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.     

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.     

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.     

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.     

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.     

Cardiac‐specific Mst1 deficiency inhibits ROS‐mediated JNK signalling to alleviate Ang II‐induced cardiomyocyte apoptosis

Apoptosis is associated with various myocardial diseases. Angiotensin II (Ang II) plays a central role in the pathogenesis of RAAS‐triggered cardiac apoptosis. Our previous studies showed that mammalian Ste20‐like kinase 1 (Mst1) aggravates cardiac dysfunction in cardiomyocyte under pathological conditions, but its role in Ang II‐mediated cardiomyocyte apoptosis is not known. We addressed this in the present study by investigating whether cardiac‐specific Mst1 knockout can alleviate Ang II‐induced cardiomyocyte apoptosis along with the underlying mechanisms. In vitro and in vivo experiments showed that Ang II increased intracellular reactive oxygen species (ROS) production and cardiomyocyte apoptosis; these were reversed by administration of the ROS scavenger N‐acetylcysteine and by Mst1 deficiency, which suppressed c‐Jun N‐terminal kinase (JNK) phosphorylation and downstream signaling. Interestingly, Mst1 knockout failed to alleviate Ang II‐induced phosphorylation of extracellular signal‐regulated kinase 1/2, and inactivated apoptosis signal‐regulating kinase1 (ASK1) by promoting its association with thioredoxin (Trx), which reversed the Ang II‐induced activation of the ASK1–JNK pathway and suppressed Ang II‐induced cardiomyocyte apoptosis. Thus, cardiac‐specific Mst1 knockout inhibits ROS‐mediated JNK signalling to block Ang II‐induced cardiomyocyte apoptosis, suggesting Mst1 as a potential therapeutic target for treatment of RAAS‐activated heart failure.     

Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet‐like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five‐step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti‐hyperglycaemic hormone‐Glp1 (glucagon‐like peptide 1) analogue Liraglutide with prolonged t_½ and Exendin 4. The differentiated islet‐like 3D clusters expressed bonafide mature and functional β‐cell markers‐PDX1 (pancreatic and duodenal homoeobox‐1), C‐peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C‐peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non‐obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long‐term in vivo functionally viable islets from hESC.     

Deletion of pancreatic β‐cell adenosine kinase improves glucose homeostasis in young mice and ameliorates streptozotocin‐induced hyperglycaemia

Severe reduction in the β‐cell number (collectively known as the β‐cell mass) contributes to the development of both type 1 and type 2 diabetes. Recent pharmacological studies have suggested that increased pancreatic β‐cell proliferation could be due to specific inhibition of adenosine kinase (ADK). However, genetic evidence for the function of pancreatic β‐cell ADK under physiological conditions or in a pathological context is still lacking. In this study, we crossed mice carrying LoxP‐flanked Adk gene with Ins2‐Cre mice to acquire pancreatic β ‐cell ADK deficiency (Ins2‐Cre^±Adk^fl/fl) mice. Our results revealed that Ins2‐Cre^+/‐Adk^fl/fl mice showed improved glucose metabolism and β‐cell mass in younger mice, but showed normal activity in adult mice. Moreover, Ins2‐Cre^±Adk^fl/fl mice were more resistant to streptozotocin (STZ) induced hyperglycaemia and pancreatic β‐cell damage in adult mice. In conclusion, we found that ADK negatively regulates β‐cell replication in young mice as well as under pathological conditions, such as STZ induced pancreatic β‐cell damage. Our study provided genetic evidence that specific inhibition of pancreatic β‐cell ADK has potential for anti‐diabetic therapy.     

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.     

Role and impact of the extracellular matrix on integrin‐mediated pancreatic β‐cell functions

Understanding the organisation and role of the extracellular matrix (ECM) in islets of Langerhans is critical for maintaining pancreatic β‐cells, and to recognise and revert the physiopathology of diabetes. Indeed, integrin‐mediated adhesion signalling in response to the pancreatic ECM plays crucial roles in β‐cell survival and insulin secretion, two major functions, which are affected in diabetes. Here, we would like to present an update on the major components of the pancreatic ECM, their role during integrin‐mediated cell‐matrix adhesions and how they are affected during diabetes. To treat diabetes, a promising approach consists in replacing β‐cells by transplantation. However, efficiency is low, because β‐cells suffer of anoikis, due to enzymatic digestion of the pancreatic ECM, which affects the survival of insulin‐secreting β‐cells. The strategy of adding ECM components during transplantation, to reproduce the pancreatic microenvironment, is a challenging task, as many of the regulatory mechanisms that control ECM deposition and turnover are not sufficiently understood. A better comprehension of the impact of the ECM on the adhesion and integrin‐dependent signalling in β‐cells is primordial to improve the healthy state of islets to prevent the onset of diabetes as well as for enhancing the efficiency of the islet transplantation therapy.     

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.     

β‐Carotene Induces Apoptosis in Human Esophageal Squamous Cell Carcinoma Cell Lines via the Cav‐1/AKT/NF‐κB Signaling Pathway

β‐carotene, a type of terpenoid, has many metabolic and physiological functions. In particular, β‐carotene has an antitumor effect. However, the efficacy of β‐carotene against esophageal squamous cell carcinoma (ESCC) remains unclear. In our study, β‐carotene inhibited the growth of ESCC cells and downregulated expression of the Caveolin‐1 (Cav‐1) protein. Cav‐1 protein was expressed only in ESCC cells, not in Het‐1A cells. Moreover, β‐carotene triggered apoptosis, induced cell cycle G0?G1 phase arrest, and inhibited cell migration. To explore the mechanism involved in these processes, we further examined the effect of β‐carotene on the Cav‐1‐mediated AKT/NF‐κB pathway. The results showed that the level of AKT and NF‐κB phosphorylation was dramatically inhibited, which led to an increase in the Bax/Bcl‐2 ratio. Correspondingly, the activity of Caspase‐3 was also enhanced. These data suggest that β‐carotene has an antiproliferative role in ESCC cells and may be a promising chemotherapeutic agent for use against ESCC cells.     

A new paradigm in cell therapy for diabetes: Turning pancreatic α‐cells into β‐cells

Cell therapy means treating diseases with the body's own cells. One of the cell types most in demand for therapeutic purposes is the pancreatic β‐cell. This is because diabetes is one of the major healthcare problems in the world. Diabetes can be treated by islet transplantation but the major limitation is the shortage of organ donors. To overcome the shortfall in donors, alternative sources of pancreatic β‐cells must be found. Potential sources include embryonic or adult stem cells or, from existing β‐cells. There is now a startling new addition to this list of therapies: the pancreatic α‐cell. Thorel and colleagues recently showed that under circumstances of extreme pancreatic β‐cell loss, α‐cells may serve to replenish the insulin‐producing compartment. This conversion of α‐cells to β‐cells represents an example of transdifferentiation. Understanding the molecular basis for transdifferentiation may help to enhance the generation of β‐cells for the treatment of diabetes.     

N‐cadherin is dispensable for pancreas development but required for β‐cell granule turnover

The cadherin family of cell adhesion molecules mediates adhesive interactions that are required for the formation and maintenance of tissues. Previously, we demonstrated that N‐cadherin, which is required for numerous morphogenetic processes, is expressed in the pancreatic epithelium at E9.5, but later becomes restricted to endocrine aggregates in mice. To study the role of N‐cadherin during pancreas formation and function we generated a tissue‐specific knockout of N‐cadherin in the early pancreatic epithelium by inter‐crossing N‐cadherin‐floxed mice with Pdx1Cre mice. Analysis of pancreas‐specific ablation of N‐cadherin demonstrates that N‐cadherin is dispensable for pancreatic development, but required for β‐cell granule turnover. The number of insulin secretory granules is significantly reduced in N‐cadherin‐deficient β‐cells, and as a consequence insulin secretion is decreased. genesis 48:374–381, 2010. © 2010 Wiley‐Liss, Inc.