Corrination mitigates peptide aggregation as exemplified for Glucagon.

Pharmaceutical development of glucagon for use in acute hypoglycemia has proved challenging, due in large part to poor solubility, poor stability and aggregate formation. Herein, we describe highly soluble, low aggregating, glucagon conjugates generated through use of the commercially available vitamin B12 precursor dicyanocobinamide ('corrination'), which retain full stimulatory action at the human glucagon receptor. The modified glucagon analogs were tested in a chemical stability assay in 50 mM phosphate buffer and the percentage of original concentration retained was determined after two weeks of incubation at 37° C. Aggregate formation assays were also performed after 48 h of agitation at 37°C using a thioflavin (ThT) fluorescence-based assay. All corrinated compounds retained original concentration to a higher degree than glucagon controls and showed markedly decreased aggregation compared to their respective noncorrinated analogues. Based on the statistically significant increase in chemical stability coupled with the notably decreased tendency to form aggregates, analogues 2 and its corrinated conjugate 5 were used for a functional assay study performed after agitation at 37°C for 24-hr after which agonism was measured at the human glucagon receptor using a cAMP FRET assay. Corrinated 5 exhibited a 6.6-fold increased potency relative to glucagon, which was shown to have a 165-fold reduction in potency. The relative potency of 5 was also improved compared to that of 2 with EC50 values of 5.5 nM and 9.6 nM for 5 and 2, respectively. In conclusion, corrination of peptides mitigates aggregation, presenting a compound with prolonged stability and agonism as demonstrated for glucagon.

A peptide triple agonist of GLP-1, neuropeptide Y1, and neuropeptide Y2 receptors promotes glycemic control and weight loss.

Mechanisms underlying long-term sustained weight loss and glycemic normalization after obesity surgery include changes in gut hormone levels, including glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). We demonstrate that two peptide biased agonists (GEP44 and GEP12) of the GLP-1, neuropeptide Y1, and neuropeptide Y2 receptors (GLP-1R, Y1-R, and Y2-R, respectively) elicit Y1-R antagonist-controlled, GLP-1R-dependent stimulation of insulin secretion in both rat and human pancreatic islets, thus revealing the counteracting effects of Y1-R and GLP-1R agonism. These agonists also promote insulin-independent Y1-R-mediated glucose uptake in muscle tissue ex vivo and more profound reductions in food intake and body weight than liraglutide when administered to diet-induced obese rats. Our findings support a role for Y1-R signaling in glucoregulation and highlight the therapeutic potential of simultaneous receptor targeting to achieve long-term benefits for millions of patients.

GTS-21, a selective alpha7 nicotinic acetylcholine receptor agonist, ameliorates diabetic nephropathy in Leprdb/db mice.

Diabetic nephropathy (DN) is a serious complicating factor in human type 2 diabetes mellitus (T2DM), and it commonly results in end-stage renal disease (ESRD) that requires kidney dialysis. Here, we report that the α7 nicotinic acetylcholine receptor (α7nAChR) agonist GTS-21 exerts a novel anti-inflammatory action to ameliorate DN, as studied using an inbred strain of Leprdb/db mice in which hyperglycemia and obesity co-exist owing to defective leptin receptor (Lepr) signaling. For this analysis, GTS-21 was administered to 10-12 week-old male and female mice as a 4 mg/kg intraperitoneal injection, twice-a-day, for 8 weeks. Kidney function and injury owing to DN were monitored by determination of plasma levels of BUN, creatinine, KIM-1 and NGAL. Histologic analysis of glomerular hypertrophy and mesangial matrix expansion were also used to assess DN in these mice. Concurrently, renal inflammation was assessed by measuring IL-6 and HMGB1, while also quantifying renal cell apoptosis, and apoptotic signaling pathways. We found that Leprdb/db mice exhibited increased markers of BUN, creatinine, NGAL, KIM-1, IL-6, cytochrome C, and HMGB-1. These abnormalities were also accompanied by histologic kidney injury (mesangial matrix expansion and apoptosis). Remarkably, all such pathologies were significantly reduced by GTS-21. Collectively, our results provide new evidence that the α7nAChR agonist GTS-21 has the ability to attenuate diabetes-induced kidney injury. Additional studies are warranted to further investigate the involvement of the vagal cholinergic anti-inflammatory reflex pathway (CAP) in ameliorating diabetic nephropathy.

The alpha-7 nicotinic acetylcholine receptor agonist GTS-21 engages the glucagon-like peptide-1 incretin hormone axis to lower levels of blood glucose in db/db mice.

AIM: To establish if alpha-7 nicotinic acetylcholine receptor (α7nAChR) agonist GTS-21 exerts a blood glucose-lowering action in db/db mice, and to test if this action requires coordinate α7nAChR and GLP-1 receptor (GLP-1R) stimulation by GTS-21 and endogenous GLP-1, respectively. MATERIALS AND METHODS: Blood glucose levels were measured during an oral glucose tolerance test (OGTT) using db/db mice administered intraperitoneal GTS-21. Plasma GLP-1, peptide tyrosine tyrosine 1-36 (PYY1-36), glucose-dependent insulinotropic peptide (GIP), glucagon, and insulin levels were measured by ELISA. A GLP-1R-mediated action of GTS-21 that is secondary to α7nAChR stimulation was evaluated using α7nAChR and GLP-1R knockout (KO) mice, or by co-administration of GTS-21 with the dipeptidyl peptidase-4 inhibitor, sitagliptin, or the GLP-1R antagonist, exendin (9-39). Insulin sensitivity was assessed in an insulin tolerance test. RESULTS: Single or multiple dose GTS-21 (0.5-8.0 mg/kg) acted in a dose-dependent manner to lower levels of blood glucose in the OGTT using 10-14 week-old male and female db/db mice. This action of GTS-21 was reproduced by the α7nAChR agonist, PNU-282987, was enhanced by sitagliptin, was counteracted by exendin (9-39), and was absent in α7nAChR and GLP-1R KO mice. Plasma GLP-1, PYY1-36, GIP, glucagon, and insulin levels increased in response to GTS-21, but insulin sensitivity, body weight, and food intake were unchanged. CONCLUSIONS: α7nAChR agonists improve oral glucose tolerance in db/db mice. This action is contingent to coordinate α7nAChR and GLP-1R stimulation. Thus α7nAChR agonists administered in combination with sitagliptin might serve as a new treatment for type 2 diabetes.

Nonpeptidic Z360-Analogs Tagged with Trivalent Radiometals as Anti-CCK2R Cancer Theranostic Agents: A Preclinical Study.

(1) Background: Theranostic approaches in the management of cholecystokinin subtype 2 receptor (CCK2R)-positive tumors include radiolabeled gastrin and CCK motifs. Moving toward antagonist-based CCK2R-radioligands instead, we herein present three analogs of the nonpeptidic CCK2R-antagonist Z360, GAS1/2/3. Each was conjugated to a different chelator (DOTA, NODAGA or DOTAGA) for labeling with medically relevant trivalent radiometals (e.g., Ga-68, In-111, Lu-177) for potential use as anti-CCK2R cancer agents; (2) Methods: The in vitro properties of the thee analogs were compared in stably transfected HEK293-CCK2R cells. Biodistribution profiles were compared in SCID mice bearing twin HEK293-CCK2R and wtHEK293 tumors; (3) Results: The GAS1/2/3 analogs displayed high CCK2R-affinity (lower nM-range). The radioligands were fairly stable in vivo and selectively targeted the HEK293-CCK2R, but not the CCK2R-negative wtHEK293 tumors in mice. Their overall pharmacokinetic profile was found strongly dependent on the radiometal-chelate. Results could be visualized by SPECT/CT for the [111In]In-analogs; (4) Conclusions: The present study highlighted the high impact of the radiometal-chelate on the end-pharmacokinetics of a new series of Z360-based radioligands, revealing candidates with promising properties for clinical translation. It also provided the impetus for the development of a new class of nonpeptidic radioligands for CCK2R-targeted theranostics of human cancer.

Cyclic AMP-dependent activation of ERK via GLP-1 receptor signalling requires the neuroendocrine cell-specific guanine nucleotide exchanger NCS-RapGEF2.

Cyclic AMP activation of the Rap-Braf-MEK-ERK pathway after signalling initiated by the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), via the Gs -protein coupled receptor (Gs PCR) PAC1, occurs uniquely through the neuritogenic cAMP sensor Rap guanine nucleotide exchange factor 2 (NCS-RapGEF2) in Neuroscreen-1 (NS-1) neuroendocrine cells. We examined the expression of other Family B Gs PCRs in this cell line and assessed cAMP elevation and neuritogenesis after treatment with their cognate peptide ligands. Exposure of NS-1 cells to the VIPR1/2 agonist vasoactive intestinal polypeptide, or the GLP1R agonist exendin-4, did not induce neuritogenesis, or elevation of cAMP, presumably as a result of insufficient receptor protein expression. Vasoactive intestinal polypeptide and exendin-4 did induce neuritogenesis after transduction of human VIPR1, VIPR2 and GLP1R into NS-1 cells. Exendin-4/GLP1R-stimulated neuritogenesis was MEK-ERK-dependent (blocked by U0126), indicating its use of the cAMP→RapGEF2→ERK neuritogenic signalling pathway previously identified for PACAP/PAC1 signalling in NS-1 cells. NCS-RapGEF2 is expressed in the rodent insulinoma cell lines MIN6 and INS-1, as well as in human pancreatic islets. As in NS-1 cells, exendin-4 caused ERK phosphorylation in INS-1 cells. Reduction in RapGEF2 expression after RapGEF2-shRNA treatment reduced exendin-4-induced ERK phosphorylation. Transcriptome analysis of INS-1 cells after 1 hour of exposure to exendin-4 revealed an immediate early-gene response that was composed of both ERK-dependent and ERK-independent signalling targets. We propose that cAMP signalling initiated by glucagon-like peptide 1 (GLP-1) in pancreatic beta cells causes parallel activation of multiple cAMP effectors, including NCS-RapGEF2, Epac and protein kinase A, to separately control various facets of GLP-1 action, including insulin secretion and transcriptional modulation.

Discovery of a stable tripeptide targeting the N-domain of CRF1 receptor.

The corticotropin-releasing factor (CRF) and its CRF1 receptor (CRF1R) play a central role in the maintenance of homeostasis. Malfunctioning of the CRF/CRF1R unit is associated with several disorders, such as anxiety and depression. Non-peptide CRF1R-selective antagonists have been shown to exert anxiolytic and antidepressant effects on experimental animals. However, none of them is in clinical use today because of several side effects, thus demonstrating the need for the development of other more suitable CRF1R antagonists. In an effort to develop novel CRF1R antagonists we designed, synthesized and chemically characterized two tripeptide analogues of CRF, namely (R)-LMI and (S)-LMI, having their Leu either in R (or D) or in S (or L) configuration, respectively. Their design was based on the crystal structure of the N-extracellular domain (N-domain) of CRF1R/CRF complex, using a relevant array of computational methods. Experimental evaluation of the stability of synthetic peptides in human plasma has revealed that (R)-LMI is proteolytically more stable than (S)-LMI. Based on this finding, (R)-LMI was selected for pharmacological characterization. We have found that (R)-LMI is a CRF antagonist, inhibiting (1) the CRF-stimulated accumulation of cAMP in HEK 293 cells expressing the CRF1R, (2) the production of interleukins by adipocytes and (3) the proliferation rate of RAW 264.7 cells. (R)-LMI likely blocked agonist actions by interacting with the N-domain of CRF1R as suggested by data using a constitutively active chimera of CRF1R. We propose that (R)-LMI can be used as an optimal lead compound in the rational design of novel CRF antagonists.

[99mTc]Tc-DGA1, a Promising CCK2R-Antagonist-Based Tracer for Tumor Diagnosis with Single-Photon Emission Computed Tomography.

Radiolabeled gastrin analogues have been proposed for theranostics of cholecystokinin subtype 2 receptor (CCK2R)-positive cancer. Peptide radioligands based on other receptor antagonists have displayed superior pharmacokinetics and higher biosafety than agonists. Here, we present DGA1, a derivative of the nonpeptidic CCK2R antagonist Z-360 carrying an acyclic tetraamine, for [99mTc]Tc labeling. Preclinical comparison of [99mTc]Tc-DGA1 with [99mTc]Tc-DG2 (CCK2R-agonist reference) was conducted in HEK293-CCK2R/CCK2i4svR cells and mice models, qualifying [99mTc]Tc-DGA1 for further study in patients with CCK2R-positive tumors and single-photon emission computed tomography/CT.

Corrination of a GLP-1 Receptor Agonist for Glycemic Control without Emesis.

Glucagon-like peptide-1 receptor (GLP-1R) agonists used to treat type 2 diabetes mellitus often produce nausea, vomiting, and in some patients, undesired anorexia. Notably, these behavioral effects are caused by direct central GLP-1R activation. Herein, we describe the creation of a GLP-1R agonist conjugate with modified brain penetrance that enhances GLP-1R-mediated glycemic control without inducing vomiting. Covalent attachment of the GLP-1R agonist exendin-4 (Ex4) to dicyanocobinamide (Cbi), a corrin ring containing precursor of vitamin B12, produces a "corrinated" Ex4 construct (Cbi-Ex4). Data collected in the musk shrew (Suncus murinus), an emetic mammal, reveal beneficial effects of Cbi-Ex4 relative to Ex4, as evidenced by improvements in glycemic responses in glucose tolerance tests and a profound reduction of emetic events. Our findings highlight the potential for clinical use of Cbi-Ex4 for millions of patients seeking improved glycemic control without common side effects (e.g., emesis) characteristic of current GLP-1 therapeutics.

"A-kinase" regulator runs amok to provide a paradigm shift in cAMP signaling.

The activity of the archetypal protein kinase A (PKA) is typically thought of in regards to the catalytic subunit, which is inhibited by the regulatory subunits in the absence of cAMP. However, it is now reported that one of the regulatory subunit isoforms (PKA-RIα) takes on a function of its own upon binding to cAMP, acting independently of this canonical cAMP signaling mechanism. PKA-RIα instead binds to and stimulates the catalytic activity of a guanine nucleotide exchange factor (P-REX1) that itself promotes Rac1 GTPase activation. This newly discovered function of PKA-RIα adds an additional layer of complexity to our understanding of cAMP signal transduction.

Nonconventional glucagon and GLP-1 receptor agonist and antagonist interplay at the GLP-1 receptor revealed in high-throughput FRET assays for cAMP.

G protein-coupled receptors (GPCRs) for glucagon (GluR) and glucagon-like peptide-1 (GLP-1R) are normally considered to be highly selective for glucagon and GLP-1, respectively. However, glucagon secreted from pancreatic α-cells may accumulate at high concentrations to exert promiscuous effects at the ß-cell GLP-1R, as may occur in the volume-restricted microenvironment of the islets of Langerhans. Furthermore, systemic administration of GluR or GLP-1R agonists and antagonists at high doses may lead to off-target effects at other receptors. Here, we used molecular modeling to evaluate data derived from FRET assays that detect cAMP as a read-out for GluR and GLP-1R activation. This analysis established that glucagon is a nonconventional GLP-1R agonist, an effect inhibited by the GLP-1R orthosteric antagonist exendin(9-39) (Ex(9-39)). The GluR allosteric inhibitors LY2409021 and MK 0893 antagonized glucagon and GLP-1 action at the GLP-1R, whereas des-His1-[Glu9]glucagon antagonized glucagon action at the GluR, while having minimal inhibitory action versus glucagon or GLP-1 at the GLP-1R. When testing Ex(9-39) in combination with des-His1-[Glu9]glucagon in INS-1 832/13 cells, we validated a dual agonist action of glucagon at the GluR and GLP-1R. Hybrid peptide GGP817 containing glucagon fused to a fragment of peptide YY (PYY) acted as a triagonist at the GluR, GLP-1R, and neuropeptide Y2 receptor (NPY2R). Collectively, these findings provide a new triagonist strategy with which to target the GluR, GLP-1R, and NPY2R. They also provide an impetus to reevaluate prior studies in which GluR and GLP-1R agonists and antagonists were assumed not to exert promiscuous actions at other GPCRs.

α7 Nicotinic Acetylcholine Receptor Regulates the Function and Viability of L Cells.

Enteroendocrine L cells secrete the incretin hormone glucagon-like peptide-1 (GLP-1), and they also express the α7 nicotinic acetylcholine receptor (α7nAChR), which may regulate GLP-1 secretion. Here, GTS-21, a selective α7nAChR agonist, was used to examine the effect of α7nAChR activation in L-cell lines, mouse intestinal primary cell cultures, and C57BL/6 mice. GTS-21 stimulated GLP-1 secretion in vitro, and this effect was attenuated by an α7nAChR antagonist or by α7nAChR-specific small interfering RNA. Under in vitro cell culture conditions of glucotoxicity, GTS-21 restored GLP-1 secretion and improved L-cell viability while also acting in vivo to raise levels of circulating GLP-1 in mice. To assess potential signaling mechanisms underlying these actions of GTS-21, we first monitored Ca2+, cAMP, and phosphatidylinositol 3-kinase (PI3K) activity. As expected for a GLP-1 secretagogue promoting Ca2+ influx through α7nAChR cation channels, [Ca2+]i increased in response to GTS-21, but [cAMP]i was unchanged. Surprisingly, pharmacological inhibition of growth factor signaling pathways revealed that GTS-21 also acts on the PI3K-protein kinase B-mammalian target of rapamycin pathway to promote L-cell viability. Moreover, the Ca2+ chelator BAPTA-AM counteracted GTS-21‒stimulated PI3K activity, thereby indicating unexpected crosstalk of L-cell Ca2+ and growth factor signaling pathways. Collectively, these data demonstrate that α7nAChR activation enhances GLP-1 secretion by increasing levels of cytosolic Ca2+ while also revealing Ca2+- and PI3K-dependent processes of α7nAChR activation that promote L-cell survival.

Restoration of Glucose-Stimulated Cdc42-Pak1 Activation and Insulin Secretion by a Selective Epac Activator in Type 2 Diabetic Human Islets.

Glucose metabolism stimulates cell division control protein 42 homolog (Cdc42)-p21-activated kinase (Pak1) activity and initiates filamentous actin (F-actin) cytoskeleton remodeling in pancreatic ß-cells so that cytoplasmic secretory granules can translocate to the plasma membrane where insulin exocytosis occurs. Since glucose metabolism also generates cAMP in ß-cells, the cross talk of cAMP signaling with Cdc42-Pak1 activation might be of fundamental importance to glucose-stimulated insulin secretion (GSIS). Previously, the type-2 isoform of cAMP-regulated guanine nucleotide exchange factor 2 (Epac2) was established to mediate a potentiation of GSIS by cAMP-elevating agents. Here we report that nondiabetic human islets and INS-1 832/13 ß-cells treated with the selective Epac activator 8-pCPT-2'-O-Me-cAMP-AM exhibited Cdc42-Pak1 activation at 1 mmol/L glucose and that the magnitude of this effect was equivalent to that which was measured during stimulation with 20 mmol/L glucose in the absence of 8-pCPT-2'-O-Me-cAMP-AM. Conversely, the cAMP antagonist Rp-8-Br-cAMPS-pAB prevented glucose-stimulated Cdc42-Pak1 activation, thereby blocking GSIS while also increasing cellular F-actin content. Although islets from donors with type 2 diabetes had profound defects in glucose-stimulated Cdc42-Pak1 activation and insulin secretion, these defects were rescued by the Epac activator so that GSIS was restored. Collectively, these findings indicate an unexpected role for cAMP as a permissive or direct metabolic coupling factor in support of GSIS that is Epac2 and Cdc42-Pak1 regulated.

Chimeric peptide EP45 as a dual agonist at GLP-1 and NPY2R receptors.

We report the design and target validation of chimeric peptide EP45, a novel 45 amino acid monomeric dual agonist peptide that contains amino acid sequence motifs present within the blood glucose-lowering agent exendin-4 (Ex-4) and the appetite-suppressing agent PYY(3-36). In a new high-throughput FRET assay that provides real-time kinetic information concerning levels of cAMP in living cells, EP45 recapitulates the action of Ex-4 to stimulate cAMP production via the glucagon-like peptide-1 receptor (GLP-1R), while also recapitulating the action of PYY(3-36) to inhibit cAMP production via the neuropeptide Y2 receptor (NPY2R). EP45 fails to activate glucagon or GIP receptors, whereas for cells that co-express NPY2R and adenosine A2B receptors, EP45 acts in an NPY2R-mediated manner to suppress stimulatory effects of adenosine on cAMP production. Collectively, such findings are remarkable in that they suggest a new strategy in which the co-existing metabolic disorders of type 2 diabetes and obesity will be treatable using a single peptide such as EP45 that lowers levels of blood glucose by virtue of its GLP-1R-mediated effect, while simultaneously suppressing appetite by virtue of its NPY2R-mediated effect.

GPR119 Agonist AS1269574 Activates TRPA1 Cation Channels to Stimulate GLP-1 Secretion.

GPR119 is a G protein-coupled receptor expressed on intestinal L cells that synthesize and secrete the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). GPR119 agonists stimulate the release of GLP-1 from L cells, and for this reason there is interest in their potential use as a new treatment for type 2 diabetes mellitus. AS1269574 is one such GPR119 agonist, and it is the prototype of a series of 2,4,6 trisubstituted pyrimidines that exert positive glucoregulatory actions in mice. Here we report the unexpected finding that AS1269574 stimulates GLP-1 release from the STC-1 intestinal cell line by directly promoting Ca(2+) influx through transient receptor potential ankyrin 1 (TRPA1) cation channels. These GPR119-independent actions of AS1269574 are inhibited by TRPA1 channel blockers (AP-18, A967079, HC030031) and are not secondary to intracellular Ca(2+) release or cAMP production. Patch clamp studies reveal that AS1269574 activates an outwardly rectifying membrane current with properties expected of TRPA1 channels. However, the TRPA1 channel-mediated action of AS1269574 to increase intracellular free calcium concentration is not replicated by GPR119 agonists (AR231453, oleoylethanolamide) unrelated in structure to AS1269574. Using human embryonic kidney-293 cells expressing recombinant rat TRPA1 channels but not GPR119, direct TRPA1 channel activating properties of AS1269574 are validated. Because we find that AS1269574 also acts in a conventional GPR119-mediated manner to stimulate proglucagon gene promoter activity in the GLUTag intestinal L cell line, new findings reported here reveal the surprising capacity of AS1269574 to act as a dual agonist at two molecular targets (GPR119/TRPA1) important to the control of L-cell function and type 2 diabetes mellitus drug discovery research.

Modeling analysis of inositol 1,4,5-trisphosphate receptor-mediated Ca2+ mobilization under the control of glucagon-like peptide-1 in mouse pancreatic ß-cells.

Glucagon-like peptide-1 (GLP-1) is an intestinally derived blood glucose-lowering hormone that potentiates glucose-stimulated insulin secretion from pancreatic ß-cells. The secretagogue action of GLP-1 is explained, at least in part, by its ability to stimulate cAMP production so that cAMP may facilitate the release of Ca(2+) from inositol trisphosphate receptor (IP3R)-regulated Ca(2+) stores. However, a quantitative model has yet to be provided that explains the molecular mechanisms and dynamic processes linking GLP-1-stimulated cAMP production to Ca(2+) mobilization. Here, we performed simulation studies to investigate how GLP-1 alters the abilities of Ca(2+) and IP3 to act as coagonists at IP3R Ca(2+) release channels. A new dynamic model was constructed based on the Kaftan model, which demonstrates dual steady-state allosteric regulation of the IP3R by Ca(2+) and IP3. Data obtained from ß-cells were then analyzed to understand how GLP-1 facilitates IP3R-mediated Ca(2+) mobilization when UV flash photolysis is used to uncage Ca(2+) and IP3 intracellularly. When the dynamic model for IP3R activation was incorporated into a minimal cell model, the Ca(2+) transients and oscillations induced by GLP-1 were successfully reconstructed. Simulation studies indicated that transient and oscillatory responses to GLP-1 were produced by sequential positive and negative feedback regulation due to fast activation and slow inhibition of the IP3R by Ca(2+). The slow rate of Ca(2+)-dependent inhibition was revealed to provide a remarkable contribution to the time course of the decay of cytosolic Ca(2+) transients. It also served to drive and pace Ca(2+) oscillations that are significant when evaluating how GLP-1 stimulates insulin secretion.

Enhanced Peptide Stability Against Protease Digestion Induced by Intrinsic Factor Binding of a Vitamin B12 Conjugate of Exendin-4.

Peptide digestion from proteases is a significant limitation in peptide therapeutic development. It has been hypothesized that the dietary pathway of vitamin B12 (B12) may be exploited in this area, but an open question is whether B12-peptide conjugates bound to the B12 gastric uptake protein intrinsic factor (IF) can provide any stability against proteases. Herein, we describe a new conjugate of B12 with the incretin peptide exendin 4 that demonstrates picomolar agonism of the glugacon-like peptide-1 receptor (GLP1-R). Stability studies reveal that Ex-4 is digested by pancreatic proteases trypsin and chymotrypsin and by the kidney endopeptidase meprin ß. Prebinding the B12 conjugate to IF, however, resulted in up to a 4-fold greater activity of the B12-Ex-4 conjugate relative to Ex-4, when the IF-B12-Ex-4 complex was exposed to 22 µg/mL of trypsin, 2.3-fold greater activity when exposed to 1.25 µg/mL of chymotrypsin, and there was no decrease in function at up to 5 µg/mL of meprin ß.

Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion.

cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells.

Leptin-stimulated KATP channel trafficking: a new paradigm for ß-cell stimulus-secretion coupling?

Insulin secretion from pancreatic ß-cells is initiated by the closure of ATP-sensitive K+ channels (KATP) in response to high concentrations of glucose, and this action of glucose is counteracted by the hormone leptin, an adipokine that signals through the Ob-Rb receptor to increase KATP channel activity. Despite intensive investigations, the molecular basis for KATP channel regulation remains uncertain, particularly from the standpoint of whether fluctuations in plasma membrane KATP channel content underlie alterations of KATP channel activity in response to glucose or leptin. Surprisingly, newly published findings reveal that leptin stimulates AMP-activated protein kinase (AMPK) in order to promote trafficking of KATP channels from cytosolic vesicles to the plasma membrane of ß-cells. This action of leptin is mimicked by low concentrations of glucose that also activate AMPK and that inhibit insulin secretion. Thus, a new paradigm for ß-cell stimulus-secretion coupling is suggested in which leptin exerts a tonic inhibitory effect on ß-cell excitability by virtue of its ability to increase plasma membrane KATP channel density and whole-cell KATP channel current. One important issue that remains unresolved is whether high concentrations of glucose suppress AMPK activity in order to shift the balance of membrane cycling so that KATP channel endocytosis predominates over vesicular KATP channel insertion into the plasma membrane. If so, high concentrations of glucose might transiently reduce KATP channel density/current, thereby favoring ß-cell depolarization and insulin secretion. Such an AMPK-dependent action of glucose would complement its established ability to generate an increase of ATP/ADP concentration ratio that directly closes KATP channels in the plasma membrane.

CO2/HCO3(-)- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor.

The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In ß cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.