a pilot clinical study of PD. Incretin analogues were originally developed to treat type 2 diabetes and several are currently on the market. In this study, we tested novel incretin analogues on the dopaminergic SH-SY5Y neuroblastoma cells against a toxic mitochondrial complex I inhibitor, Rotenone. Here, we investigate for the first time the effects of six different incretin receptor agonists - Liraglutide, D-Ser2-Oxyntomodulin, a GLP-1/GIP Dual receptor agonist, dAla(2)-GIP-GluPal, Val(8)GLP-1-GluPal and exendin-4. Post-treatment with doses of 1, 10 or 100 nM of incretin analogues for 12 h increased the survival of SH-SY5Y cells treated with 1 µM Rotenone for 12 h. Furthermore, we studied the post-treatment effect of 100 nM incretin analogues against 1 µM Rotenone stress on apoptosis, mitochondrial stress and autophagy markers. We found significant protective effects of the analogues against Rotenone stress on cell survival and on mitochondrial and autophagy-associated markers. The novel GLP-1/GIP Dual receptor agonist was superior and effective at a tenfold lower concentration compared to the other analogues. Using the Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, we further show that the neuroprotective effects are partially PI3K-independent. Our data suggest that the neuroprotective properties exhibited by incretin analogues against Rotenone stress involve enhanced autophagy, increased Akt-mediated cell survival and amelioration of mitochondrial dysfunction. These mechanisms can explain the neuroprotective effects of incretin analogues reported in clinical trials. GLP-1, GIP and dual incretin receptor agonists showed protective effects in SH-SY5Y cells treated with the stressor Rotenone. The novel GLP-1/GIP dual receptor agonist was superior and effective at a tenfold lower concentration compared to the other analogues. The drugs protected the cells from rotenone-induced impairment in cell growth and Akt activation, mitochondrial damage, impairments of autophagy and apoptotic cell signalling. See paper for details.

Alternative form of glucose-dependent insulinotropic polypepide and its physiology.

Glucose-dependent insulinotropic polypepide (GIP) was first extracted from porcine gut mucosa and identified as "incretin" decades ago. Though early studies have shown the possible GIP isoforms by gel filtration profiles from porcine or human intestinal extracts analyzed by radioimmunoassay (RIA), GIP is currently believed to consist of 42 amino acids (GIP1-42), which are released from gut K-cells and promote postprandial insulin release. In fact, GIP1-42 is usually processed from proGIP by the action of prohormone convertase (PC) 1/3 in the gut. GIP expression is occasionally found in the intestinal glucagon-like peptide-1-secreting cells, suggesting gene expression of both GIP and proglucagon can co-exist in identical cells. However, GIP1-42 immunoreactivity is rarely found in α-cells or other pancreatic endocrine cells of wild-type mammals. Interestingly, we found that short-form GIP1-30 is expressed in and released from pancreatic α-cells and a subset of enteroendocrine cells through proGIP processing by PC2. GIP1-30 is also insulinotropic and modulates glucose-stimulated insulin secretion in a paracrine manner. It is also suggested that short-form GIP1-30 possibly plays a crucial role for the islet development. It has not been well elucidated whether expression of GIP1-30 is modulated in the diabetic status, and whether GIP1-30 might have therapeutic potentials. Our preliminary data suggest that short-form GIP1-30 might play important roles in glucose metabolism.

Optical Control of Insulin Secretion Using an Incretin Switch.

Incretin mimetics are set to become a mainstay of type 2 diabetes treatment. By acting on the pancreas and brain, they potentiate insulin secretion and induce weight loss to preserve normoglycemia. Despite this, incretin therapy has been associated with off-target effects, including nausea and gastrointestinal disturbance. A novel photoswitchable incretin mimetic based upon the specific glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide was designed, synthesized, and tested. This peptidic compound, termed LirAzo, possesses an azobenzene photoresponsive element, affording isomer-biased GLP-1R signaling as a result of differential activation of second messenger pathways in response to light. While the trans isomer primarily engages calcium influx, the cis isomer favors cAMP generation. LirAzo thus allows optical control of insulin secretion and cell survival.

Transformation of oligomers of lipidated peptide induced by change in pH.

Oligomerization of lipidated peptides is of general scientific interest and is important in biomedical and pharmaceutical applications. We investigated the solution properties of a lipidated peptide, Liraglutide, which is one of the glucagon-like peptide-1 (GLP-1) agonists used for the treatment of type II diabetes. Liraglutide can serve as a model system for studying biophysical and biochemical properties of micelle-like self-assemblies of the lipidated peptides. Here, we report a transformation induced in Liraglutide oligomers by changing pH in the vicinity of pH 7. This fully reversible transformation is characterized by changes in the size and aggregation number of the oligomer and an associated change in the secondary structure of the constituent peptides. This transformation has quite slow kinetics: the equilibrium is reached in a course of several days. Interestingly, while the transformation is induced by changing pH, its kinetics is essentially independent of the final pH. We interpreted these findings using a model in which desorption of the monomer from the oligomer is the rate-limiting step in the transformation, and we determined the rate constant of the monomer desorption.

Measurement of the incretin hormones: glucagon-like peptide-1 and glucose-dependent insulinotropic peptide.

The two incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), are secreted from the gastrointestinal tract in response to meals and contribute to the regulation of glucose homeostasis by increasing insulin secretion. Assessment of plasma concentrations of GLP-1 and GIP is often an important endpoint in both clinical and preclinical studies and, therefore, accurate measurement of these hormones is important. Here, we provide an overview of current approaches for the measurement of the incretin hormones, with particular focus on immunological methods.

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 ß 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 ß-sheet, random coil and reverse ß-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.

Unsaturated glycoceramides as molecular carriers for mucosal drug delivery of GLP-1.

The incretin hormone Glucagon-like peptide 1 (GLP-1) requires delivery by injection for the treatment of Type 2 diabetes mellitus. Here, we test if the properties of glycosphingolipid trafficking in epithelial cells can be applied to convert GLP-1 into a molecule suitable for mucosal absorption. GLP-1 was coupled to the extracellular oligosaccharide domain of GM1 species containing ceramides with different fatty acids and with minimal loss of incretin bioactivity. When applied to apical surfaces of polarized epithelial cells in monolayer culture, only GLP-1 coupled to GM1-ceramides with short- or cis-unsaturated fatty acids trafficked efficiently across the cell to the basolateral membrane by transcytosis. In vivo studies showed mucosal absorption after nasal administration. The results substantiate our recently reported dependence on ceramide structure for trafficking the GM1 across polarized epithelial cells and support the idea that specific glycosphingolipids can be harnessed as molecular vehicles for mucosal delivery of therapeutic peptides.

Latest research and development trends in non-insulin anti-diabetics.

Type 2 diabetes mellitus, also called non-insulin dependent diabetes mellitus, is a chronic endocrine disease characterized by insulin resistance in tissues such as fat, liver and skeletal muscle, and impaired insulin secretion in pancreatic ß cells. The prevalence and incidence of type 2 diabetes exploded over last decades along with increased population obesity owing to western lifestyle factors such as lack of exercise and high calorie diets. As diabetes progresses without appropriate treatment, many micro- and macro-vascular complications occur, leading to increased risk of mortality. Although lifestyle modifications including a healthier diet and more frequent exercise are suggested as initial therapy for type 2 diabetes, pharmacotherapy is required in many cases. Currently, several anti-diabetic drugs with different mechanisms of action are available, but increased effectiveness and tolerability are a still unmet need for diabetes pharmacotherapy. Thus, the development of new anti-diabetic drugs is an active research area in both academia and the pharmaceutical industry. This review focuses on the targets in the latest developments of non-insulin anti-diabetics that attract the most interest in this disease area.

[Incretin modulators, a new perspective in diabetes mellitus treatment]. / Modulatorii incretinelor, o noua perspectiva în tratamentul diabetului zaharat.

AIM: The study was designed to present the modern therapy used for the treatment of type 2 diabetes mellitus (T2DM). MATERIAL AND METHODS: Diabetes mellitus and the pharmacological and the pharmacokinetic characteristics of the incretins and of the incretin modulators are presented. RESULTS: Diabetes mellitus is a chronic metabolic disorder, which is considered to be a major health issue all over the world. World Health Organization indicates that world-wide almost 3 million deaths per year are caused by diabetes and 1,5 million new cases are diagnosed annually, T2DM representing approximately 90-95% of the diagnosed cases. This form of diabetes mellitus is characterized by several path physiological defects including insulin resistance at peripheral target tissues, excess hepatic glucose production and progressive pancreatic beta cell dysfunction. The newest direction in the treatment of T2DM mellitus is the incretin modulators. The incretins (GLP-1--glucagon-like-peptide-1 and GIP--glucose-dependent insulin tropic peptide), are natural hormones that contribute to glucose homeostasis by acting on the pancreas, gastrointestinal tract, muscle and brain tissue. After secretion, GLP-1 and GIP are immediately metabolized by dipeptidyl peptidase IV enzyme (DPP IV), which limits their therapeutic use. CONCLUSIONS: In order to achieve optimal glycemic control, by using the therapeutic effects of incretins, research has been directed to the development of synthetic analogues of GLP-1 resistant to DPP IV enzyme inactivation and DPP IV inhibitors.