Presence of a functional receptor for GLP‐1 in osteoblastic cells,independent of the cAMP‐linked GLP‐1 receptor

Glucagon‐like peptide 1 (GLP‐1) controls glucose metabolism in extrapancreatic tissues through receptors other than the pancreatic cAMP‐linked GLP‐1 receptor; also, GLP‐1 induces an insulin‐ and PTH‐independent bone anabolic action in insulin‐resistant and type‐2 diabetic rats. Here we searched for the presence and characteristics of GLP‐1 receptors in osteoblastic MC3T3‐E1 cells. [¹²⁵I]‐GLP‐1 specific binding to MC3T3‐E1 cells was time‐ and temperature‐dependent, reaching maximal value at 30 min at 25°C; in these conditions, [¹²⁵I]‐GLP‐1 binding was dissociable, and displaced by GLP‐1, partially by GLP‐2, but not by exendin‐4 (Ex‐4), exendin‐9 (Ex‐9), glucagon or insulin; Scatchard analysis of the unlabeled GLP‐1 data showed high and low affinity binding sites; cross‐linking of GLP‐1 binding revealed an estimated 70 kDa band, almost undetectable in the presence of 10^?6 M GLP‐1. GLP‐1, Ex‐9, insulin or glucagon failed to modify cellular cAMP content, while GLP‐2 and Ex‐4 increased it. However, GLP‐1 induced an immediate hydrolysis of glycosylphosphatidylinositols (GPIs) generating short‐lived inositolphosphoglycans (IPGs), and an increase in phosphatidylinositol‐3 kinase (PI3K) and mitogen activated protein kinase (MAPK) activities; Ex‐4 also affected GPIs, but its action was delayed with respect to that of GLP‐1. This incretin was found to decrease Runx2 but increased osteocalcin gene expression, without affecting that of osteoprotegerin or the canonical Wnt pathway activity in MC3T3‐E1 cells which do not express the pancreatic GLP‐1 receptor. Our data demonstrate for the first time that GLP‐1 can directly and functionally interact with osteoblastic cells, possibly through a GPI/IPG‐coupled receptor. J. Cell. Physiol. 225: 585–592, 2010. © 2010 Wiley‐Liss, Inc.     

Dual factor delivery of CXCL12 and Exendin‐4 for improved survival and function of encapsulated beta cells under hypoxic conditions

A bioartifical pancreas (BAP) remains a promising approach for treating insulin‐dependent diabetes. Several obstacles to the clinical implementation of a BAP remain, including hypoxia following implantation. Within native pancreatic islets, CXCL12 and glucagon‐like peptide‐1 (GLP‐1) act in a paracrine fashion to promote the survival, function, and proliferation of β‐cells. This work sought to investigate if the presentation of CXCL12 and delivery of a GLP‐1 receptor analog, Exendin‐4 (Ex‐4), alone and in combination, conferred pro‐survival and insulinotropic effects on an encapsulated β‐cell line, βTC‐tet, cultured under hypoxic conditions of 7.6 mmHg O₂. Our findings indicate that presentation of CXCL12 in the encapsulation matrix completely abrogated apoptosis under hypoxic conditions. Delivery of Ex‐4 increased insulin secretion rate under both normoxic and hypoxic conditions, and additionally reduced apoptosis under hypoxic conditions. Furthermore, presentation of CXCL12 combined with Ex‐4 delivery significantly increased insulin secretion rate under hypoxic conditions compared to delivery of Ex‐4 alone. These findings demonstrate that the presentation of CXCL12 combined with the delivery of Ex‐4 may constitute a promising strategy for supporting β‐cell function and survival following transplantation. Biotechnol. Bioeng. 2013; 110: 2292–2300. © 2013 Wiley Periodicals, Inc.     

Oral delivery of bioencapsulated exendin‐4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta‐TC6 cells

Glucagon‐like peptide (GLP‐1) increases insulin secretion but is rapidly degraded (half‐life: 2 min in circulation). GLP‐1 analogue, exenatide (Byetta) has a longer half‐life (3.3–4 h) with potent insulinotropic effects but requires cold storage, daily abdominal injections with short shelf life. Because patients with diabetes take >60 000 injections in their life time, alternative delivery methods are highly desired. Exenatide is ideal for oral delivery because insulinotropism is glucose dependent, with reduced risk of hypoglycaemia even at higher doses. Therefore, exendin‐4 (EX4) was expressed as a cholera toxin B subunit (CTB)–fusion protein in tobacco chloroplasts to facilitate bioencapsulation within plant cells and transmucosal delivery in the gut via GM1 receptors present in the intestinal epithelium. The transgene integration was confirmed by PCR and Southern blot analysis. Expression level of CTB‐EX4 reached up to 14.3% of total leaf protein (TLP). Lyophilization of leaf material increased therapeutic protein concentration by 12‐ to 24‐fold, extended their shelf life up to 15 months when stored at room temperature and eliminated microbes present in fresh leaves. The pentameric structure, disulphide bonds and functionality of CTB‐EX4 were well preserved in lyophilized materials. Chloroplast‐derived CTB‐EX4 showed increased insulin secretion similar to the commercial EX4 in beta‐TC6, a mouse pancreatic cell line. Even when 5000‐fold excess dose of CTB‐EX4 was orally delivered, it stimulated insulin secretion similar to the intraperitoneal injection of commercial EX4 but did not cause hypoglycaemia in mice. Oral delivery of the bioencapsulated EX4 should eliminate injections, increase patient compliance/convenience and significantly lower their cost.     

Excess cholesterol inhibits glucose‐stimulated fusion pore dynamics in insulin exocytosis

Type 2 diabetes is caused by defects in both insulin sensitivity and insulin secretion. Glucose triggers insulin secretion by causing exocytosis of insulin granules from pancreatic β‐cells. High circulating cholesterol levels and a diminished capacity of serum to remove cholesterol from β‐cells are observed in diabetic individuals. Both of these effects can lead to cholesterol accumulation in β‐cells and contribute to β‐cell dysfunction. However, the molecular mechanisms by which cholesterol accumulation impairs β‐cell function remain largely unknown. Here, we used total internal reflection fluorescence microscopy to address, at the single‐granule level, the role of cholesterol in regulating fusion pore dynamics during insulin exocytosis. We focused particularly on the effects of cholesterol overload, which is relevant to type 2 diabetes. We show that excess cholesterol reduced the number of glucose‐stimulated fusion events, and modulated the proportion of full fusion and kiss‐and‐run fusion events. Analysis of single exocytic events revealed distinct fusion kinetics, with more clustered and compound exocytosis observed in cholesterol‐overloaded β‐cells. We provide evidence for the involvement of the GTPase dynamin, which is regulated in part by cholesterol‐induced phosphatidylinositol 4,5‐bisphosphate enrichment in the plasma membrane, in the switch between full fusion and kiss‐and‐run fusion. Characterization of insulin exocytosis offers insights into the role that elevated cholesterol may play in the development of type 2 diabetes.     

Characterization of an insulinotropic peptide from skin secretions of Odorrana andersonii

Insulinotropic peptide agents are regarded as potential candidates for anti‐diabetic treatment. In the present study, a novel insulinotropic peptide, termed OA‐A1, was purified from frog skin secretions of Odorrana andersonii. Mature OA‐A1 was determined to be a 1965.049 Da peptide with an amino acid sequence of LVGKLLKGAVGDVCGLLPIC, in which an intramolecular disulfide bridge was formed by two cysteine residues. At the cellular level, OA‐A1 exhibited potent proliferation promoting effects on mouse‐derived pancreatic β‐TC‐6 cells and significantly stimulated insulin release in β‐TC‐6 cells at a minimum concentration of 1 nM. In the animal model, OA‐A1 also showed a dose‐dependent insulin‐releasing role in mice. At concentrations ranging from 1 nmol/kg to 1 μmol/kg, OA‐A1 had a significant acute hypoglycemic effect on streptozotocin (STZ)‐induced diabetic mice. The pancreatic islet areas of diabetic mice increased dose‐dependently after 21 days of OA‐A1 treatment (1–100 nmol/kg) compared with those of the saline control group. Moreover, OA‐A1 significantly improved the oral glucose tolerance of STZ‐induced diabetic mice. Taken together, these results suggest that OA‐A1 provides an excellent template for the development of novel anti‐diabetic therapeutic agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

β cell membrane remodelling and procoagulant events occur in inflammation‐driven insulin impairment: a GLP‐1 receptor dependent and independent control

Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell‐derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon‐like peptide (GLP)‐1 analogue, is known to promote insulin secretion and β‐cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin‐m5f β‐cell function, TF activity mediated by MPs and their modulation by 1 μM liraglutide were examined in a cell cross‐talk model. Methyl‐β‐cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N‐éthylmaleimide‐sensitive‐factor Attachment protein Receptor (SNARE)‐dependent exocytosis. Cytokines induced a two‐fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two‐fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD‐treated cells showed similar patterns. Cells pre‐treated by saturating concentration of the GLP‐1r antagonist exendin (9‐39), showed a partial abolishment of the liraglutide‐driven insulin secretion and liraglutide‐decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP‐1r‐dependent and ‐independent pathways. Our results confirm an integrative β‐cell response to GLP‐1 that targets receptor‐mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation‐driven procoagulant events.     

β‐Methylphenylalanine exerts neuroprotective effects in a Parkinson's disease model by protecting against tyrosine hydroxylase depletion

We evaluated the neuroprotective effects of β‐methylphenylalanine in an experimental model of rotenone‐induced Parkinson's disease (PD) in SH‐SY5Y cells and rats. Cells were pre‐treated with rotenone (2.5 µg/mL) for 24 hours followed by β‐methylphenylalanine (1, 10 and 100 mg/L) for 72 hours. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), mitochondrial fragmentation, apoptosis, and mRNA and protein levels of tyrosine hydroxylase were determined. In a rat model of PD, dopamine (DA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) levels, bradykinesia and tyrosine hydroxylase expression were determined. In rotenone–pre‐treated cells, β‐methylphenylalanine significantly increased cell viability and MMP, whereas ROS levels, apoptosis and fragmented mitochondria were reduced. β‐Methylphenylalanine significantly increased the mRNA and protein levels of tyrosine hydroxylase in SH‐SY5Y cells. In the rotenone‐induced rat model of PD, oral administration of β‐methylphenylalanine recovered DA and DOPAC levels and bradykinesia. β‐Methylphenylalanine significantly increased the protein expression of tyrosine hydroxylase in the striatum and substantia nigra of rats. In addition, in silico molecular docking confirmed binding between tyrosine hydroxylase and β‐methylphenylalanine. Our experimental results show neuroprotective effects of β‐methylphenylalanine via the recovery of mitochondrial damage and protection against the depletion of tyrosine hydroxylase. We propose that β‐methylphenylalanine may be useful in the treatment of PD.     

Obesity‐Induced Diabetes in Mouse Strains Treated With Gold Thioglucose: A Novel Animal Model for Studying β‐Cell Dysfunction

An obesity‐induced diabetes model using genetically normal mouse strains would be invaluable but remains to be established. One reason is that several normal mouse strains are resistant to high‐fat diet‐induced obesity. In the present study, we show the effectiveness of gold thioglucose (GTG) in inducing hyperphagia and severe obesity in mice, and demonstrate the development of obesity‐induced diabetes in genetically normal mouse strains. GTG treated DBA/2, C57BLKs, and BDF1 mice gained weight rapidly and exhibited significant increases in nonfasting plasma glucose levels 8–12 weeks after GTG treatment. These mice showed significantly impaired insulin secretion, particularly in the early phase after glucose load, and reduced insulin content in pancreatic islets. Interestingly, GTG treated C57BL/6 mice did not become diabetic and retained normal early insulin secretion and islet insulin content despite being as severely obese and insulin resistant as the other mice. These results suggest that the pathogenesis of obesity‐induced diabetes in GTG‐treated mice is attributable to the inability of their pancreatic β‐cells to secrete enough insulin to compensate for insulin resistance. Mice developing obesity‐induced diabetes after GTG treatment might be a valuable tool for investigating obesity‐induced diabetes. Furthermore, comparing the genetic backgrounds of mice with different susceptibilities to diabetes may lead to the identification of novel genetic factors influencing the ability of pancreatic β‐cells to secrete insulin.     

Oral administration of a cholera toxin B subunit-insulin fusion protein produced in silkworm protects against autoimmune diabetes

The oral administration of disease-specific autoantigens can induce oral immune tolerance and prevent or delay the onset of autoimmune disease symptoms. Here, we describe the construction of an edible vaccine consisting of a fusion protein composed of cholera toxin B subunit (CTB) and insulin that is produced in silkworm larvae at levels of up to 0.3 mg/ml of hemolymph. The silkworm bioreactor produced this fusion protein vaccine as the pentameric CTB-insulin form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and insulin. Non-obese diabetic mice fed hemolymph containing microgram quantities of the CTB-insulin fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of symptoms of clinical diabetes. These results demonstrate that the silkworm bioreactor is a feasible production and delivery system for an oral protein vaccine designed to develop immunological tolerance against T-cell-mediated autoimmune diabetes by regulatory T-cell induction.     

Effects of Exendin‐4 Alone and With Peptide YY3–36 on Food Intake and Body Weight in Diet‐Induced Obese Rats

Significant weight loss following Roux‐en‐Y gastric bypass surgery (RYGB) in obese humans correlates with enhanced secretion of anorexigenic gut hormones glucagon‐like peptide‐1 (GLP‐1) and peptide YY_3–36 (PYY_3–36). Our aim here was to identify a dosing strategy for intraperitoneal (IP) infusion of GLP‐1 homologue exendin‐4 alone and with PYY_3–36 that produces a sustained reduction in daily food intake and body weight in diet‐induced obese (DIO) rats. We tested 12 exendin‐4 strategies over 10 weeks. Exendin‐4 infused during the first and last 3 h of the dark period at 15–20 pmol/h (0.15 nmol/kg/day) produced a sustained 24 ± 1% reduction in daily food intake for 17 days, and decreased body weight by 7%. In a separate group of DIO rats, none of seven dosing strategies combining exendin‐4 and PYY_3–36 produced a similar reduction in daily food intake for >10 days. The subsequent decline in efficacies of exendin‐4 alone and with PYY_3–36 on food intake and body weight in each experiment suggested possible receptor downregulation and tolerance to treatments. However, when treatments were discontinued for 1 day following losses in efficacies, daily food intake significantly increased. Together, these results demonstrate that (i) intermittent IP infusion of a low dose of exendin‐4 can produce a relatively prolonged reduction in daily food intake and body weight in DIO rats, (ii) co‐infusion of exendin‐4 and PYY_3–36 does not further prolong this response, and (iii) activation of an orexigenic mechanism gradually occurs to counteract the inhibitory effects of exendin‐4 alone and with PYY_3–36 on food intake and body weight.     

Assessment of the potential of temporin peptides from the frog Rana temporaria (Ranidae) as anti‐diabetic agents

Temporin A (FLPLIGRVLSGIL‐NH₂), temporin F (FLPLIGKVLSGIL‐NH₂), and temporin G (FFPVIGRILNGIL‐NH₂), first identified in skin secretions of the frog Rana temporaria, produced concentration‐dependent stimulation of insulin release from BRIN‐BD11 rat clonal β‐cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 μM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Ca²⁺ concentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration‐dependent stimulation of insulin release from 1.1B4 human‐derived pancreatic β‐cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 μM), but not temporin G, protected BRIN‐BD11 cells against cytokine‐induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon‐like peptide‐1. Intraperitoneal injection of temporin G (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion whereas temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment.     

A Spatial Model of Insulin‐Granule Dynamics in Pancreatic β‐Cells

Insulin secretion from pancreatic β‐cells in response to sudden glucose stimulation is biphasic. Prolonged secretion in vivo requires synthesis, delivery to the plasma membrane (PM) and exocytosis of insulin secretory granules (SGs). Here, we provide the first agent‐based space‐resolved model for SG dynamics in pancreatic β‐cells. Using recent experimental data, we consider a single β‐cell with identical SGs moving on a phenomenologically represented cytoskeleton network. A single exocytotic machinery mediates SG exocytosis on the PM. This novel model reproduces the measured spatial organization of SGs and insulin secretion patterns under different stimulation protocols. It proposes that the insulin potentiation effect and the rising second‐phase secretion are mainly due to the increasing number of docking sites on the PM. Furthermore, it shows that 6 min after glucose stimulation, the ‘newcomer’ SGs are recruited from a region within less than 600 nm from the PM.      

Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse β‐cell lines, human islets and CB1R‐null (CB1R^?/?) mice, we have now investigated the role of CB1Rs in modulating β‐cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP‐1‐mediated cAMP accumulation and insulin secretion as well as glucose‐stimulated insulin secretion in mouse β‐cell lines and human islets. In addition, silencing CB1R in mouse β cells resulted in an increased expression of pro‐insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in β cells lacking insulin receptor. Furthermore, CB1R^?/? mice had increased pro‐insulin, GCK and GLUT2 expression in β cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve β‐cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to β‐cell function in type 2 diabetes.     

Expression of cholera toxin B subunit and the B chain of human insulin as a fusion protein in transgenic tobacco plants

A DNA construct containing the cholera toxin B subunit (CTB) gene genetically fused to a nucleotide sequence encoding three copies of tandemly repeated diabetes-associated autoantigen, the B chain of human insulin, was produced and transferred into low-nicotine tobaccos by Agrobacterium. Integration of the fusion gene into the plant genome was confirmed by polymerase chain reaction (PCR). The results of immunoblot analysis verified the synthesis and assembly of the fusion protein into pentamers in transgenic tobacco. GM1–ELISA showed that the plant-derived fusion protein retained GM1–ganglioside receptor binding specificity. The fusion protein accounted for 0.11% of the total leaf protein. The production of transgenic plants expressing CTB–InsB₃ offers a new opportunity to test plant-based oral antigen therapy against autoimmune diabetes by inducing oral tolerance.     

Lentinan protects pancreatic β cells from STZ‐induced damage

Pancreatic β‐cell death or dysfunction mediated by oxidative stress underlies the development and progression of diabetes mellitus (DM). In this study, we evaluated the effect of lentinan (LNT), an active ingredient purified from the bodies of Lentinus edodes, on pancreatic β‐cell apoptosis and dysfunction caused by streptozotocin (STZ) and the possible mechanisms implicated. The rat insulinoma cell line INS‐1 were pre‐treated with the indicated concentration of LNT for 30 min. and then incubated for 24 hrs with or without 0.5 mM STZ. We found that STZ treatment causes apoptosis of INS‐1 cells by enhancement of intracellular reactive oxygen species (ROS) accumulation, inducible nitric oxide synthase (iNOS) expression and nitric oxide release and activation of the c‐jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK) signalling pathways. However, LNT significantly increased cell viability and effectively attenuated STZ‐induced ROS production, iNOS expression and nitric oxide release and the activation of JNK and p38 MAPK in a dose‐dependent manner in vitro. Moreover, LNT dose‐dependently prevented STZ‐induced inhibition of insulin synthesis by blocking the activation of nuclear factor kappa beta and increasing the level of Pdx‐1 in INS‐1 cells. Together these findings suggest that LNT could protect against pancreatic β‐cell apoptosis and dysfunction caused by STZ and therefore may be a potential pharmacological agent for preventing pancreatic β‐cell damage caused by oxidative stress associated with diabetes.