Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments.

Bacteria equipped with genetically encoded lactate biosensors are promising tools for biopharmaceutical production, diagnostics, and cellular therapies. However, many applications involve glucose-rich and anoxic environments, in which current whole-cell lactate biosensors show low performance. Here we engineer an optimized, synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite and anoxic repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expressions to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia), can operate in glucose-rich, aerobic and anoxic conditions. We show that ALPaGA works reliably in the probiotic chassisEscherichia coliNissle 1917 and can detect endogenous l-lactate produced by 3D tumor spheroids with an improved dynamic range. In the future, the ALPaGA system could be used to monitor bioproduction processes and improve the specificity of engineered bacterial cancer therapies by restricting their activity to the lactate-rich microenvironment of solid tumors.

Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring.

Stimulation of numerous G protein-coupled receptors leads to the elevation of intracellular concentrations of cAMP, which subsequently activates the PKA pathway. Specificity of the PKA signaling module is determined by a sophisticated subcellular targeting network that directs the spatiotemporal activation of the kinase. This specific compartmentalization mechanism occurs through high-affinity interactions of PKA with A-kinase anchoring proteins (AKAPs), the role of which is to target the kinase to discrete subcellular microdomains. Recently, a peptide designated "AKAPis" has been proposed to competitively inhibit PKA-AKAP interactions in vitro. We therefore sought to characterize a cell-permeable construct of the AKAPis inhibitor and use it as a tool to characterize the impact of PKA compartmentalization by AKAPs. Using insulin-secreting pancreatic beta-cells (INS-1 cells), we showed that TAT-AKAPis (at a micromolar range) dose dependently disrupted a significant fraction of endogenous PKA-AKAP interactions. Immunoflurescent analysis also indicated that TAT-AKAPis significantly affected PKA subcellular localization. Furthermore, TAT-AKAPis markedly attenuated glucagon-induced phosphorylations of p44/p42 MAPKs and cAMP response element binding protein, which are downstream effectors of PKA. In parallel, TAT-AKAPis dose dependently inhibited the glucagon-induced potentiation of insulin release. Therefore, AKAP-mediated subcellular compartmentalization of PKA represents a key mechanism for PKA-dependent phosphorylation events and potentiation of insulin secretion in intact pancreatic beta-cells. More interestingly, our data highlight the effectiveness of the cell-permeable peptide-mediated approach to monitoring in cellulo PKA-AKAP interactions and delineating PKA-dependent phosphorylation events underlying specific cellular responses.

Adult muscle-derived stem cells engraft and differentiate into insulin-expressing cells in pancreatic islets of diabetic mice.

BACKGROUND: Pancreatic beta cells are unique effectors in the control of glucose homeostasis and their deficiency results in impaired insulin production leading to severe diabetic diseases. Here, we investigated the potential of a population of nonadherent muscle-derived stem cells (MDSC) from adult mouse muscle to differentiate in vitro into beta cells when transplanted as undifferentiated stem cells in vivo to compensate for beta-cell deficiency. RESULTS: In vitro, cultured MDSC spontaneously differentiated into insulin-expressing islet-like cell clusters as revealed using MDSC from transgenic mice expressing GFP or mCherry under the control of an insulin promoter. Differentiated clusters of beta-like cells co-expressed insulin with the transcription factors Pdx1, Nkx2.2, Nkx6.1, and MafA, and secreted significant levels of insulin in response to glucose challenges. In vivo, undifferentiated MDSC injected into streptozotocin (STZ)-treated mice engrafted within 48 h specifically to damaged pancreatic islets and were shown to differentiate and express insulin 10-12 days after injection. In addition, injection of MDSC into hyperglycemic diabetic mice reduced their blood glucose levels for 2-4 weeks. CONCLUSION: These data show that MDSC are capable of differentiating into mature pancreatic beta islet-like cells, not only upon culture in vitro, but also in vivo after systemic injection in STZ-induced diabetic mouse models. Being nonteratogenic, MDSC can be used directly by systemic injection, and this potential reveals a promising alternative avenue in stem cell-based treatment of beta-cell deficiencies.

Type II PKAs are anchored to mature insulin secretory granules in INS-1 ß-cells and required for cAMP-dependent potentiation of exocytosis.

Specificity of the cAMP-dependent protein kinase (PKA) pathway relies on an extremely sophisticated compartmentalization mechanism of the kinase within a given cell, based on high-affinity binding of PKA tetramer pools to different A-Kinase Anchoring Proteins (AKAPs). We and others have previously shown that AKAPs-dependent PKA subcellular targeting is a requisite for optimal cAMP-dependent potentiation of insulin exocytosis. We thus hypothesized that a PKA pool may directly anchor to the secretory compartment to potentiate insulin exocytosis. Here, using immunofluorescence analyses combined to subcellular fractionations and purification of insulin secretory granules (ISGs), we identified discrete subpools of type II PKAs, RIIα and RIIß PKAs, along with the catalytic subunit, physically associated with ISGs within pancreatic insulin-secreting ß-cells. Ultrastructural analysis of native rodent ß-cells confirmed in vivo the occurrence of PKA on dense-core ISGs. Isoform-selective disruption of binding of PKAs to AKAPs reinforced the requirement of type II PKA isoforms for cAMP potentiation of insulin exocytosis. This granular localization of PKA was of critical importance since siRNA-mediated depletion of either RIIα or RIIß PKAs resulted in a significant reduction of cAMP-dependent potentiation of insulin release. The present work provides evidence for a previously unrecognized pool of type II PKAs physically anchored to the ß-cell ISGs compartment and supports a non-redundant function for type II PKAs during cAMP potentiation of exocytosis.

Extracellularly regulated kinases 1/2 (p44/42 mitogen-activated protein kinases) phosphorylate synapsin I and regulate insulin secretion in the MIN6 beta-cell line and islets of Langerhans.

The p44/p42 MAPKs (ERK1/2) cascade regulates beta-cell nuclear events, which modulates cell differentiation and gene transcription, whereas its implication in processes occurring in the cytoplasm, such as activation of the exocytotic machinery, is still unclear. Using the MIN6 beta-cell line and isolated rat islets of Langerhans, we investigated whether glucose, by activating the ERK1/2 cascade, induces phosphorylation of cytoplasmic proteins implicated in exocytosis of insulin granules such as synapsin I. We observed that the majority of ERK1/2 activity induced by glucose remains in the cytoplasm and physically interacts with synapsin I, allowing phosphorylation of the substrate. Therefore, we reexamined the potential requirement of ERK1/2 for insulin secretion. Blocking activation of ERK1/2 using MEK1/2, the MAPK kinase inhibitor PD98059 or using small interfering RNA-mediated silencing of ERK1 and ERK2 expressions resulted in partial inhibition of glucose-induced insulin release, indicating that ERK1/2 pathway participates also in the regulation of insulin secretion. Moreover, using the pancreatic islet perifusion model, we found that the ERK1/2 activity participates in the first and second phases of insulin release induced by glucose. Taken together, our results demonstrate new aspects of the glucose-dependent actions of ERK1/2 in beta-cells exerted on cytoplasmic proteins, including synapsin I, and participating in the overall glucose-induced insulin secretion.

Cooperative effects between protein kinase A and p44/p42 mitogen-activated protein kinase to promote cAMP-responsive element binding protein activation after beta cell stimulation by glucose and its alteration due to glucotoxicity.

Long-term hyperglycemia, a major characteristic of the diabetic state, contributes to the deterioration of the beta cell function, a concept known as beta cell glucotoxicity. We used the MIN6 beta cell line and isolated rat islets to clarify the signaling mechanism(s) used by glucose to activate cAMP-responsive element binding protein (CREB), a transcription factor crucial for beta cell biology, and to evaluate the possible downregulation of this mechanism mediated by long-term hyperglycemia. We report that glucose (10 mM) induces an increase in cytosolic calcium concentration that leads to cAMP-induced protein kinase A (PKA) activation, promoting nuclear translocation of activated ERK1/2. The observation that glucose-induced CREB phosphorylation was totally inhibited by the PKA inhibitor H89 (2 microM) and reduced by 50% with the ERK1/2 inhibitor PD98059 (20 microM) indicates that ERK1/2, located downstream of PKA, cooperates with PKA and is responsible for half of the PKA-mediated CREB phosphorylation elicited by glucose in MIN6 beta cells. We also found that exposure of mu cells for 24 h to high glucose (25 mM) induced a 70% decrease in cellular ERK1/2 and a 50% decrease in CREB content. In high-glucose-treated, ERK1/2- and CREB-downregulated beta cells, there was a loss of glucose (10 mM, 5 min)-stimulated ERK1/2 and CREB phosphorylation that was associated with nuclear apoptotic characteristics. Since we have shown that activation of ERK1/2 is crucial for CREB phosphorylation, loss of the ERK1/2-CREB signaling pathway in beta cells due to long-term hyperglycemia is likely to exacerbate beta cell failure in diabetic states by affecting physiologically relevant gene expression and by inducing apoptosis.

Glucagon promotes cAMP-response element-binding protein phosphorylation via activation of ERK1/2 in MIN6 cell line and isolated islets of Langerhans.

By using the MIN6 cell line and pancreatic islets, we show that in the presence of a low glucose concentration, corresponding to physiological glucagon release from alpha cells, glucagon treatment of the beta cell caused a rapid, time-dependent phosphorylation and activation of p44/p42 mitogen-activated protein kinase (ERK1/2) independently from extracellular calcium influx. Inhibition of either cAMP-dependent protein kinase (PKA) or MEK completely blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Shc-p21(Ras) and phosphatidylinositol 3-kinase, was observed in response to glucagon treatment. Chelation of intracellular calcium (intracellular [Ca(2+)]) reduced glucagon-mediated ERK1/2 activation. In addition, internalization of glucagon receptors through clathrin-coated pits formation is required for ERK1/2 activation. Remarkably, glucagon promotes the nuclear translocation of ERK1/2 and induces the phosphorylation of cAMP-response element-binding protein (CREB). Miniglucagon, produced from glucagon and released together with the mother hormone from the alpha cells in low glucose situations, blocks the insulinotropic effect of glucagon, whereas it does not inhibit the glucagon-induced PKA/ERK1/2/CREB pathway. We conclude that glucagon-induced ERK1/2 activation is mediated by PKA and that an increase in [Ca(2+)](i) is required for maximal ERK activation. Our results uncover a novel mechanism by which the PKA/ERK1/2 signaling network engaged by glucagon, in situation of low glucose concentration, regulates phosphorylation of CREB, a transcription factor crucial for normal beta cell function and survival.