Considerations in the Preclinical Assessment of the Safety of Antisense Oligonucleotides.

The nucleic acid therapeutics field has made tremendous progress in the past decades. Continuous advances in chemistry and design have led to many successful clinical applications, eliciting even more interest from researchers including both academic groups and drug development companies. Many preclinical studies in the field focus on improving the delivery of antisense oligonucleotide drugs (ONDs) and/or assessing their efficacy in target tissues, often neglecting the evaluation of toxicity, at least in early phases of development. A series of consensus recommendations regarding regulatory considerations and expectations have been generated by the Oligonucleotide Safety Working Group and the Japanese Research Working Group for the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S6 and Related Issues (WGS6) in several white papers. However, safety aspects should also be kept in sight in earlier phases while screening and designing OND to avoid subsequent failure in the development phase. Experts and members of the network "DARTER," a COST Action funded by the Cooperation in Science and Technology of the EU, have utilized their collective experience working with OND, as well as their insights into OND-mediated toxicities, to generate a series of consensus recommendations to assess OND toxicity in early stages of preclinical research. In the past few years, several publications have described predictive assays, which can be used to assess OND-mediated toxicity in vitro or ex vivo to filter out potential toxic candidates before moving to in vivo phases of preclinical development, that is, animal toxicity studies. These assays also have the potential to provide translational insight since they allow a safety evaluation in human in vitro systems. Yet, small preliminary in vivo studies should also be considered to complement this early assessment. In this study, we summarize the state of the art and provide guidelines and recommendations on the different tests available for these early stage preclinical assessments.

Investigating discovery strategies and pharmacological properties of stereodefined phosphorodithioate LNA gapmers.

The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS2) introduces second sulfur into the phosphorothioate linkage to remove the chirality of phosphorus atom. Here, we describe the application of symmetrical non-bridging PS2 linkages in the context of stereodefined locked nucleic acids (LNAs) antisense oligonucleotides with the goal of reducing chiral complexity and, ultimately, resulting in single molecules. In addition, we propose a rather simple strategy to rapidly identify stereodefined gapmers, combining PS2 and a preferred stereochemistry motif (RSSR), which supports RNase-H-mediated target knockdown. Pharmacological efficacy and metabolic stability are investigated systematically using ApoB as a target sequence, where in vivo data correlate well to what is observed in vitro.

Safety, Tissue Distribution, and Metabolism of LNA-Containing Antisense Oligonucleotides in Rats.

Antisense oligonucleotides (ASOs) are chemically modified nucleic acids with therapeutic potential, some of which have been approved for marketing. We performed a study in rats to investigate mechanisms of toxicity after administration of 3 tool locked nucleic acid (LNA)-containing ASOs with differing established safety profiles. Four male rats per group were dosed once, 3, or 6 times subcutaneously, with 7 days between dosing, and sacrificed 3 days after the last dose. These ASOs were either unconjugated (naked) or conjugated with N-acetylgalactosamine for hepatocyte-targeted delivery. The main readouts were in-life monitoring, clinical and anatomic pathology, exposure assessment and metabolite identification in liver and kidney by liquid chromatography coupled to tandem mass spectrometry, ASO detection in liver and kidney by immunohistochemistry, in situ hybridization, immune electron microscopy, and matrix-assisted laser desorption/ionization mass spectrometry imaging. The highly toxic compounds showed the greatest amount of metabolites and a low degree of tissue accumulation. This study reveals different patterns of cell death associated with toxicity in liver (apoptosis and necrosis) and kidney (necrosis only) and provides new ultrastructural insights on the tissue accumulation of ASOs. We observed that the immunostimulatory properties of ASOs can be either primary from sequence-dependent properties or secondary to cell necrosis.

Evaluation of bovine milk extracellular vesicles for the delivery of locked nucleic acid antisense oligonucleotides.

The natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs. We found that additional purification by density gradients efficiently reduced levels of non-EV associated proteins. The potential of BMEVs to functionally transfer LNA ASOs was tested using advanced in vitro systems (i.e. hPSC-derived neurons and primary human cells). A slight increase in cellular LNA ASO internalization and target gene reduction was observed when LNA ASOs were delivered using BMEVs. When dosed orally in mice, only a small fraction (about 1% of total administered dose) of LNA ASOs was recovered in the peripheral tissues liver and kidney, however, no significant reduction in target gene expression (i.e. functional knockdown) was observed.

Refining LNA safety profile by controlling phosphorothioate stereochemistry.

Drug discovery with phosphorothioate oligonucleotides is an area of intensive research. In this study we have controlled the stereochemistry of the phosphorothioate backbone of LNA oligonucleotides to investigate the differences in safety profile, target mRNA knock down, and cellular uptake in vitro. The study reveals that controlling only four stereocenters in an isomeric phosphorothioate mixture can improve the therapeutic index significantly by improving safety without compromising activity.

Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator.

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.

In Vitro Assessment of the Hepatotoxicity Potential of Therapeutic Oligonucleotides.

The mechanisms of antisense oligonucleotide-induced liver toxicity are still poorly understood. Assessment of the hepatic safety profile is currently mostly investigated directly in rodent studies. A predictive preclinical in vitro model that is capturing liver liabilities of antisense oligonucleotides can be of great help to be used as a first filter in the screening process of therapeutic oligonucleotides. We describe here an in vitro cytotoxicity assay using freshly isolated mouse hepatocytes or cryopreserved human hepatocytes that recapitulates the hepatotoxic profile of antisense oligonucleotides previously observed in rodents and can be used for the prioritization of molecules prior to in vivo testing.

GalNAc Conjugation Attenuates the Cytotoxicity of Antisense Oligonucleotide Drugs in Renal Tubular Cells.

Targeted delivery of antisense oligonucleotide (AON) drugs is a promising strategy to increase their concentration in the desired tissues and cell types while reducing access to other organs. Conjugation of AONs to N-acetylgalactosamine (GalNAc) has been shown to efficiently shift their biodistribution toward the liver via high-affinity binding to the asialoglycoprotein receptor (ASGPR) expressed at the surface of hepatocytes. Nevertheless, GalNAc conjugation does not prevent accumulation of AONs in the kidney cortex, and GalNAc-conjugated AONs might cause kidney toxicities, for example, under conditions of ASGPR saturation. Here, we investigated the nephrotoxicity potential of GalNAc-conjugated AONs by in vitro profiling of AON libraries in renal proximal tubule epithelial cells (PTECs) and in vivo testing of selected candidates. Whereas GalNAc-conjugated AONs appeared generally innocuous to PTECs, some caused mild-to-moderate nephrotoxicity in rats. Interestingly, the in vivo kidney liabilities could be recapitulated in vitro by treating PTECs with the unconjugated (or naked) parental AONs. An in vitro mechanistic study revealed that GalNAc conjugation attenuated AON-induced renal cell toxicity despite intracellular accumulation similar to that of naked AONs and independent of target knockdown. Overall, our in vitro findings reveal ASGPR-independent properties of GalNAc AONs that confer a favorable safety profile at the cellular level, which may variably translate in vivo due to catabolic transformation of circulating AONs.

Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia.

Single-stranded oligonucleotides (ON) comprise a promising therapeutic platform that enables selective modulation of currently undruggable targets. The development of novel ON drug candidates has demonstrated excellent efficacy, but in certain cases also some safety liabilities were reported. Among them are events of thrombocytopenia, which have recently been evident in late stage trials with ON drugs. The underlying mechanisms are poorly understood and the risk for ON candidates causing such events cannot be sufficiently assessed pre-clinically. We investigated potential thrombocytopenia risk factors of ONs and implemented a set of in vitro assays to assess these risks. Our findings support previous observations that phosphorothioate (PS)-ONs can bind to platelet proteins such as platelet collagen receptor glycoprotein VI (GPVI) and activate human platelets in vitro to various extents. We also show that these PS-ONs can bind to platelet factor 4 (PF4). Binding to platelet proteins and subsequent activation correlates with ON length and connected to this, the number of PS in the backbone of the molecule. Moreover, we demonstrate that locked nucleic acid (LNA) ribosyl modifications in the wings of the PS-ONs strongly suppress binding to GPVI and PF4, paralleled by markedly reduced platelet activation. In addition, we provide evidence that PS-ONs do not directly affect hematopoietic cell differentiation in culture but at higher concentrations show a pro-inflammatory potential, which might contribute to platelet activation. Overall, our data confirm that certain molecular attributes of ONs are associated with a higher risk for thrombocytopenia. We propose that applying the in vitro assays discussed here during the lead optimization phase may aid in deprioritizing ONs with a potential to induce thrombocytopenia.

Inhibition of EGF Uptake by Nephrotoxic Antisense Drugs In Vitro and Implications for Preclinical Safety Profiling.

Antisense oligonucleotide (AON) therapeutics offer new avenues to pursue clinically relevant targets inaccessible with other technologies. Advances in improving AON affinity and stability by incorporation of high affinity nucleotides, such as locked nucleic acids (LNA), have sometimes been stifled by safety liabilities related to their accumulation in the kidney tubule. In an attempt to predict and understand the mechanisms of LNA-AON-induced renal tubular toxicity, we established human cell models that recapitulate in vivo behavior of pre-clinically and clinically unfavorable LNA-AON drug candidates. We identified elevation of extracellular epidermal growth factor (EGF) as a robust and sensitive in vitro biomarker of LNA-AON-induced cytotoxicity in human kidney tubule epithelial cells. We report the time-dependent negative regulation of EGF uptake and EGF receptor (EGFR) signaling by toxic but not innocuous LNA-AONs and revealed the importance of EGFR signaling in LNA-AON-mediated decrease in cellular activity. The robust EGF-based in vitro safety profiling of LNA-AON drug candidates presented here, together with a better understanding of the underlying molecular mechanisms, constitutes a significant step toward developing safer antisense therapeutics.

Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs.

Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development.

Incretin-like effects of small molecule trace amine-associated receptor 1 agonists.

OBJECTIVE: Type 2 diabetes and obesity are emerging pandemics in the 21st century creating worldwide urgency for the development of novel and safe therapies. We investigated trace amine-associated receptor 1 (TAAR1) as a novel target contributing to the control of glucose homeostasis and body weight. METHODS: We investigated the peripheral human tissue distribution of TAAR1 by immunohistochemistry and tested the effect of a small molecule TAAR1 agonist on insulin secretion in vitro using INS1E cells and human islets and on glucose tolerance in C57Bl6, and db/db mice. Body weight effects were investigated in obese DIO mice. RESULTS: TAAR1 activation by a selective small molecule agonist increased glucose-dependent insulin secretion in INS1E cells and human islets and elevated plasma PYY and GLP-1 levels in mice. In diabetic db/db mice, the TAAR1 agonist normalized glucose excursion during an oral glucose tolerance test. Sub-chronic treatment of diet-induced obese (DIO) mice with the TAAR1 agonist resulted in reduced food intake and body weight. Furthermore insulin sensitivity was improved and plasma triglyceride levels and liver triglyceride content were lower than in controls. CONCLUSIONS: We have identified TAAR1 as a novel integrator of metabolic control, which acts on gastrointestinal and pancreatic islet hormone secretion. Thus TAAR1 qualifies as a novel and promising target for the treatment of type 2 diabetes and obesity.

A Transcriptome-Led Exploration of Molecular Mechanisms Regulating Somatostatin-Producing D-Cells in the Gastric Epithelium.

The stomach epithelium contains a myriad of enteroendocrine cells that modulate a range of physiological functions, including postprandial secretion of regulatory peptides, gastric motility, and nutrient absorption. Somatostatin (SST)-producing D-cells are present in the oxyntic and pyloric regions of the stomach, and provide a tonic inhibitory tone that regulates activity of neighboring enteroendocrine cells and gastric acid secretion. Cellular mechanisms underlying the effects of regulatory factors on gastric D-cells are poorly defined due to problems in identifying primary D-cells, and uncertainty remains about which stimuli influence D-cells directly. In this study, we introduce a transgenic mouse line, SST-Cre, which upon crossing with Cre reporter strains, facilitates the identification and purification of gastric D-cells, or cell-specific expression of genetically encoded calcium indicators. Populations of D-cells from the gastric antrum and corpus were isolated and analyzed by RNA sequencing and quantitative RT-PCR. The expression of hormones, hormone receptors, neurotransmitter receptors, and nutrient receptors was quantified. Pyy, Gipr, Chrm4, Calcrl, Taar1, and Casr were identified as genes that are highly enriched in D-cells compared with SST-negative cells. Hormone secretion assays performed in mixed gastric epithelial cultures confirmed that SST secretion is regulated by incretin hormones, cholecystokinin, acetylcholine, vasoactive intestinal polypeptide, calcitonin gene-related polypeptide, oligopetides, and trace amines. Cholecystokinin and oligopeptides elicited increases in intracellular calcium in single-cell imaging experiments performed using cultured D-cells. Our data provide the first transcriptomic analysis and functional characterization of gastric D-cells, and identify regulatory pathways that underlie the direct detection of stimuli by this cell type.

Discovery and optimisation of 1-hydroxyimino-3,3-diphenylpropanes, a new class of orally active GPBAR1 (TGR5) agonists.

A series of non-steroidal GPBAR1 (TGR5) agonists was developed from a hit in a high-throughput screening campaign. Lead identification efforts produced biphenyl-4-carboxylic acid derivative (R)-22, which displayed a robust secretion of PYY after oral administration in a degree that can be correlated with the unbound plasma concentration. Further optimisation work focusing on reduction of the lipophilicity provided the 1-phenylpiperidine-4-carboxylic acid derivative (R)-29 (RO5527239), which showed an improved secretion of PYY and GLP-1, translating into a significant reduction of postprandial blood glucose excursion in an oral glucose tolerance test in DIO mice.

Taspoglutide, an analog of human glucagon-like Peptide-1 with enhanced stability and in vivo potency.

Taspoglutide is a novel analog of human glucagon-like peptide-1 [hGLP-1(7-36)NH2] in clinical development for the treatment of type 2 diabetes. Taspoglutide contains alpha-aminoisobutyric acid substitutions replacing Ala(8) and Gly(35) of hGLP-1(7-36)NH2. The binding affinity [radioligand binding assay using [(125)I]hGLP-1(7-36)NH2], potency (cAMP production in CHO cells stably overexpressing hGLP-1 receptor), and in vitro plasma stability of taspoglutide compared with hGLP-1(7-36)NH2 have been evaluated. Effects on basal and glucose-stimulated insulin secretion were determined in vitro in INS-1E cells and in vivo in normal rats. Taspoglutide has comparable affinity (affinity constant 1.1 +/- 0.2 nm) to the natural ligand (affinity constant 1.5 +/- 0.3 nm) for the hGLP-1 receptor and exhibits comparable potency in stimulating cAMP production (EC(50) Taspo 0.06 nm and EC(50) hGLP-1(7-36)NH2 0.08 nm). Taspoglutide exerts insulinotropic action in vitro and in vivo and retains the glucoincretin property of hGLP-1(7-36)NH2. Stimulation of insulin secretion is concentration dependent and evident in the presence of high-glucose concentrations (16.7 mm) with a taspoglutide concentration as low as 0.001 nm. Taspoglutide is fully resistant to dipeptidyl peptidase-4 cleavage (during 1 h incubation at room temperature with purified enzyme) and has an extended in vitro plasma half-life relative to hGLP-1(7-36)NH2 (9.8 h vs. 50 min). In vitro, taspoglutide does not inhibit dipeptidyl peptidase-4 activity. This study provides the biochemical and pharmacological basis for the sustained plasma drug levels and prolonged therapeutic activity seen in early clinical trials of taspoglutide. Excellent stability and potency with substantial glucoincretin effects position taspoglutide as a promising new agent for treatment of type 2 diabetes.

Fibroblast growth factor-21 improves pancreatic beta-cell function and survival by activation of extracellular signal-regulated kinase 1/2 and Akt signaling pathways.

Fibroblast growth factor-21 (FGF-21) is a recently discovered metabolic regulator. Here, we investigated the effects of FGF-21 in the pancreatic beta-cell. In rat islets and INS-1E cells, FGF-21 activated extracellular signal-regulated kinase 1/2 and Akt signaling pathways. In islets isolated from healthy rats, FGF-21 increased insulin mRNA and protein levels but did not potentiate glucose-induced insulin secretion. Islets and INS-1E cells treated with FGF-21 were partially protected from glucolipotoxicity and cytokine-induced apoptosis. In islets isolated from diabetic rodents, FGF-21 treatment increased islet insulin content and glucose-induced insulin secretion. Short-term treatment of normal or db/db mice with FGF-21 lowered plasma levels of insulin and improved glucose clearance compared with vehicle after oral glucose tolerance testing. Constant infusion of FGF-21 for 8 weeks in db/db mice nearly normalized fed blood glucose levels and increased plasma insulin levels. Immunohistochemistry of pancreata from db/db mice showed a substantial increase in the intensity of insulin staining in islets from FGF-21-treated animals as well as a higher number of islets per pancreas section and of insulin-positive cells per islet compared with control. No effect of FGF-21 was observed on islet cell proliferation. In conclusion, preservation of beta-cell function and survival by FGF-21 may contribute to the beneficial effects of this protein on glucose homeostasis observed in diabetic animals.

Sterol regulatory element-binding protein 1 mediates liver X receptor-beta-induced increases in insulin secretion and insulin messenger ribonucleic acid levels.

Liver X receptors (LXRalpha and LXRbeta) regulate glucose and lipid metabolism. Pancreatic beta-cells and INS-1E insulinoma cells express only the LXRbeta isoform. Activation of LXRbeta with the synthetic agonist T0901317 increased glucose-induced insulin secretion and insulin content, whereas deletion of the receptor in LXRbeta knockout mice severely blunted insulin secretion. Analysis of gene expression in LXR agonist-treated INS-1E cells and islets from LXRbeta-deficient mice revealed that LXRbeta positively regulated expression of ATP-binding cassette transporter A1 (ABCA1), sterol regulatory element-binding protein 1 (SREBP-1), insulin, PDX-1, glucokinase, and glucose transporter 2 (Glut2). Down-regulation of SREBP-1 expression with the specific small interfering RNA blocked basal and LXRbeta-induced expression of pancreatic duodenal homeobox 1 (PDX-1), insulin, and Glut2 genes. SREBP-1 small interfering RNA also prevented an increase in insulin secretion and insulin content induced by T0901317. Moreover, 5-(tetradecyloxy)-2-furoic acid, an inhibitor of the SREBP-1 target gene acetyl-coenzyme A carboxylase, blocked T0901317-induced stimulation of insulin secretion. In conclusion, activation of LXRbeta in pancreatic beta-cells increases insulin secretion and insulin mRNA expression via SREBP-1-regulated pathway. These data support the role of LXRbeta, SREBP-1, and cataplerosis/anaplerosis pathways in the control of insulin secretion in pancreatic beta-cells.

A novel glucokinase activator modulates pancreatic islet and hepatocyte function.

The glucose-sensing enzyme glucokinase (GK) plays a key role in glucose metabolism. We report here the effects of a novel glucokinase activator, LY2121260. The activator enhanced GK activity via binding to the allosteric site located in the hinge region of the enzyme. LY2121260 stimulated insulin secretion in a glucose-dependent manner in pancreatic beta-cells and increased glucose use in rat hepatocytes. In addition, incubation of beta-cells with the GK activator resulted in increased GK protein levels, suggesting that enhanced insulin secretion on chronic treatment with a GK activator may be due to not only changed enzyme kinetics but also elevated enzyme levels. Animals treated with LY2121260 showed an improved glucose tolerance after oral glucose challenge. These results support the concept that GK activators represent a new class of compounds that increase both insulin secretion and hepatic glucose use and in doing so may prove to be effective agents for the control of blood glucose levels in patients with type 2 diabetes.

Glucagon stimulates exocytosis in mouse and rat pancreatic alpha-cells by binding to glucagon receptors.

Glucagon, secreted by the pancreatic alpha-cells, stimulates insulin secretion from neighboring beta-cells by cAMP- and protein kinase A (PKA)-dependent mechanisms, but it is not known whether glucagon also modulates its own secretion. We have addressed this issue by combining recordings of membrane capacitance (to monitor exocytosis) in individual alpha-cells with biochemical assays of glucagon secretion and cAMP content in intact pancreatic islets, as well as analyses of glucagon receptor expression in pure alpha-cell fractions by RT-PCR. Glucagon stimulated cAMP generation and exocytosis dose dependently with an EC50 of 1.6-1.7 nm. The stimulation of both parameters plateaued at concentrations beyond 10 nm of glucagon where a more than 3-fold enhancement was observed. The actions of glucagon were unaffected by the GLP-1 receptor antagonist exendin-(9-39) but abolished by des-His1-[Glu9]-glucagon-amide, a specific blocker of the glucagon receptor. The effects of glucagon on alpha-cell exocytosis were mimicked by forskolin and the stimulatory actions of glucagon and forskolin on exocytosis were both reproduced by intracellular application of 0.1 mm cAMP. cAMP-potentiated exocytosis involved both PKA-dependent and -independent (resistant to Rp-cAMPS, an Rp-isomer of cAMP) mechanisms. The presence of the cAMP-binding protein cAMP-guanidine nucleotide exchange factor II in alpha-cells was documented by a combination of immunocytochemistry and RT-PCR and 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP, a cAMP-guanidine nucleotide exchange factor II-selective agonist, mimicked the effect of cAMP and augmented rapid exocytosis in a PKA-independent manner. We conclude that glucagon released from the alpha-cells, in addition to its well-documented systemic effects and paracrine actions within the islet, also represents an autocrine regulator of alpha-cell function.

Liver X receptor activation stimulates insulin secretion via modulation of glucose and lipid metabolism in pancreatic beta-cells.

Liver X receptors (LXRs) alpha and beta, transcription factors of a nuclear hormone receptor family, are expressed in pancreatic islets as well as glucagon-secreting and insulin-secreting cell lines. Culture of pancreatic islets or insulin-secreting MIN6 cells with a LXR specific agonist T0901317 caused an increase in glucose-dependent insulin secretion and islet insulin content. The stimulatory effect of T0901317 on insulin secretion was observed only after >72 h of islet culture with the compound. In MIN6 cells, T0901317 increased protein expression of lipogenic enzymes, fatty acid synthase, and acetyl-CoA carboxylase. LXR activation also produced an increase in glucokinase protein and pyruvate carboxylase (PC) activity levels. The PC inhibitor phenylacetic acid abolished the increase in insulin secretion in cells treated with T0901317. The results suggest that LXRs can control insulin secretion and biosynthesis via regulation of glucose and lipid metabolism in pancreatic beta-cells.