Intact COS-7 Cells Research Articles

Highly Cooperative Dependence of Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) 2a Pump Activity on Cytosolic Calcium in Living Cells

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT(1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT(1) receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28((1.32))-Ile-53((1.57)) fragment of TMD1 and Leu-143((4.40))-Phe-170((4.67)) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C((1.34))-AT(1) and T33C((1.37))-AT(1) and TMD4 mutant V169C((4.66))-AT(1). Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT(1)) background, we found that V169C((4.66))-AT(1) remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C((4.64))-AT(1) was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30((1.34)) and Thr-33((1.37)), as well as residues Arg-167((4.64)) and Val-169((4.66)) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT(1) receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process.

The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

Identification of the Non-lysosomal Glucosylceramidase as β-Glucosidase 2

The primary catabolic pathway for glucosylceramide is catalyzed by the lysosomal enzyme glucocerebrosidase that is defective in Gaucher disease patients. A distinct non-lysosomal glucosylceramidase has been described but its identity remained enigmatic for years. We here report that the non-lysosomal glucosylceramidase is identical to the earlier described bile acid beta-glucosidase, being beta-glucosidase 2 (GBA2). Expressed GBA2 is identical to the native non-lysosomal glucosylceramidase in various enzymatic features such as substrate specificity and inhibitor sensitivity. Expression of GBA2 coincides with increased non-lysosomal glucosylceramidase activity, and GBA2-targeted RNA interference reduces endogenous non-lysosomal glucosylceramidase activity in cells. GBA2 is found to be located at or close to the cell surface, and its activity is linked to sphingomyelin generation. Hydrophobic deoxynojirimycins are extremely potent inhibitors for GBA2. In mice pharmacological inhibition of GBA2 activity is associated with impaired spermatogenesis, a phenomenon also very recently reported for GBA2 knock-out mice (Yildiz, Y., Matern, H., Thompson, B., Allegood, J. C., Warren, R. L., Ramirez, D. M., Hammer, R. E., Hamra, F. K., Matern, S., and Russell, D. W. (2006) J. Clin. Invest. 116, 2985-2994). In conclusion, GBA2 plays a role in cellular glucosylceramide metabolism.

Identification and functional characterization of hCLS1, a human cardiolipin synthase localized in mitochondria

In eukaryotic cells, CLS (cardiolipin synthase) is involved in the final step of cardiolipin synthesis by catalysing the transfer of a phosphatidyl residue from CDP-DAG (diacylglycerol) to PG (phosphatidylglycerol). Despite an important role of cardiolipin in regulating mitochondrial function, a gene encoding the mammalian CLS has not been identified so far. We report in the present study the identification and characterization of a human cDNA encoding the first mammalian CLS [hCLS1 (human CLS1)]. The predicted hCLS1 peptide sequence shares significant homology with the yeast and plant CLS proteins. The recombinant hCLS1 enzyme expressed in COS-7 cells catalysed efficiently the synthesis of cardiolipin in vitro using CDP-DAG and PG as substrates. Furthermore, overexpression of hCLS1 cDNA in COS-7 cells resulted in a significant increase in cardiolipin synthesis in intact COS-7 cells without any significant effects on the activity of the endogenous phosphatidylglycerophosphate synthase of the transfected COS-7 cells. Immunohistochemical analysis demonstrated that the recombinant hCLS1 protein was localized to the mitochondria when transiently expressed in COS-7 cells, which was further corroborated by results from subcellular fractionation analyses of the recombinant hCLS1 protein. Northern-blot analysis showed that the hCLS1 gene was predominantly expressed in tissues that require high levels of mitochondrial activities for energy metabolism, with the highest expression in skeletal and cardiac muscles. High levels of hCLS1 expression were also detected in liver, pancreas, kidney and small intestine, implying a functional role of hCLS1 in these tissues.

The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters

The final step of triacylglycerol biosynthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes. The two known DGATs, DGAT1 and DGAT2, are encoded by unrelated genes. Although both DGAT1 and DGAT2 knockout mice have reduced tissue triacylglycerol contents, they have disparate phenotypes, prompting us to investigate whether the two enzymes have unrecognized functional differences. We now report that DGAT1 exhibits additional acyltransferase activities in vitro, including those of acyl CoA:monoacylglycerol acyltransferase (MGAT), wax monoester and wax diester synthases, and acyl CoA:retinol acyltransferase (ARAT), which catalyze the synthesis of diacylglycerols, wax esters, and retinyl esters, respectively. These activities were demonstrated in in vitro assays with membranes from insect cells or homogenates from COS7 cells overexpressing DGAT1. Wax synthase and ARAT activities were also demonstrated in intact COS7 cells expressing DGAT1. Additionally, cells and tissues from DGAT1-deficient mice exhibited reduced ARAT activity, and the mice had increased levels of unesterified retinol in their livers on a high-retinol diet. Our findings indicate that DGAT1 can utilize a variety of acyl acceptors as substrates in vitro and suggest that these activities may be relevant to the in vivo functions of DGAT1.

Characterization of human SCD2, an oligomeric desaturase with improved stability and enzyme activity by cross-linking in intact cells

SCD (stearoyl-CoA desaturase) catalyses the conversion of saturated fatty acids into mono-unsaturated fatty acids, a critical step involved in lipid metabolism and various other biological functions. In the present study, we report the identification and characterization of a human gene that encodes a novel SCD enzyme (hSCD2). The hSCD2 gene codes for a 37.5-kDa protein that shares 61% and 57% sequence identity with the human SCD1 and mouse SCD2 enzymes respectively. The recombinant hSCD2 enzyme expressed in mammalian and Sf9 insect cells efficiently catalysed desaturation of both stearoyl- and palmitoyl-CoAs to the corresponding mono-unsaturated fatty acids. In comparison with the hSCD1 gene that is predominantly expressed in liver, hSCD2 is most abundantly expressed in pancreas and brain. Additionally, hSCD2 transcripts from adult and foetal tissues exhibit different sizes because of alternative splicing in the non-coding region, suggesting that hSCD2 expression is developmentally regulated. The recombinant human SCD2 and SCD1 transiently expressed in COS-7 cells exhibited as oligomeric proteins that consist of homodimers and oligomers when resolved by SDS/PAGE. The complex formation was independent of SCD protein expression levels, as supported by a relatively constant ratio of the level of dimers and oligomers to that of the monomers from COS-7 cells transiently transfected with different amounts of SCD expression vectors. Furthermore, treatment of intact COS-7 cells with a cross-linking reagent resulted in dose-dependent increases in the levels of SCD protein and activity, suggesting that oligomerization may play an important role in regulating the stability of SCD enzymes.

Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-3

We have cloned and characterized the nucleoside triphosphate diphosphohydrolase-3 (NTPDase3) from mouse spleen. Analysis of cDNA shows an open reading frame of 1587 base pairs encoding a protein of 529 amino acids with a predicted molecular mass of 58 953 Da and an estimated isoelectric point of 5.78. The translated amino acid sequence shows the presence of two transmembrane domains, eight potential N-glycosylation sites and the five apyrase conserved regions. The genomic sequence is located on chromosome 9F4 and is comprised of 11 exons. Intact COS-7 cells transfected with an expression vector containing the coding sequence for mouse NTPDase3 hydrolyzed P2 receptor agonists (ATP, UTP, ADP and UDP) but not AMP. NTPDase3 required divalent cations (Ca 2+>Mg 2+) for enzymatic activity. Interestingly, the enzyme had two optimum pHs for ATPase activity (pH 5.0 and 7.4) and one for ADPase activity (pH 8.0). Consequently, the ATP/ADP and UTP/UDP hydrolysis ratios were two to four folds higher at pH 5.0 than at pH 7.4, for both, intact cells and protein extracts. At pH 7.4 mouse NTPDase3 hydrolyzed ATP, UTP, ADP and UDP according to Michaelis–Menten kinetics with apparent K ms of 11, 10, 19 and 27 μM, respectively. In agreement with the K m values, the pattern of triphosphonucleoside hydrolysis showed a transient accumulation of the corresponding diphosphonucleoside and similar affinity for uracil and adenine nucleotides. NTPDase3 hydrolyzes nucleotides in a distinct manner than other plasma membrane bound NTPDases that may be relevant for the fine tuning of the concentration of P2 receptor agonists.

Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R 1 in Manduca sexta: : involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis

Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R 1 (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K d ∼ 1 nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R 1 revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R 1 cDNA, subsequently killing the cells. Recruitment of BT-R 1 by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.

Expression of a Cloned Full-Length cDNA Encoding Bovine Luteinizing Hormone Receptor in COS-7 Cells.

A cloned full-length cDNA encoding bovine luteinizing hormone (LH) receptor was expressed in COS-7 cells and the binding activity of the protein to human chorionic gonadotropin (hCG) was examined. Molecular cloning of the bovine LH receptor cDNA was carried out by reverse transcription-polymerase chain reaction using total RNA extracted from a bovine corpus luteum. Three overlapping partial fragments of the receptor cDNA were amplified, sequenced, and engineered to generate the entire cDNA coding sequence and it was subcloned into a mammalian expression vector. Sequence analysis showed a 2,103-nucleotide open reading frame encoding the bovine LH receptor, predicting a putative 26-amino acid signal peptide, and a 675-amino acid mature receptor protein that is 94, 88, 86 and 85% identical to the porcine, human, rat and mouse homologs, respectively. Scatchard analysis of the intact COS-7 cells transfected with the bovine LH receptor cDNA showed high affinity and low capacity of [125I]-hCG binding sites on the cells. These results suggest that the protein expressed from the cloned bovine LH receptor cDNA is located on the cell surface with its binding activity to LH/hCG.

Agonist-independent and -dependent oligomerization of dopamine D 2 receptors by fusion to fluorescent proteins

Oligomerization of the short (D 2S) and long (D 2L) isoforms of the dopamine D 2 receptor was explored in transfected Cos-7 cells by their C-terminal fusion to either an enhanced cyan or enhanced yellow fluorescent protein (ECFP or EYFP) and the fluorescent fusion protein interaction was monitored by a fluorescence resonance energy transfer (FRET) assay. The pharmacological properties of the fluorescent fusion proteins, as measured by both displacement of [ 3H]nemonapride binding and agonist-mediated stimulation of [ 35S]GTPγS binding upon co-expression with a G αoCys 351Ile protein, were not different from the respective wild-type D 2S and D 2L receptors. Co-expression of D2S:ECFP+D2S:EYFP in a 1:1 ratio and D2L:ECFP+D2L:EYFP in a 27:1 ratio resulted, respectively, in an increase of 26% and 16% in the EYFP-specific fluorescent signal. These data are consistent with a close proximity of both D 2S and D 2L receptor pairs of fluorescent fusion proteins in the absence of ligand. The agonist-independent D 2S receptor oligomerization could be attenuated by co-expression with either a wild-type, non-fluorescent D 2S or D 2L receptor subtype, but not with a distinct β 2-adrenoceptor. Incubation with the agonist (−)-norpropylapomorphine dose-dependently (EC 50: 0.23±0.06 nM) increased the FRET signal for the co-expression of D2S:ECFP and D2S:EYFP, in support of agonist-dependent D 2S receptor oligomerization. In conclusion, our data strongly suggest the occurrence of dopamine D 2 receptor oligomers in intact Cos-7 cells.

Protein Kinase C Phosphorylates RGS2 and Modulates Its Capacity for Negative Regulation of Gα11 Signaling

RGS proteins (regulators of G protein signaling) attenuate heterotrimeric G protein signaling by functioning as both GTPase-activating proteins (GAPs) and inhibitors of G protein/effector interaction. RGS2 has been shown to regulate Galpha(q)-mediated inositol lipid signaling. Although purified RGS2 blocks PLC-beta activation by the nonhydrolyzable GTP analog guanosine 5'-O-thiophosphate (GTPgammaS), its capacity to regulate inositol lipid signaling under conditions where GTPase-promoted hydrolysis of GTP is operative has not been fully explored. Utilizing the turkey erythrocyte membrane model of inositol lipid signaling, we investigated regulation by RGS2 of both GTP and GTPgammaS-stimulated Galpha(11) signaling. Different inhibitory potencies of RGS2 were observed under conditions assessing its activity as a GAP versus as an effector antagonist; i.e. RGS2 was a 10-20-fold more potent inhibitor of aluminum fluoride and GTP-stimulated PLC-betat activity than of GTPgammaS-promoted PLC-betat activity. We also examined whether RGS2 was regulated by downstream components of the inositol lipid signaling pathway. RGS2 was phosphorylated by PKC in vitro to a stoichiometry of approximately unity by both a mixture of PKC isozymes and individual calcium and phospholipid-dependent PKC isoforms. Moreover, RGS2 was phosphorylated in intact COS7 cells in response to PKC activation by 4beta-phorbol 12beta-myristate 13alpha-acetate and, to a lesser extent, by the P2Y(2) receptor agonist UTP. In vitro phosphorylation of RGS2 by PKC decreased its capacity to attenuate both GTP and GTPgammaS-stimulated PLC-betat activation, with the extent of attenuation correlating with the level of RGS2 phosphorylation. A phosphorylation-dependent inhibition of RGS2 GAP activity was also observed in proteoliposomes reconstituted with purified P2Y(1) receptor and Galpha(q)betagamma. These results identify for the first time a phosphorylation-induced change in the activity of an RGS protein and suggest a mechanism for potentiation of inositol lipid signaling by PKC.

Association of CD44 with OTS-8 in tumor vascular endothelial cells

Endothelial cells in solid tumors play an important role in tumor growth, invasion and metastasis through angiogenesis. We have recently cloned two tumor vascular antigens from isolated rat tumor vascular endothelial cells (TEC). One is CD44, a family of cell surface proteins implicated in adhesion interactions and tumor metastasis. The other is OTS-8, a marker for osteoblast into osteocyte transition and type I alveolar epithelial cells termed as E11 antigen and RTI40, respectively. To test for a possible interaction between the two antigens on endothelial cells in tumor angiogenesis, we examined in vivo association of CD44 with OTS-8 using lysates of isolated rat TEC and COS-7 cells cotransfected with CD44 and OTS-8 expression plasmids. The association was detected by direct co-immunoprecipitation of the two types of cells lysed with digitonin, whereas the detection was lost when lysed with Nonidet P-40. To confirm this association, intact COS-7 cells cotransfected were reacted with homobifunctional N-hydroxysuccinimide ester crosslinking reagents. Immunoblot analysis showed a crosslinked CD44/OTS-8 protein complex of 120 kDa, suggesting the proximity of the two proteins. These findings provide evidence of a weak physical association between CD44 and OTS-8 in TEC, and suggest that OTS-8 may alter the mode of endothelial cell growth and/or migration induced by CD44 in tumor angiogenesis.

Cloning and Characterization of Phospholipase D from Rat Brain

The regulation of phospholipase D cloned from rat brain (rPLD) was examined in vivo and in vitro. The enzyme was a shorter splice variant of human phospholipase D 1 (Hammond, S. M., Altshuller, Y. M. , Sung, T.-C., Rudge, S. M., Rose, K., Engebrecht, J. A., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). Its expression in COS-7 cells led to increased phospholipase D (PLD) activity that was further stimulated by constitutively active V14RhoA. V14RhoA had no effect on the endogenous PLD of the COS-7 cells, but constitutively active L71ARF3 increased its activity. In contrast, L71ARF3 did not activate rPLD expressed in the cells. Addition of phorbol ester markedly increased the endogenous PLD activity of COS-7 cells, and there was a further increase in the cells expressing rPLD. In membranes from COS-7 cells expressing rPLD, addition of myristoylated ADP-ribosylation factor (ARF) and RhoA in vitro stimulated PLD activity. The effect of ARF was greater than that of RhoA, although the concentrations for half-maximal stimulation (0.08-0.2 microM) were similar. Membranes isolated from cells expressing rPLD plus L71ARF3 and/or V14RhoA also showed higher PLD activity but no synergism between the two G proteins. Addition of phorbol ester and protein kinase C alpha (PKCalpha) also stimulated PLD activity in membranes from COS-7 cells expressing rPLD, but it had no effect on the activity in control (vector) membranes and did not enhance the effects of constitutively active ARF or Rho. The stimulation by PKCalpha did not require ATP and was not increased by addition of this nucleotide. No synergism between ARF and Rho and between these and PKCalpha on PLD activity was observed when these were added to membranes from cells expressing rPLD. Oleate inhibited the PLD activity of membranes from both control and rPLD-expressing cells. In summary, these results indicate that in vitro, rPLD is stimulated by ARF, RhoA, and PKCalpha and inhibited by oleate. However, in intact COS-7 cells, ARF activates endogenous PLD but not rPLD, whereas the reverse is true for RhoA. In addition, the effects of phorbol ester are much greater in the intact cells. It is concluded that the regulation of rPLD in intact COS-7 cells differs significantly from that seen in vitro; possible reasons for this are discussed.

Activation of a cAMP-regulated Ca2+-Signaling Pathway in Pancreatic β-Cells by the Insulinotropic Hormone Glucagon-like Peptide-1

Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone that is currently under investigation for use in the treatment of diabetes mellitus. To investigate the Ca2+ signaling pathways by which GLP-1 may stimulate the secretion of insulin from pancreatic beta-cells, we examined its effects on the concentration of free intracellular Ca2+ ([Ca2+]i) while simultaneously determining what action it exerts on ion channel function. Measurements of [Ca2+]i were obtained from single rat beta-cells and from beta TC6 and HIT-T15 insulinoma cells loaded with the Ca2+ indicator fura-2, and changes in membrane potential and current were monitored using the perforated patch clamp technique. We report a previously undocumented action of GLP-1 and analogs of cAMP (8-bromo-cAMP, Sp- or Rp-adenosine 3',5'-cyclic monophosphothionate triethylamine) to raise [Ca2+]i that is attributable to the activation of a prolonged inward current designated here as IcAMP. Activation of IcAMP is associated with an increased membrane conductance, membrane depolarization, and triggers large increases of [Ca2+]i. IcAMP is primarily a Na+ current that is blocked by extracellularly applied La3+ or by intracellular administration of Ca2+ chelators (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl, EGTA) and which exhibits a reversal potential of about -26 mV. We propose that IcAMP results from the opening of nonselective cation channels that are activated by intracellular Ca2+ and cAMP and which might play an important role in the regulation of insulin secretion from pancreatic beta-cells.