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.     

Charge inversion at position 68 of the glucagon and glucagon‐like peptide‐1 receptors supports selectivity in hormone action

Glucagon and glucagon‐like peptide‐1 (GLP‐1)are two structurally related hormones that acutely regulate glucose control in opposite directions through homologous receptors. The molecular basis for selectivity between these two hormones and their receptors is of physiological and medicinal importance. The application of co‐agonists to enhance body weight reduction and correct multiple abnormalities associated with the metabolic syndrome has recently been reported. Substitution of amino acids 16, 18, and 20 in glucagon with those found in GLP‐1 and exendin‐4 were identified as partial contributors to balanced, high potency receptor action. The amidation of the C‐terminus was an additional glucagon‐based structural change observed to be of seminal importance to discriminate recognition by both receptors. In this work, the molecular basis for receptor selectivity associated with differences in C‐terminal peptide sequence has been determined. A single charge inversion in glucagon and GLP‐1 receptor sequence at position 68* was determined to significantly alter hormone action. Changing E68* in GLP‐1R to the corresponding Lys of GCGR reduced receptor activity for natural GLP‐1 hormones by eightfold. The enhanced C‐terminal positive charges in GLP‐1 peptides favor the native receptor's negative charge at position 68*, while the unfavorable interaction with the C‐terminal acid of native glucagon is minimized by amidation. The extension of these observations to other glucagon‐related hormones such as oxyntomodulin and exendin, as well as other related receptors such as GIPR, should assist in the assembly of additional hormones with broadened pharmacology. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

SREBP‐1c,Pdx‐1, and GLP‐1R involved in palmitate–EPA regulated glucose‐stimulated insulin secretion in INS‐1 cells

Impairment of glucose‐stimulated insulin secretion (GSIS) caused by glucolipotoxicity is an essential feature in type 2 diabetes mellitus (T2DM). Palmitate and eicosapentaenoate (EPA), because of their lipotoxicity and protection effect, were found to impair or restore the GSIS in beta cells. Furthermore, palmitate was found to up‐regulate the expression level of sterol regulatory element‐binding protein (SREBP)‐1c and down‐regulate the levels of pancreatic and duodenal homeobox (Pdx)‐1 and glucagon‐like peptide (GLP)‐1 receptor (GLP‐1R) in INS‐1 cells. To investigate the underlying mechanism, the lentiviral system was used to knock‐down or over‐express SREBP‐1c and Pdx‐1, respectively. It was found that palmitate failed to suppress the expression of Pdx‐1 and GLP‐1R in SREBP‐1c‐deficient INS‐1 cells. Moreover, down‐regulation of Pdx‐1 could cause the low expression of GLP‐1R with/without palmitate treatment. Additionally, either SREBP‐1c down‐regulation or Pdx‐1 over‐expression could partially alleviate palmitate‐induced GSIS impairment. These results suggested that sequent SREBP‐1c‐Pdx‐1‐GLP‐1R signal pathway was involved in the palmitate‐caused GSIS impairment in beta cells. J. Cell. Biochem. 111: 634–642, 2010. © 2010 Wiley‐Liss, Inc.     

A surface plasmon resonance assay for characterisation and epitope mapping of anti‐GLP‐1 antibodies

The incretin hormone glucagon‐like peptide‐1 (GLP‐1) has been subject to substantial pharmaceutical research regarding the treatment of type 2 diabetes mellitus. However, quantification of GLP‐1 levels remains complicated due to the low circulation concentration and concurrent existence of numerous metabolites, homologous peptides, and potentially introduced GLP‐1 receptor agonists. Surface plasmon resonance (SPR) facilitates real‐time monitoring allowing a more detailed characterisation of the interaction compared with conventional enzyme‐linked immunosorbent assays (ELISA). In this paper, we describe the development of the first SPR assays for characterisation of anti‐GLP‐1 antibodies for ELISA purposes. Binding responses were obtained on covalently immobilised anti‐GLP‐1 antibodies at 12°C, 25°C, and 40°C and fitted to a biomolecular (1:1) interaction model showing association rates of 1.01 × 10³ to 4.54 × 10³ M^?1 s^?1 and dissociation rates of 3.56 × 10^?5 to 1.56 × 10^?3 s^?1 leading to affinities of 35.2 to 344 nM, depending on the temperature. Determination of thermodynamic properties revealed an enthalpy driven interaction (ΔH < ΔS < 0) with higher affinities at lower temperatures due to the formation and stabilisation of hydrogen bonds within the binding site primarily composed of polar amino acids (ΔC_p < 0). Pair‐wise epitope mapping was performed on captured anti‐GLP‐1 antibodies followed by subsequent interaction with GLP‐1 (7‐36) and other anti‐GLP‐1 antibodies. A global evaluation of every binding response led to an epitope map elucidating the potential of various anti‐GLP‐1 antibody pairs for sandwich ELISA and hence pinpointing the optimal antibody combinations. The SPR assays proved capable of providing vital information for ELISA development endorsing it as a useful optimisation tool.     

Hyperglycemia induces NF‐κB activation and MCP‐1 expression via downregulating GLP‐1R expression in rat mesangial cells: inhibition by metformin

Hyperglycemia impairs glucagon‐like peptide‐1 receptor (GLP‐1R) signaling in multiple cell types and thereby potentially attenuates the therapeutic effects of GLP‐1R agonists. We hypothesized that the downregulation of GLP‐1R by hyperglycemia might reduce the renal‐protective effects of GLP‐1R agonists in diabetic nephropathy (DN). In this study, we examined the effects of high glucose on the expression of GLP‐1R and its signaling pathways in the HBZY‐1 rat mesangial cell line. We found that high glucose reduced GLP‐1R messenger RNA (mRNA) levels in HBZY‐1 cells and in the renal cortex in db/db mice comparing with control groups. In consistence, GLP‐1R agonist exendin‐4 induced CREB phosphorylation was attenuated by high glucose but not low glucose treatment, which is paralleled with abrogated anti‐inflammatory functions in HBZY‐1 cells linked with nuclear factor‐κB (NF‐κB) activation. In consistence, GLP‐1R inhibition aggravated the high glucose‐induced activation of NF‐κB and MCP‐1 protein levels in cultured HBZY‐1 cells while overexpression of GLP‐1R opposite effects. We further proved that metformin restored high glucose‐inhibited GLP‐1R mRNA expression and decreased high glucose evoked inflammation in HBZY‐1 cells. On the basis of these findings, we conclude that high glucose lowers GLP‐1R expression and leads to inflammatory responses in mesangial cells, which can be reversed by metformin. These data support the rationale of combinative therapy of metformin with GLP‐1R agonists in DN.     

Sitagliptin improves functional recovery via GLP‐1R‐induced anti‐apoptosis and facilitation of axonal regeneration after spinal cord injury

Axon growth and neuronal apoptosis are considered to be crucial therapeutic targets against spinal cord injury (SCI). Growing evidences have reported stimulation of glucagon‐like peptide‐1 (GLP‐1)/GLP‐1 receptor (GLP‐1R) signalling axis provides neuroprotection in experimental models of neurodegeneration disease. Endogenous GLP‐1 is rapidly degraded by dipeptidyl peptidase‐IV (DPP4), resulting in blocking of GLP‐1/GLP1R signalling process. Sitagliptin, a highly selective inhibitor of DPP4, has approved to have beneficial effects on diseases in which neurons damaged. However, the roles and the underlying mechanisms of sitagliptin in SCI repairing remain unclear. In this study, we used a rat model of SCI and PC12 cells/primary cortical neurons to explore the mechanism of sitagliptin underlying SCI recovery. We discovered the expression of GLP‐1R decreased in the SCI model. Administration of sitagliptin significantly increased GLP‐1R protein level, alleviated neuronal apoptosis, enhanced axon regeneration and improved functional recovery following SCI. Nevertheless, treatment with exendin9‐39, a GLP‐1R inhibitor, remarkably reversed the protective effect of sitagliptin. Additionally, we detected the AMPK/PGC‐1α signalling pathway was activated by sitagliptin stimulating GLP‐1R. Taken together, sitagliptin may be a potential agent for axon regrowth and locomotor functional repair via GLP‐1R‐induced AMPK/ PGC‐1α signalling pathway after SCI.     

Neuroprotective actions of Glucagon‐like peptide‐1 in differentiated human neuroprogenitor cells

Glucagon‐like peptide‐1 (GLP‐1)‐based therapies are currently available for the treatment of type 2 diabetes, based on their actions on pancreatic β cells. GLP‐1 is also known to exert neuroprotective actions. To determine its mechanism of action, we developed a neuron‐rich cell culture system by differentiating human neuroprogenitor cells in the presence of a combination of neurotrophins and retinoic acid. The neuronal nature of these cells was characterized by neurogenesis pathway‐specific array. GLP‐1 receptor expression was seen mainly in the neuronal population. Culture of neurons in the presence of Aβ oligomers resulted in the induction of apoptosis as shown by the activation of caspase‐3 and caspase‐6. Exendin‐4, a long‐acting analog of GLP‐1, protected the neurons from apoptosis induced by Aβ oligomers. Exendin‐4 stimulated cyclic AMP response element binding protein phosphorylation, a regulatory step in its activation. A transient transfection assay showed induction of a reporter linked to CRE site‐containing human brain‐derived neurotrophic factor promoter IV, by the growth factor through multiple signaling pathways. The anti‐apoptotic action of exendin‐4 was lost following down‐regulation of cAMP response element binding protein. Withdrawal of neurotrophins resulted in the loss of neuronal phenotype of differentiated neuroprogenitor cells, which was prevented by incubation in the presence of exendin‐4. Diabetes is a risk factor in the pathogenesis of Alzheimer's disease. Our findings suggest that GLP‐1‐based therapies can decrease the incidence of Alzheimer's disease among aging diabetic population.     

Functional association of the N‐terminal residues with the central region in glucagon‐related peptides

GLP‐1 is an incretin peptide involved in the regulation of glucose metabolism and the glucose‐dependent stimulation of insulin secretion. Ex‐4 is a paralog of GLP‐1 that has comparable GLP‐1R potency but extended physiological action. GLP‐1 and Ex‐4 are helical peptides that share ～50% sequence homology but differ at several residues, notably the second amino acid which controls susceptibility to DPP‐IV cleavage. This single amino acid difference yields divergent receptor potency when studied in the context of the two hormone sequences. Ex‐4 uniquely tolerates Gly2 through select amino acid differences in the middle region of the peptide that are absent in GLP‐1. We report that substitution of Ex‐4 amino acids Glu16, Leu21, and Glu24 to the GLP‐1 sequence enabled Gly2 tolerance. The coordination of the N‐terminus with these central residues shows an interaction of substantial importance not only to DPP‐IV stability but also to receptor activation. Extension of this observation to glucagon‐based co‐agonist peptides showed different structural requirements for effective communication between the N‐terminus and the mid‐section of these peptides in achieving high potency agonism at the GLP‐1 and GCGRs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A fusion protein derived from plants holds promising potential as a new oral therapy for type 2 diabetes

The incretin hormone glucagon‐like peptide‐1 (GLP‐1) is recognized as a promising candidate for the treatment of type 2 diabetes (T2D), with one of its mimetics, exenatide (synthetic exendin‐4) having already been licensed for clinical use. We seek to further improve the therapeutic efficacy of exendin‐4 (Ex‐4) using innovative fusion protein technology. Here, we report the production in plants a fusion protein containing Ex‐4 coupled with human transferrin (Ex‐4‐Tf) and its characterization. We demonstrated that plant‐made Ex‐4‐Tf retained the activity of both proteins. In particular, the fusion protein stimulated insulin release from pancreatic β‐cells, promoted β‐cell proliferation, stimulated differentiation of pancreatic precursor cells into insulin‐producing cells, retained the ability to internalize into human intestinal cells and resisted stomach acid and proteolytic enzymes. Importantly, oral administration of partially purified Ex‐4‐Tf significantly improved glucose tolerance, whereas commercial Ex‐4 administered by the same oral route failed to show any significant improvement in glucose tolerance in mice. Furthermore, intraperitoneal (IP) injection of Ex‐4‐Tf showed a beneficial effect in mice similar to IP‐injected Ex‐4. We also showed that plants provide a robust system for the expression of Ex‐4‐Tf, producing up to 37 μg prEx‐4‐Tf/g fresh leaf weight in transgenic tobacco and 137 μg prEx‐4‐Tf/g freshweight in transiently transformed leaves of N. benthamiana. These results indicate that Ex‐4‐Tf holds substantial promise as a new oral therapy for type 2 diabetes. The production of prEx‐4‐Tf in plants may offer a convenient and cost‐effective method to deliver the antidiabetic medicine in partially processed plant food products.     

Novel signal transduction and peptide specificity of glucagon-like peptide receptor in 3T3-L1 adipocytes

Glucagon-like peptide-1 (7–36) amide (GLP-1), in addition to its well known effect of enhancing glucose-mediated insulin release, has been shown to have insulinomimetic effects and to enhance insulin-mediated glucose uptake and lipid synthesis in 3T3-L1 adipocytes. To elucidate the mechanisms of GLP-1 action in these cells, we studied the signal transduction and peptide specificity of the GLP-1 response. In 3T3-L1 adipocytes, GLP-1 caused a decrease in intracellular cAMP levels which is the opposite to the response observed in pancreatic beta cells in response to the same peptide. In 3T3-L1 adipocytes, free intracellular calcium was not modified by GLP-1. Peptide specificity was examined to help determine if a different GLP receptor isoform was expressed in 3T3-L1 adipocytes vs. beta cells. Peptides with partial homology to GLP-1 such as GLP-2, GLP-1 (1–36), and glucagon all lowered cAMP levels in 3T3-L1 adipocytes. In addition, an antagonist of pancreatic GLP-1 receptor, exendin-4 (9–39), acted as an agonist to decrease cAMP levels in 3T3-L1 adipocytes as did exendin-4 (1–39), a known agonist for the pancreatic GLP-1 receptor. Binding studies using ¹²⁵I-GLP-1 also suggest that pancreatic GLP-1 receptor isoform is not responsible for the effect of GLP-1 and related peptides in 3T3-L1 adipocytes. Based on these results, we propose that the major form of the GLP receptor in 3T3-L1 adipocytes is functionally different from the pancreatic GLP-1 receptor. J. Cell. Physiol. 172:275–283, 1997. Published 1997 Wiley-Liss, Inc.

1 This article was prepared by a group of United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

     

Glucagon‐like peptide‐1 ameliorates cardiac lipotoxicity in diabetic cardiomyopathy via the PPARα pathway

Lipotoxicity cardiomyopathy is the result of excessive accumulation and oxidation of toxic lipids in the heart. It is a major threat to patients with diabetes. Glucagon‐like peptide‐1 (GLP‐1) has aroused considerable interest as a novel therapeutic target for diabetes mellitus because it stimulates insulin secretion. Here, we investigated the effects and mechanisms of the GLP‐1 analog exendin‐4 and the dipeptidyl peptidase‐4 inhibitor saxagliptin on cardiac lipid metabolism in diabetic mice (DM). The increased myocardial lipid accumulation, oxidative stress, apoptosis, and cardiac remodeling and dysfunction induced in DM by low streptozotocin doses and high‐fat diets were significantly reversed by exendin‐4 and saxagliptin treatments for 8 weeks. We found that exendin‐4 inhibited abnormal activation of the (PPARα)‐CD36 pathway by stimulating protein kinase A (PKA) but suppressing the Rho‐associated protein kinase (ROCK) pathway in DM hearts, palmitic acid (PA)‐treated rat h9c2 cardiomyocytes (CMs), and isolated adult mouse CMs. Cardioprotection in DM mediated by exendin‐4 was abolished by combination therapy with the PPARα agonist wy‐14643 but mimicked by PPARα gene deficiency. Therefore, the PPARα pathway accounted for the effects of exendin‐4. This conclusion was confirmed in cardiac‐restricted overexpression of PPARα mediated by adeno‐associated virus serotype‐9 containing a cardiac troponin T promoter. Our results provide the first direct evidence that GLP‐1 protects cardiac function by inhibiting the ROCK/PPARα pathway, thereby ameliorating lipotoxicity in diabetic cardiomyopathy.     

PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis

The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3-E1 preosteoblast-like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and β-glycerophosphate, and samples were collected from 0–26 days of culture. DNA levels as determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-E1 and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3-E1 and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using ¹²⁵I-PTHrP. The numbers of receptors per μg DNA were greatest at days 3 and 5 for MC3T3-E1 and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP-stimulated cAMP levels increased 5-fold to peak values at day 5 for MC3T3-E1 cells and 6-fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH-dependent cAMP response since ascorbic acid-treated MC3T3-E1 cells had twice the PTH-stimulated cAMP levels as non-treated cells. When the collagen synthesis inhibitor 3,4-dehydroproline was administered to MC3T3-E1 cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix-producing osteoblastic cell in vitro. © 1996 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.     

A novel peptide Phylloseptin‐PBu from Phyllomedusa burmeisteri possesses insulinotropic activity via potassium channel and GLP‐1 receptor signalling

Insulin, as one of the most important hormones regulating energy metabolism, plays an essential role in maintaining glucose and lipid homeostasis in vivo. Failure or insufficiency of insulin secretion from pancreatic beta‐cells increases glucose and free fatty acid level in circulation and subsequently contributes to the emergence of hyperglycaemia and dyslipidaemia. Therefore, stimulating the insulin release benefits the treatment of type 2 diabetes and obesity significantly. Frog skin peptides have been extensively studied for their biological functions, among which, Phylloseptin peptides discovered in Phyllomedusinae frogs have been found to exert antimicrobial, antiproliferative and insulinotropic activities, while the mechanism associated with Phylloseptin‐induced insulin secretion remains elusive. In this study, we reported a novel peptide named Phylloseptin‐PBu, isolated and identified from Phyllomedusa burmeisteri, exhibited dose‐dependent insulinotropic property in rat pancreatic beta BRIN‐BD11 cells without altering cell membrane integrity. Further mechanism investigations revealed that Phylloseptin‐PBu‐induced insulin output is predominantly modulated by K_ATP‐[K⁺] channel depolarization triggered extracellular calcium influx and GLP‐1 receptor initiated PKA signalling activation. Overall, our study highlighted that this novel Phylloseptin‐PBu peptide has clear potential to be developed as a potent antidiabetic agent with established function‐traced mechanism and low risk of cytotoxicity.     

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.     

Fluid shear stress induces Runx‐2 expression via upregulation of PIEZO1 in MC3T3‐E1 cells

Mechanically induced biological responses in bone cells involve a complex biophysical process. Although various mechanosensors have been identified, the precise mechanotransduction pathway remains poorly understood. PIEZO1 is a newly discovered mechanically activated ion channel in bone cells. This study aimed to explore the involvement of PIEZO1 in mechanical loading (fluid shear stress)‐induced signaling cascades that control osteogenesis. The results showed that fluid shear stress increased PIEZO1 expression in MC3T3‐E1 cells. The fluid shear stress elicited the key osteoblastic gene Runx‐2 expression; however, PIEZO1 silencing using small interference RNA blocked these effects. The AKT/GSK‐3β/β‐catenin pathway was activated in this process. PIEZO1 silencing impaired mechanically induced activation of the AKT/GSK‐3β/β‐catenin pathway. Therefore, the results demonstrated that MC3T3‐E1 osteoblasts required PIEZO1 to adapt to the external mechanical fluid shear stress, thereby inducing osteoblastic Runx‐2 gene expression, partly through the AKT/GSK‐3β/β‐catenin pathway.     

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.     

Stimulatory effect of a phorbol ester on expression of insulin-like growth factor (IGF) binding protein-2 and level of IGF-I receptors in mouse osteoblastic MC3T3-E1 cells

We examined the relationship between signal transduction and the expression of insulin-like growth factor I (IGF-I), IGF-I receptor level, and IGF binding proteins (IGFBPs) in murine clonal osteoblastic MC3T3-E1 cells. 12–O-Tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, decreased the secretion of immunoreactive IGF-I into the medium, whereas dibutyryl cAMP (Bt₂cAMP) augmented the secretion In contrast, TPA increased the level of type IIGF receptor on the cells. Furthermore, MC3T3-E1 cells produced and secreted at least three different IGFBPs with molecular masses of 24, 30, and 34 kDa, and the 24-kDa IGFBP was predominant under normal conditions. However, TPA specifically increased the secretion of the 34-kDa IGFBP. The N-terminal amino acid sequence of the purified 34-kDa IGFBP was nearly identical with that of rat IGFBP-2. Furthermore, the 34-kDa IGFBP was immunoreactive to anti-IGFBP-2 antiserum. The level of IGFBP-2 mRNA in the cells was increased by TPA, indicating that the increase in IGFBP-2 secretion results from the stimulation of IGFBP-2 production. In contrast, Bt₂cAMP affected neither IGF-l receptor number nor the IGFBP secretion. These results indicate that the production of IGF-l and the expression of IGF-l receptors and IGFBP-2 are up-regulated by the activation of adenylate cyclase and protein kinase C, respectively, in osteoblastic MC3T3-E1 cells. © 1994 Willey-Liss, Inc.     

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.