Abnormalities of pancreatic exocrine function in obesity: studies in the obese mouse

Insulin is known to play a specific role in the biosynthesis of pancreatic amylase. In the insulin resistant adult C57 BL/6J--ob/ob mouse there is a reduction of pancreatic amylase content. The differences of enzyme content could not be explained by differences of food intake between obese and lean mice, but are more likely to be the consequence of insulin resistance at the level of the exocrine pancreas. By contrast, greater pancreatic content of amylase and lipase seen in young obese mice (less than 2-months old) was associated with the greater food intake of these mice with respect to lean controls.     

Dissection of the nucleotide and metal-phosphate binding sites in cAMP-dependent protein kinase.

The catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is more stable by several criteria when it is part of a holoenzyme complex. By measuring the thermal stability of the free C subunit in the presence and absence of nucleotides and/or divalent metal ions, it was found that most of the stabilizing effects associated with the type I holoenzyme could be attributed to the nucleotide. The specific requirements for this enhanced stability were further dissected: Adenosine stabilized the C subunit up to 5 degrees C; however, divalent cations (i.e., Mg2+, Ca2+, and Mn2+) do not increase heat stability in combination with adenosine and adenine (1). Divalent cations as well as ATP and ADP have no effect by themselves (2). The enhanced stability derived from both ATP and ADP requires divalent cations. MnATP (12 degrees C) shows a much stronger effect than CaATP (7 degrees C) and MgATP (5 degrees C) (3). In the holoenzyme complex or the protein kinase inhibitor/C subunit complex, metal/ATP is also required for enhanced stability; neither the RI or RII subunits nor PKI alone stabilize the C subunit significantly (4). For high thermal stability, the occupation of the second, low-affinity metal-binding site is necessary (5). From these results, we concluded that the adenine moiety works independently from the metal-binding sites, stabilizing the free C subunit by itself. When the beta- and gamma-phosphates are present, divalent metals are required for positioning these phosphates, and two metals are required to achieve thermostability comparable to adenosine alone. The complex containing two metals is the most stable. A comparison of several conformations of the C subunit derived from different crystal structures is given attributing open and closed forms of the C subunit to less and more thermostable enzymes, respectively.     

Comparative thermodynamic analysis of cyclic nucleotide binding to protein kinase A

We have investigated the thermodynamic parameters and binding of a regulatory subunit of cAMP-dependent protein kinase (PKA) to its natural low-molecular-weight ligand, cAMP, and analogues thereof. For analysis of this model system, we compared side-by-side isothermal titration calorimetry (ITC) with surface plasmon resonance (SPR). Both ITC and SPR analyses revealed that binding of the protein to cAMP or its analogues was enthalpically driven and characterised by similar free energy values (DeltaG=-9.4 to -10.7 kcal mol-1) for all interactions. Despite the similar affinities, binding of the cyclic nucleotides used here was characterised by significant differences in the contribution of entropy (-TDeltaS) and enthalpy (DeltaH) to DeltaG. The comparison of ITC and SPR data for one cAMP analogue further revealed deviations caused by the method. These equilibrium parameters could be complemented by thermodynamic data of the transition state (DeltaHnot equal, DeltaGnot equal, DeltaSnot equal) for both association and dissociation measured by SPR. This direct comparison of ITC and SPR highlights method-specific advantages and drawbacks for thermodynamic analyses of protein/ligand interactions.     

Cyclic nucleotides as affinity tools: phosphorothioate cAMP analogues address specific PKA subproteomes

cAMP (adenosine-3',5'-cyclic monophosphate) is a general second messenger controlling distinct targets in eukaryotic cells. In a (sub)proteomic approach, two classes of phosphorothioate cAMP affinity tools were used to isolate and to identify signalling complexes of the main cAMP target, cAMP dependent protein kinase (PKA). Agonist analogues (here: Sp-cAMPS) bind to the regulatory subunits of PKA (PKA-R), together with their interaction partners, and cause dissociation of a holoenzyme complex comprising PKA-R and catalytic subunits of PKA (PKA-C). Antagonist analogues (here: Rp-cAMPS) bind to the holoenzyme without dissociating the complex and were developed to identify interaction partners that bind to the entire complex or to PKA-C. More than 80 different proteins were isolated from tissue extracts including several PKA isoforms and known as well as potentially new interaction partners. Nevertheless, unspecific binding of general nucleotide binding proteins limited the outcome of this chemical proteomics approach. Surface plasmon resonance (SPR) was employed to optimise the entire workflow of pull down proteomics and to quantify the effects of different nucleotides (ATP, ADP, GTP and NADH) on PKA-R binding to affinity material. We could demonstrate that the addition of NADH to lysates improved specificity in pull down experiments. Using a combination of SPR studies and pull down experiments it was shown unambiguously that it is possible to specifically elute protein complexes with cAMP or cGMP from cAMPS analogue matrices. The side-by-side analysis of the PKA-R interactome and the holoenzyme complexed with interacting proteins will contribute to a further dissection of the multifaceted PKA signalling network.     

Identification of a novel A-kinase anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin-2 shuttle in renal principal cells

Arginine vasopressin (AVP) increases the water permeability of renal collecting duct principal cells by inducing the fusion of vesicles containing the water channel aquaporin-2 (AQP2) with the plasma membrane (AQP2 shuttle). This event is initiated by activation of vasopressin V2 receptors, followed by an elevation of cAMP and the activation of protein kinase A (PKA). The tethering of PKA to subcellular compartments by protein kinase A anchoring proteins (AKAPs) is a prerequisite for the AQP2 shuttle. During the search for AKAP(s) involved in the shuttle, a new splice variant of AKAP18, AKAP18delta, was identified. AKAP18delta functions as an AKAP in vitro and in vivo. In the kidney, it is mainly expressed in principal cells of the inner medullary collecting duct, closely resembling the distribution of AQP2. It is present in both the soluble and particulate fractions derived from renal inner medullary tissue. Within the particulate fraction, AKAP18delta was identified on the same intracellular vesicles as AQP2 and PKA. AVP not only recruited AQP2, but also AKAP18delta to the plasma membrane. The elevation of cAMP caused the dissociation of AKAP18delta and PKA. The data suggest that AKAP18delta is involved in the AQP2 shuttle.     

Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding

Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIalpha very well (apparent K(D) values of 0.5 and 2 nM, respectively). Both proteins also bind RIIbeta quite well, but with three- to fourfold lower affinities than those observed versus RIIalpha. However, only S-AKAP84/D-AKAP1 interacts with RIalpha at a nanomolar affinity (apparent K(D) of 185 nM). In comparison, AKAP95 binds RIIalpha (apparent K(D) of 5.9 nM) with a tenfold higher affinity than RIIbeta and has no detectable binding to RIalpha. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIalpha and RIIbeta to all the AKAPs examined (EC(50)-values from 6 to 360 nM). Furthermore, RIalpha interaction with S-AKAP84/D-AKAP1 was competed (EC(50) 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.     

Glucagon receptor-mediated activation of Gs is accompanied by subunit dissociation

The effect of the glucagon receptor on the activation of the stimulatory GTP-binding protein of adenylyl cyclase (Gs) in the native rat liver membrane environment was studied. The activated state of Gs was assessed by its ability to reconstitute the cyc- S49 cell membrane adenylyl cyclase. The Gs protein was activated by saturating concentrations of guanosine 5'-thiotriphosphate (GTP gamma S) or guanyl-5'-yl imidodiphosphate in a hormone-dependent manner at 0.4 mM Mg2+ in native membranes or in membranes that had been treated with 1 mM N-ethylmaleimide to eliminate the catalytic activity of adenylyl cyclase. At 50 mM Mg2+, Gs was fully activated by GTP gamma S in the absence of hormone. The unactivated Gs protein migrates around 4 S, whereas activated Gs migrates around 2 S on sucrose density gradients. When pure Gs is analyzed on sucrose density gradients, it is found that the unactivated protein migrates at 4.1 S. Gs was activated by saturating concentrations of GTP gamma S and Mg2+, and the alpha subunit of Gs was chromatographically purified. The resolved alpha subunit of Gs that is capable of stimulating the cyc- adenylyl cyclase migrates at 2.1 S. From these data, we conclude that activation of Gs results in the dissociation of this protein in the membrane environment and that the hormone-occupied receptor promotes this dissociation process under conditions where Mg2+ ions are limiting.     

The hepatic glucagon receptor. Solubilization, characterization, and development of an affinity adsorption assay for the soluble receptor

The hepatic glucagon receptor was covalently labeled with [125I-Try10]monoiodoglucagon [( 125I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the Mr = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [125I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f0 = 1.8, and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees C for 15 min prior to the addition of [125I]MIG permitted us to identify the high molecular weight form (Mr = approximately 113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The Mr = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [125I] MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [125I]MIG binding activity. [125I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [125I]MIG with the soluble receptor is insensitive to the presence of GTP. IC50 for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC50 in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the Mr = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.     

Impaired FAD-glycerophosphate dehydrogenase activity in islet and liver homogenates of fa/fa rats

The mitochondrial FAD-linked enzyme glycerophosphate dehydrogenase plays a key role in the pancreatic B-cell glucose sensing device. In the present study, the activity of this enzyme was examined in islets of fa/fa rats in which inherited diabetes mellitus is associated with obesity, hyperinsulinism and severe insulin resistance. The specific activity of both FAD-linked glycerophosphate dehydrogenase and glutamate dehydrogenase were decreased in islet and liver homogenates prepared from fa/fa, as compared to Fa/Fa, rats, this coinciding with a low ratio between glutamateoxalacetate and glutamate-pyruvate transaminase activity in both islet and liver extracts, islet hyperplasia, hyperinsulinemia and hepatic steatosis in the hyperglycemic fa/fa rats. It is speculated that a low activity of FAD-linked glycerophosphate dehydrogenase in the pancreatic B-cell may participate to the perturbation of glucose homeostasis in fa/fa rats, like in other animal models of non-insulin-dependent diabetes mellitus.     

Excitation-contraction coupling in a pre-vertebrate twitch muscle: The myotomes of Branchiostoma lanceolatum

Summary The segmented trunk muscle (myotome muscle) of the lancelet (Branchiostoma lanceolatum), a pre-vertebrate chordate, was studied in order to gain information regarding the evolution of excitation-contraction (EC) coupling.Myotome membrane vesicles could be separated on isopycnic sucrose gradients into two main fractions, probably comprising solitary microsomes and diads of plasma membrane and sarcoplasmic reticulum, respectively. Both fractions bound the dihydropyridine PN 200/110 and the phenylalkylamine (–)D888 (devapamil) while specific ryanodine binding was observed in the diad preparation only. Pharmacological effects on Ca²⁺ currents measured under voltage-clamp conditions in single myotome fibers included a weak block by the dihydropyridine nifedipine and a shift of the voltage dependences of inactivation and restoration to more negative potentials by (–)D888. After blocking the Ca²⁺ current by cadmium in voltage-clamped single fibers, the contractile response persisted and a rapid intramembrane charge movement could be demonstrated. Both responses exhibited a voltage sensitivity very similar to the one of the voltage-activated Ca²⁺ channels.Our biochemical and electrophysiological results indicate that the EC coupling mechanism of the protochordate myotome cell is similar to that of the vertebrate skeletal muscle fiber: Intracellular Ca²⁺ release, presumably taking place via the ryanodine receptor complex, is under control of the cell membrane potential. The sarcolemmal Ca²⁺ channels might serve as voltage sensors for this process.We thank Drs. H.Ch. Lüttgau and L.M.G. Heilmeyer, Jr. for stimulating discussions during the work, Dr. N.R. Brandt for helpful suggestions, and Drs. A.H. Caswell and M. Michalak for their generous gifts of antibodies. We also thank Ms. P. Goldmann, Mr. R. Schwalm, and Mr. U. Siemen for technical support and Ms. E. Linnepe for editorial help. This work was supported by grant G1 72/1-5 of the Deutsche Forschungsgemeinschaft. R. Benterbusch was recipient of a scholarship by the Studienstiftung des Deutschen Volkes.