Encapsulated, genetically engineered cells, secreting glucagon-like peptide-1 for the treatment of non-insulin-dependent diabetes mellitus.

Non-insulin-dependent, or type II, diabetes mellitus is characterized by a progressive impairment of glucose-induced insulin secretion by pancreatic beta cells and by a relative decreased sensitivity of target tissues to the action of this hormone. About one third of type II diabetic patients are treated with oral hypoglycemic agents to stimulate insulin secretion. These drugs however risk inducing hypoglycemia and, over time, lose their efficacy. An alternative treatment is the use of glucagon-like peptide-1 (GLP-1), a gut peptidic hormone with a strong insulinotropic activity. Its activity depends of the presence of normal blood glucose concentrations and therefore does not risk inducing hypoglycemia. GLP-1 can correct hyperglycemia in diabetic patients, even in those no longer responding to hypoglycemic agents. Because it is a peptide, GLP-1 must be administered by injection; this may prevent its wide therapeutic use. Here we propose to use cell lines genetically engineered to secrete a mutant form of GLP-1 which has a longer half-life in vivo but which is as potent as the wild-type peptide. The genetically engineered cells are then encapsulated in semi-permeable hollow fibers for implantation in diabetic hosts for constant, long-term, in situ delivery of the peptide. This approach may be a novel therapy for type II diabetes.

Deletion of mu- and kappa-opioid receptors in mice changes epidermal hypertrophy, density of peripheral nerve endings, and itch behavior.

The mu- (MOR) and kappa- (KOR) opioid receptors have been implicated in the regulation of homeostasis of non-neuronal cells, such as keratinocytes, and sensations like pain and chronic pruritus. Therefore, we have studied the phenotype of skin after deletion of MOR and KOR. In addition, we applied a dry skin model in these knockout mice and compared the different mice before and after induction of the dermatitis in terms of epidermal thickness, epidermal peripheral nerve ending distribution, dermal inflammatory infiltrate (mast cells, CD4 positive lymphocytes), and scratching behavior. MOR knockout mice reveal as phenotype a significantly thinner epidermis and a higher density of epidermal fiber staining by protein gene product 9.5 than the wild-type counterparts. Epidermal hypertrophy, induced by the dry skin dermatitis, was significantly less developed in MOR knockout than in wild-type mice. Neither mast cells nor CD4 T(h)-lymphocytes are involved in the changes of epidermal nerve endings and epidermal homeostasis. Finally, behavior experiments revealed that MOR and KOR knockout mice scratch less after induction of dry skin dermatitis than wild-type mice. These results indicate that MOR and KOR are important in skin homeostasis, epidermal nerve fiber regulation, and pathophysiology of itching.

Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4

Rapport de synthèse : L'immunité innée regroupe les mécanismes moléculaires et cellulaires formant la première ligne de défense contre les infections microbiennes. La détection des micro-organismes pathogènes est assurée par des cellules sentinelles (cellules dendritiques et macrophages) qui jouent un rôle fondamental dans l'initiation des mécanismes de défense de l'hôte. Au contact de produits microbiens, ces cellules produisent un large échantillonnage de molécules, dont des cytokines, impliquées dans le développement de la réponse inflammatoire. La régulation de cette réponse relève d'un équilibre délicat, son insuffisance tant que son excès pouvant compromettre le devenir des patients infectés. La sepsis sévère et le choc septique représentent les formes les plus sévères d'infection, et leur mortalité demeure élevée (25 à 30% pour la sepsis sévère et 50 à 60% pour le choc septique). De plus, l'incidence de la sepsis tend à augmenter, atteignant en 2000 plus de 240 cas pour 100'000 personnes en Grande-Bretagne. La sepsis est caractérisée dans sa phase aiguë par une réponse inflammatoire exubérante. La plupart des thérapies visant à la bloquer ont toutefois montré des bénéfices incertains lors de leur application clinique. Il est donc impératif d'identifier de nouvelles cibles thérapeutiques. Les "Toll-like receptors" (TLRs) sont une famille de récepteurs qui jouent un rôle fondamental dans la détection des micro-organismes par les cellules du système immunitaire inné. Parmi eux, TLR4 est indispensable à la reconnaissance du lipopolysaccharide (LPS) des bactéries Gram-négatives. L'interaction entre TLR4 et le LPS représentant un élément précoce de la réponse de l'hôte à l'infection, nous avons émit l'hypothèse que TLR4 pourrait représenter une cible de choix en vue du développement de nouvelles thérapies contre la sepsis. Dans l'objectif de valider ce concept, nous avons, dans un premier temps, démontré que des souris génétiquement déficientes en TLR4 étaient totalement résistantes au choc septique induit par Escherichia coli (E. coli), une bactérie Gram-négative fréquemment responsable de sepsis. Forts de cette observation, nous avons développé une molécule recombinante composée du domaine extracellulaire de TLR4 fusionné à la partie IgGi-Fc. Cette molécule soluble, qui inhibait la réponse des macrophages au LPS in vitro, a été utilisée pour générer des anticorps anti-TLR4 chez le lapin. La spécificité et l'efficacité de ces anticorps ont été prouvées en démontrant que les anti-TLR4 bloquaient les signaux d'activation intracellulaire et la production de TNF et d'IL-6 en réponse au LPS et aux bactéries Gram-négatives in vitro et in vivo. Enfin, l'efficacité des ces anticorps a été testée dans des modèles de sepsis chez la souris. Ainsi, l'injection prophylactique (-lh) ou thérapeutique (+3h) d'anticorps anti-TLR4 réduisait la production de TNF et protégeait les animaux de la mort. De manière spectaculaire, ces anticorps réduisaient également la production de TNF et protégeaient de la sepsis à E. coli lorsqu'ils étaient administrés de manière prophylactique (-4h) et thérapeutique, jusqu'à 13 heures après l'initiation de l'infection. Ces résultats indiquent donc qu'il est possible de bloquer le développement de la réponse inflammatoire et de protéger du choc septique à bactéries Gram-négatives en utilisant des thérapies ciblant TLR4. Par ailleurs, ils suggèrent qu'une fenêtre d'opportunité de plusieurs heures pourrait être mise à profit pour initier un traitement chez les patients septiques. Ces résultats devraient encourager la poursuite des essais cliniques en cours qui visent à tester l'efficacité de thérapies dirigées contre TLR4 comme traitement complémentaire de la sepsis.

Islands of euchromatin-like sequence and expressed polymorphic sequences within the short arm of human chromosome 21.

The goals of the human genome project did not include sequencing of the heterochromatic regions. We describe here an initial sequence of 1.1 Mb of the short arm of human chromosome 21 (HSA21p), estimated to be 10% of 21p. This region contains extensive euchromatic-like sequence and includes on average one transcript every 100 kb. These transcripts show multiple inter- and intrachromosomal copies, and extensive copy number and sequence variability. The sequencing of the "heterochromatic" regions of the human genome is likely to reveal many additional functional elements and provide important evolutionary information.

Receptor activator for NF-κB ligand in acute myeloid leukemia: expression, function, and modulation of NK cell immunosurveillance.

The TNF family member receptor activator for NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-γ production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK-RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.

The role of the NKG2D receptor for tumor immunity.

Natural killer (NK) cells have originally been identified based on their capacity to kill transformed cells in a seemingly non-specific fashion. Over the last 15 years, knowledge on receptor ligand systems used by NK cells to specifically detect transformed cells has been accumulating rapidly. One of these receptor ligand systems, the NKG2D pathway, has received particular attention, and now serves as a paradigm for how the immune system is able to gather information about the health status of autologous host cells. In addition to its significance on NK cells, NKG2D, as well as other NK cell receptors, play significant roles on T cells. This review aims at summarizing recent insights into the regulation of NKG2D function, the control over NKG2D ligand expression and the role of NKG2D in tumor immunity. Finally, we will discuss first attempts to exploit NKG2D function to improve immunity to tumors.

Clinical proteomics : a focus on transformed human T and B lymphocytes

Résumé large public La protéomique clinique est une discipline qui vise l'étude des protéines dans un but diagnostique ou thérapeutique. Nous avons utilisé cette approche pour étudier les lymphocytes T «tueurs » ou cytotoxiques qui font partie des globules blancs du système sanguin et agissent dans la lutte contre les infections et les tumeurs. Ces cellules sont impliquées dans l'immunothérapie cellulaire qui se fonde sur la capacité naturelle des ces lymphocytes à repérer les cellules tumorales et à les détruire. L'introduction du gène de la télomérase dans les lymphocytes T résulte en une prolongation de leur longévité, ce qui en ferait des candidats intéressants pour l'immunothérapie cellulaire. Il subsiste cependant des doutes quant aux conséquences de l'utilisation de ces lymphocytes «immortalisés ». Pour répondre à cette question, nous avons comparé le profile protéique de lymphocytes T cytotoxiques «jeunes » et vieux » avec celui des lymphocytes «immortalisés ». Nous avons trouvé que ces derniers présentent une double face et partagent à la fois les caractéristiques de la jeunesse et de la vieillesse. Dans une seconde étude de protéomique clinique, nous nous sommes penchés sur les lymphocytes B «immortalisés » cette fois-ci non pas avec la télomérase, mais avec le virus d'Epstein-Barr. Ces derniers sont utilisés comme modèle dans l'étude de la leucodystrophie, une maladie génétique rare qui affecte le cerveau. Notre but est d'identifier des marqueurs biologiques potentiels qui pourraient aider le diagnostic et le traitement de cette maladie neurodégénérative. Nous avons pour ce faire comparé les profiles protéiques des lymphocytes B «immortalisés » provenant d'individus sains et malades. Malheureusement, notre analyse n'a pas révélé de différences notoires entre ces deux classes de lymphocytes. Ceci nous permet toutefois de conclure que la maladie n'affecte pas la synthèse des protéines de manière prépondérante dans ces cellules sanguines. En résumé, le travail présenté dans cette thèse montre à la fois le potentiel et les limites de l'analyse des protéines lymphocytaires, dans différentes situations biologiques. Résumé La protéomique clinique ouvre la porte vers de multiples horizons relatifs au traitement de diverses maladies. Ce domaine particulier alliant la protéomique à la médecine, implique l'intervention de la biologie moléculaire et cellulaire. Dans notre étude, nous nous sommes d'abord intéressés aux lymphocytes T CD8+ cytotoxiques dans le contexte de l'immunothérapie adoptive. Le fondement de cette thérapie repose sur la capacité naturelle de ces lymphocytes à reconnaître les cellules tumorales et à les détruire chez les patients atteints de cancer. L'introduction du gène de la transcriptase réverse de la télomérase (hTERT) dans les lymphocytes T humains permet de rallonger leur durée de vie, sans toutefois induire d'altérations liées à la transformation. Cependant, des incertitudes subsistent quant à la ressemblance physiologique et biochimique entre ces cellules surexprirnant la télomérase et les cellules normales. Afin de répondre à cette question, nous avons comparé l'expression des protéines de lymphocytes humains T CD8+ «jeunes » et «vieux »avec celle de lymphocytes transduits avec hTERT. Nous avons trouvé que les lymphocytes T surexprimant la télomérase ont un profile protéique intermédiaire, avec certaines expressions protéiques similaires aux jeunes cellules T et d'autres se rapprochant des cellules vieilles. Dans la seconde partie de notre étude, nous nous sommes intéressés aux lymphocytes B transformés avec le virus d'Epstein-Barr provenant de patients atteints d'une maladie génétique rare du cerveau, la leucodystrophie. Dans cette maladie, des mutations dans le facteur de transcription eIF2B, impliqué dans la synthèse protéique, ont été trouvées. Afin d'analyser les conséquences de ces mutations et de trouver des biomarqueurs spécifiques à cette maladie, nous avons effectué une analyse protéomique des lymphoblastes provenant de malades et d'individus sains. Nous avons trouvé que les mutations dans le complexe ubiquitaire eIF2B n'affectent pas de manière significative l'expression des protéines des lymphoblastes mutés. En conclusion, notre travail illustre le potentiel et les limitations des technologies protéomiques utilisées pour disséquer l'implication des protéines dans différentes situations biologiques. Summary Clinical proteomics opens the door to multiple applications related to the treatment of diseases. This particular field is at the crossroad of proteomics and medicine and involves tools from cellular and molecular biology. We focused first our investigations on cytotoxic T cells in the context of adoptive immunotherapy, which is an interesting and evolving field. The basis of this therapy relies on the natural capacity of cytotoxic CD8+ T lymphocytes in recognizing tumor cells and destroying them in cancer patients. As their number is reduced, the idea would be to use transformed T lymphocytes with extended life span. Overexpression of telomerase into human T lymphocytes results in the extension of their replicative life span, but it still remains unclear whether these cells are physiologically indistinguishable from normal ones. To address this question, we compared the proteome of young and aged CD8+ T lymphocytes with that of T cells transduced with hTERT and found that the latter cells displayed an intermediate protein pattern, sharing similar protein expression with young, but also with aged T cells. We were then interested in studying Epstein-Barr virus transformed B lymphocytes in the context of a rare human brain genetic disorder called leukodystrophy. In this disease, mutations in the ubiquitous factor eIF2B involved in protein synthesis and its regulation have been reported. In order to analyze the functional consequences of the mutations and to find out specific biomarkers of eIF2B-related disorders, proteomic and peptidomic studies were carried out on lymphoblasts from eIF2Bmutated patients versus healthy patients. Following two-dimensional gel electrophoresis and mass fingerprints, mutations in the eIF2B complex did not appear to significantly affect the proteome of the mutated lymphoblasts extracts. To conclude, our work emphasizes the potentials and the limitations of the proteomic technologies used to analyze the role of lymphocyte proteins in different biological situations.

Targeting Cancer Stem-like Cells as an Approach to Defeating Cellular Heterogeneity in Ewing Sarcoma.

Plasticity in cancer stem-like cells (CSC) may provide a key basis for cancer heterogeneity and therapeutic response. In this study, we assessed the effect of combining a drug that abrogates CSC properties with standard-of-care therapy in a Ewing sarcoma family tumor (ESFT). Emergence of CSC in this setting has been shown to arise from a defect in TARBP2-dependent microRNA maturation, which can be corrected by exposure to the fluoroquinolone enoxacin. In the present work, primary ESFT from four patients containing CD133(+) CSC subpopulations ranging from 3% to 17% of total tumor cells were subjected to treatment with enoxacin, doxorubicin, or both drugs. Primary ESFT CSC and bulk tumor cells displayed divergent responses to standard-of-care chemotherapy and enoxacin. Doxorubicin, which targets the tumor bulk, displayed toxicity toward primary adherent ESFT cells in culture but not to CSC-enriched ESFT spheres. Conversely, enoxacin, which enhances miRNA maturation by stimulating TARBP2 function, induced apoptosis but only in ESFT spheres. In combination, the two drugs markedly depleted CSCs and strongly reduced primary ESFTs in xenograft assays. Our results identify a potentially attractive therapeutic strategy for ESFT that combines mechanism-based targeting of CSC using a low-toxicity antibiotic with a standard-of-care cytotoxic drug, offering immediate applications for clinical evaluation.

c-Myc controls the development of CD8alphaalpha TCRalphabeta intestinal intraepithelial lymphocytes from thymic precursors by regulating IL-15-dependent survival.

The murine gut epithelium contains a large population of thymus-derived intraepithelial lymphocytes (IELs), including both conventional CD4(+) and CD8alphabeta(+) T cells (expressing T-cell receptor alphabeta [TCRalphabeta]) and unconventional CD8alphaalpha(+) T cells (expressing either TCRalphabeta or TCRgammadelta). Whereas conventional IELs are widely accepted to arise from recirculation of activated CD4(+) and CD8alphabeta(+) T cells from the secondary lymphoid organs to the gut, the origin and developmental pathway of unconventional CD8alphaalpha IELs remain controversial. We show here that CD4-Cre-mediated inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biologic activities, selectively impairs the development of CD8alphaalpha TCRalphabeta IELs. In the absence of c-Myc, CD4(-) CD8(-) TCRalphabeta(+) thymic precursors of CD8alphaalpha TCRalphabeta IELs are present but fail to develop on adoptive transfer in immunoincompetent hosts. Residual c-Myc-deficient CD8alphaalpha TCRalphabeta IEL display reduced proliferation and increased apoptosis, which correlate with significantly decreased expression of interleukin-15 receptor subunits and lower levels of the antiapoptotic protein Bcl-2. Transgenic overexpression of human BCL-2 resulted in a pronounced rescue of CD8alphaalpha TCRalphabeta IEL in c-Myc-deficient mice. Taken together, our data support a model in which c-Myc controls the development of CD8alphaalpha TCRalphabeta IELs from thymic precursors by regulating interleukin-15 receptor expression and consequently Bcl-2-dependent survival.

A Y-like social chromosome causes alternative colony organization in fire ants.

Intraspecific variability in social organization is common, yet the underlying causes are rarely known. In the fire ant Solenopsis invicta, the existence of two divergent forms of social organization is under the control of a single Mendelian genomic element marked by two variants of an odorant-binding protein gene. Here we characterize the genomic region responsible for this important social polymorphism, and show that it is part of a pair of heteromorphic chromosomes that have many of the key properties of sex chromosomes. The two variants, hereafter referred to as the social B and social b (SB and Sb) chromosomes, are characterized by a large region of approximately 13 megabases (55% of the chromosome) in which recombination is completely suppressed between SB and Sb. Recombination seems to occur normally between the SB chromosomes but not between Sb chromosomes because Sb/Sb individuals are non-viable. Genomic comparisons revealed limited differentiation between SB and Sb, and the vast majority of the 616 genes identified in the non-recombining region are present in the two variants. The lack of recombination over more than half of the two heteromorphic social chromosomes can be explained by at least one large inversion of around 9 megabases, and this absence of recombination has led to the accumulation of deleterious mutations, including repetitive elements in the non-recombining region of Sb compared with the homologous region of SB. Importantly, most of the genes with demonstrated expression differences between individuals of the two social forms reside in the non-recombining region. These findings highlight how genomic rearrangements can maintain divergent adaptive social phenotypes involving many genes acting together by locally limiting recombination.

NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8⁺ T cells.

Memory T cells exert antigen-independent effector functions, but how these responses are regulated is unclear. We discovered an in vivo link between flagellin-induced NLRC4 inflammasome activation in splenic dendritic cells (DCs) and host protective interferon-γ (IFN-γ) secretion by noncognate memory CD8(+) T cells, which could be activated by Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that CD8α(+) DCs were particularly efficient at sensing bacterial flagellin through NLRC4 inflammasomes. Although this activation released interleukin 18 (IL-18) and IL-1β, only IL-18 was required for IFN-γ production by memory CD8(+) T cells. Conversely, only the release of IL-1β, but not IL-18, depended on priming signals mediated by Toll-like receptors. These findings provide a comprehensive mechanistic framework for the regulation of noncognate memory T cell responses during bacterial immunity.

Hypoxia-inducible miR-210 regulates the susceptibility of tumor cells to lysis by cytotoxic T cells.

Hypoxia in the tumor microenvironment plays a central role in the evolution of immune escape mechanisms by tumor cells. In this study, we report the definition of miR-210 as a miRNA regulated by hypoxia in lung cancer and melanoma, documenting its involvement in blunting the susceptibility of tumor cells to lysis by antigen-specific cytotoxic T lymphocytes (CTL). miR-210 was induced in hypoxic zones of human tumor tissues. Its attenuation in hypoxic cells significantly restored susceptibility to autologous CTL-mediated lysis, independent of tumor cell recognition and CTL reactivity. A comprehensive approach using transcriptome analysis, argonaute protein immunoprecipitation, and luciferase reporter assay revealed that the genes PTPN1, HOXA1, and TP53I11 were miR-210 target genes regulated in hypoxic cells. In support of their primary importance in mediating the immunosuppressive effects of miR-210, coordinate silencing of PTPN1, HOXA1, and TP53I11 dramatically decreased tumor cell susceptibility to CTL-mediated lysis. Our findings show how miR-210 induction links hypoxia to immune escape from CTL-mediated lysis, by providing a mechanistic understanding of how this miRNA mediates immunosuppression in oxygen-deprived regions of tumors where cancer stem-like cells and metastatic cellular behaviors are known to evolve.

Single cell analysis reveals similar functional competence of dominant and non-dominant CD8 T-cell clonotypes

The properties of CD8 T-cells requiredfor protection from infectiousdisease and cancer are only partiallycharacterized, and only limited data isavailable regarding T-cell clonotypes.It has been proposed that dominantT-cell clonotypes may have higherprotective potential than their nondominantcounterparts. Our objectiveswere to assess memory andeffector functions, stage of differentiationand clonotype selection of tumor-reactive T lymphocytes followingpeptide vaccination in melanomapatients.We also characterized dominantversus non-dominant clonotypesto further understand the in vivo functionof these T-cells based on theirprevalence. Using a novel single-cellapproach for simultaneous ex vivomolecular and functional analysis, wereport the preferential selection andexpansion of several tumor-specificco-dominant clonotypes of intermediateto high frequencies, irrespectiveof whether native or analog peptidewas used for vaccination. Theseclonotypes made up 40 - 95% of thedifferentiated "effector-like" T-cells,but only 25% of the less-differentiated"effector-memory" cells. Bothsubsets also contained non-dominantT-cell clonotypes, but these were significantlymore frequent in the lessdifferentiatedcells. Thus, cell differentiationwas clonotype-dependent.Surprisingly however, the acquisitionof memory and effector T-cell propertieswas clonotype independent, as wefound similar functional profiles indominant and low/ non-dominantT-cell clonotypes. In contrast to analogpeptide vaccination, native peptidevaccination induced T-cell functionsthat were more comprehensive,with more pronounced effector functionscombined with memory cellproperties. In summary, this study revealsthat T-cell functions are determinedprimarily by the antigen andthe stage of T-cell differentiation, butare similar in dominant and non-dominantclonotypes participating in aCD8 T-cell response. The identifiedclonotypic basis of T-cell responsescontributes to the rational developmentof vaccines.

Expression and functional activity of glucagon, glucagon-like peptide I, and glucose-dependent insulinotropic peptide receptors in rat pancreatic islet cells.

Rat pancreatic alpha- and beta-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in beta-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in beta-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide I(7-36) amide (GLP-I), while RNA from islet alpha-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in beta-cells and not in alpha-cells. Receptor activity, measured as cellular cAMP production after exposing islet beta-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/l GLP-I and 50 pmol/l GIP but required 1 nmol/l glucagon. EC50 values of GLP-I- and GIP-induced cAMP formation were comparable (0.2 nmol/l) and 45-fold lower than the EC50 of glucagon (9 nmol/l). Maximal stimulation of cAMP production was comparable for the three peptides. In purified alpha-cells, 1 nmol/l GLP-I failed to increase cAMP levels, while 10 pmol/l to 10 nmol/l GIP exerted similar stimulatory effects as in beta-cells. In conclusion, these data show that stimulation of glucagon, GLP-I, and GIP receptors in rat beta-cells causes cAMP production required for insulin release, while adenylate cyclase in alpha-cells is positively regulated by GIP.