Regulation of glucose-dependent insulinotropic polypeptide release by protein in the rat

Glucose-dependent insulinotropic polypeptide (GIP) release has been demonstrated predominantly after ingestion of carbohydrate and fat. These studies were conducted to determine the effects of protein on GIP expression in the rat. Whereas no significant changes in duodenal mucosal GIP mRNA levels were detected in response to peptone, the duodenal GIP concentration increased from 8.4+/-1.5 to 19.8+/-3.2 ng GIP/mg protein at 120 min (P<0.01). Plasma GIP levels also increased from 95+/-5.2 pg/ml to a peak of 289+/-56.1 pg/ml at 120 min (P<0.01). To determine whether the effects of protein on GIP were due to stimulation of acid secretion, rats were pretreated with 10 mg/kg omeprazole, after which mucosal and plasma GIP concentrations were partially attenuated. To further examine the effects of luminal acid, rats were administered intraduodenal 0.1 M HCl for 120 min, which significantly enhanced GIP expression. These studies indicate that nutrient protein provides a potent stimulus for GIP expression in the rat, an effect that occurs at the posttranslational level and may be mediated in part through the acid-stimulatory properties of protein. The effects of acid on GIP are consistent with a role for GIP as an enterogastrone in the rat.     

Endocytosis as a mechanism for tyrosine kinase-dependent suppression of a voltage-gated potassium channel

The voltage-gated potassium channel Kv1.2 undergoes tyrosine phosphorylation-dependent suppression of its ionic current. However, little is known about the physical mechanism behind that process. We have found that the Kv1.2 alpha-subunit protein undergoes endocytosis in response to the same stimuli that evoke suppression of Kv1.2 ionic current. The process is tyrosine phosphorylation-dependent because the same tyrosine to phenylalanine mutation in the N-terminus of Kv1.2 that confers resistance to channel suppression (Y132F) also confers resistance to channel endocytosis. Overexpression of a dominant negative form of dynamin blocked stimulus-induced Kv1.2 endocytosis and also blocked suppression of Kv1.2 ionic current. These data indicate that endocytosis of Kv1.2 from the cell surface is a key mechanism for channel suppression by tyrosine kinases.     

Activation of lipoprotein lipase by glucose-dependent insulinotropic polypeptide in adipocytes. A role for a protein kinase B, LKB1, and AMP-activated protein kinase cascade

Glucose-dependent insulinotropic polypeptide (GIP) has been mainly studied because of its glucose-dependent insulinotropic action and its ability to regulate beta-cell proliferation and survival. Considerably less is known about the effects of GIP on fat metabolism, and the present study was directed at identifying the mechanisms underlying its stimulatory action on lipoprotein lipase (LPL). In differentiated 3T3-L1 adipocytes, GIP, in the presence of insulin, increased LPL activity and triglyceride accumulation through a pathway involving increased phosphorylation of protein kinase B (PKB) and reductions in phosphorylated LKB1 and AMP-activated protein kinase (AMPK). Knockdown of AMPK using RNA interference and application of the AMPK inhibitor, Compound C, supported this conclusion. In contrast, the other major incretin hormone, glucagon-like peptide-1, exhibited no significant effects on LPL activity or PKB, LKB1, or AMPK phosphorylation. Cultured subcutaneous human adipocytes showed similar responses to GIP but with greater sensitivity. Chronic elevation of circulating GIP levels in the Vancouver diabetic fatty Zucker rat in vivo resulted in increased LPL activity and elevated triglyceride accumulation in epididymal fat tissue, combined with a modulation of PKB, LKB1, and AMPK phosphorylation similar to that observed in vitro. This appears to be the first demonstration of a GIP-stimulated signal transduction pathway involved in increasing fat storage in adipocytes.     

Expression of recombinant human glucose-dependent insulinotropic polypeptide in Escherichia coli by sequence-specific proteolysis of a protein A fusion protein

Glucose-dependent insulinotropic polypeptide (GIP) is a forty-two amino acid hormone that stimulates the secretion of insulin from the pancreatic B-cells in the presence of elevated glucose concentrations. The human GIP gene with the human A-fibrinopeptide sequence was synthesized and linked to the Staphylococcus aureus protein A gene in the vector pRIT2T. This plasmid was expressed in Escherichia coli, and the resulting fusion protein consisted of three domains: protein A for ease of purification, fibrinopeptide sequence for thrombin cleavage and human GIP. The GIP was subsequently cleaved from the fusion protein with -thrombin. The identity of the recombinant human GIP was confirmed by SDS-PAGE, ELISA, HPLC and amino-terminal amino acid sequence analysis. This recombinant product was shown to have comparable insulinotropic activity to porcine GIP in the isolated perfused pancreas.     

Characterization of the cellular and metabolic effects of a novel enzyme-resistant antagonist of glucose-dependent insulinotropic polypeptide

A novel N-terminally substituted Pro(3) analogue of glucose-dependent insulinotropic polypeptide (GIP) was synthesized and tested for plasma stability and biological activity both in vitro and in vivo. Native GIP was rapidly degraded by human plasma with only 39 +/- 6% remaining intact after 8 h, whereas (Pro(3))GIP was completely stable even after 24 h. In CHL cells expressing the human GIP receptor, (Pro(3))GIP antagonized the cyclic adenosine monophosphate (cAMP) stimulatory ability of 10(-7) M native GIP, with an IC(50) value of 2.6 microM. In the clonal pancreatic beta cell line BRIN-BD11, (Pro(3))GIP over the concentration range 10(-13) to 10(-8) M dose dependently inhibited GIP-stimulated (10(-7) M) insulin release (1.2- to 1.7-fold; P < 0.05 to P < 0.001). In obese diabetic (ob/ob) mice, intraperitoneal administration of (Pro(3))GIP (25 nmol/kg body wt) countered the ability of native GIP to stimulate plasma insulin (2.4-fold decrease; P < 0.001) and lower the glycemic excursion (1.5-fold decrease; P < 0.001) induced by a glucose load (18 mmol/kg body wt). Collectively these data demonstrate that (Pro(3))GIP is a novel and potent enzyme-resistant GIP receptor antagonist capable of blocking the ability of native GIP to increase cAMP, stimulate insulin secretion, and improve glucose homeostasis in a commonly employed animal model of type 2 diabetes.     

Cloning and expression of a human voltage-gated potassium channel. A novel member of the RCK potassium channel family.

We have isolated and characterized a human cDNA (HBK2) that is homologous to novel member (RCK2) of the K+ channel RCK gene family expressed in rat brain. RCK2 mRNA was detected predominantly in midbrain areas and brainstem. The primary sequences of the HBK2/RCK2 K+ channel proteins exhibit major differences to other members of the RCK gene family. The bend region between segments S1 and S2 is unusually long and does not contain the N-glycosylation site commonly found in this region. They might be O-glycosylated instead. Functional characterization of the HBK2/RCK2 K+ channels in Xenopus laevis oocytes following micro-injection in in vitro transcribed HBK2 or RCK2 cRNA showed that the HBK2/RCK2 proteins form voltage-gated K+ channels with novel functional and pharmacological properties. These channels are different to RCK1, RCK3, RCK4 and RCK5 K+ channels.     

Indirect RT-PCR in-situ hybridization: a novel non-radioactive method for detecting glucose-dependent insulinotropic peptide

To establish indirect in-situ PCR for the detection of intestinal peptide hormones, rat intestine and a murine intestinal tumor cell line, STC 1, were used. The results exhibited intensive staining of GIP-producing K-cells. Paraformaldehyde-fixed cryostat sections yielded the best results in signal to background ratio with RT-PCR in-situ hybridization. Moreover, it was possible to elevate the positive staining signal and to reduce background staining. Digoxigenin-labeled in-situ hybridization served as a control for specificity and sensitivity of GIP (glucose-dependent insulinotropic peptide) mRNA expression on cryostat as well as paraffin sections. In conclusion, this RT-PCR in-situ hybridization protocol proves to be a specific, sensitive and reliable non-radioactive technique for the detection of intestinal peptide hormone mRNA, especially in tissues or tumor cells where the application of ISH is limited.     

Prohormone convertase 1/3 is essential for processing of the glucose-dependent insulinotropic polypeptide precursor

The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor. This study investigates the processing of proGIP in PC1/3 and PC2 null mice and in cell lines using adenovirus-mediated overexpression. Supporting a role for PC1/3 in proGIP processing, we found co-localization of GIP and PC1/3 but not PC2 in intestinal sections by immunohistochemistry, and analysis of intestinal extracts from PC1/3-deficient animals demonstrated severely impaired processing to GIP, whereas processing to GIP was unaltered in PC2-deficient mice. Accordingly, overexpression of preproGIP in the neuroendocrine AtT-20 cell line that expresses high levels of endogenous PC1/3 and negligible levels of PC2 resulted in production of GIP. Similar results were obtained after co-expression of preproGIP and PC1/3 in GH4 cells that express no PC2 and only low levels of PC1/3. In addition, studies in GH4 cells and the alpha-TC1.9 cell line, expressing PC2 but not PC1/3, indicate that PC2 can mediate processing to GIP but also to other fragments not found in intestinal extracts. Taken together, our data indicate that PC1/3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells.     

A novel current pathway parallel to the central pore in a mutant voltage-gated potassium channel

Voltage-gated potassium channels are proteins composed of four subunits consisting of six membrane-spanning segments S1-S6, with S4 as the voltage sensor. The region between S5 and S6 forms the potassium-selective ion-conducting central α-pore. Recent studies showed that mutations in the voltage sensor of the Shaker channel could disclose another ion permeation pathway through the voltage-sensing domain (S1-S4) of the channel, the ω-pore. In our studies we used the voltage-gated hKv1.3 channel, and the insertion of a cysteine at position V388C (Shaker position 438) generated a current through the α-pore in high potassium outside and an inward current at hyperpolarizing potentials carried by different cations like Na(+), Li(+), Cs(+), and NH(4)(+). The observed inward current looked similar to the ω-current described for the R1C/S Shaker mutant channel and was not affected by some pore blockers like charybdotoxin and tetraethylammonium but was inhibited by a phenylalkylamine blocker (verapamil) that acts from the intracellular side. Therefore, we hypothesize that the hKv1.3_V388C mutation in the P-region generated a channel with two ion-conducting pathways. One, the α-pore allowing K(+) flux in the presence of K(+), and the second pathway, the σ-pore, functionally similar but physically distinct from the ω-pathway. The entry of this new pathway (σ-pore) is presumably located at the backside of Y395 (Shaker position 445), proceeds parallel to the α-pore in the S6-S6 interface gap, ending between S5 and S6 at the intracellular side of one α-subunit, and is blocked by verapamil.     

A novel mechanism for mitogen-activated protein kinase localization

Mitogen-activated protein kinases/extracellular signal regulated kinases (MAPKs/ERKs) are typically thought to be soluble cytoplasmic enzymes that translocate to the nucleus subsequent to their phosphorylation by their activating kinases or mitogen-activated protein/extracellular signal regulated kinase kinase. We report here the first example of nuclear translocation of a MAPK that occurs via temporally regulated exit from a membranous organelle. Confocal microscopy examining the subcellular localization of ERK3 in several cell lines indicated that this enzyme was targeted to the Golgi/endoplasmic reticulum Golgi intermediate compartment. Deletion analysis of green fluorescent protein (GFP)-ERK3 uncovered a nuclear form that was carboxy-terminally truncated and established a Golgi targeting motif at the carboxy terminus. Immunoblot analysis of cells treated with the proteasome inhibitor MG132 further revealed two cleavage products, suggesting that in vivo, carboxy-terminal cleavage of the full-length protein controls its subcellular localization. In support of this hypothesis, we found that deletion of a small region rich in acidic residues within the carboxy terminus eliminated both the cleavage and nuclear translocation of GFP-ERK3. Finally, cell cycle synchronization studies revealed that the subcellular localization of ERK3 is temporally regulated. These data suggest a novel mechanism for the localization of an MAPK family member, ERK3, in which cell cycle-regulated, site-specific proteolysis generates the nuclear form of the protein.     

Catalytic mechanism and substrate specificity of the beta-subunit of the voltage-gated potassium channel

The beta-subunits of voltage-gated potassium (Kv) channels are members of the aldo-keto reductase (AKR) superfamily. These proteins regulate inactivation and membrane localization of Kv1 and Kv4 channels. The Kvbeta proteins bind to pyridine nucleotides with high affinity; however, their catalytic properties remain unclear. Here we report that recombinant rat Kvbeta2 catalyzes the reduction of a wide range of aldehydes and ketones. The rate of catalysis was slower (0.06-0.2 min(-1)) than those of most other AKRs but displayed the expected hyperbolic dependence on substrate concentration, with no evidence of allosteric cooperativity. Catalysis was prevented by site-directed substitution of Tyr-90 with phenylalanine, indicating that the acid-base catalytic residue, identified in other AKRs, has a conserved function in Kvbeta2. The protein catalyzed the reduction of a broad range of carbonyls, including aromatic carbonyls, electrophilic aldehydes and prostaglandins, phospholipids, and sugar aldehydes. Little or no activity was detected with carbonyl steroids. Initial velocity profiles were consistent with an ordered bi-bi rapid equilibrium mechanism in which NADPH binding precedes carbonyl binding. Significant primary kinetic isotope effects (2.0-3.1) were observed under single- and multiple-turnover conditions, indicating that the bond-breaking chemical step is rate-limiting. Structure-activity relationships with a series of para-substituted benzaldehydes indicated that the electronic interactions predominate during substrate binding and that no significant charge develops during the transition state. These data strengthen the view that Kvbeta proteins are catalytically active AKRs that impart redox sensitivity to Kv channels.     

A novel mechanism for activation of the protein kinase Aurora A

Segregation of chromosomes during mitosis requires interplay between several classes of protein on the spindle, including protein kinases, protein phosphatases, and microtubule binding motor proteins [1-4]. Aurora A is an oncogenic cell cycle-regulated protein kinase that is subject to phosphorylation-dependent activation [5-11]. Aurora A localization to the mitotic spindle depends on the motor binding protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2), but the protein(s) involved in Aurora A activation are unknown [11-13]. Here, we purify an activator of Aurora A from Xenopus eggs and identify it as TPX2. Remarkably, Aurora A that has been fully deactivated by Protein Phosphatase 2A (PP2A) becomes phosphorylated and reactivated by recombinant TPX2 in an ATP-dependent manner. Increased phosphorylation and activation of Aurora A requires its own kinase activity, suggesting that TPX2 stimulates autophosphorylation and autoactivation of the enzyme. Consistently, wild-type Aurora A, but not a kinase inactive mutant, becomes autophosphorylated on the regulatory T loop residue (Thr 295) after TPX2 treatment. Active Aurora A from bacteria is further activated at least 7-fold by recombinant TPX2, and TPX2 also impairs the ability of protein phosphatases to inactivate Aurora A in vitro. This concerted mechanism of stimulation of activation and inhibition of deactivation implies that TPX2 is the likely regulator of Aurora A activity at the mitotic spindle and may explain why loss of TPX2 in model systems perturbs spindle assembly [14-16]. Our finding that a known binding protein, and not a conventional protein kinase, is the relevant activator for Aurora A suggests a biochemical model in which the dynamic localization of TPX2 on mitotic structures directly modulates the activity of Aurora A for spindle assembly.     

Deletion of protein kinase A phosphorylation sites in the HERG potassium channel inhibits activation shift by protein kinase A.

We investigated the role of protein kinase A (PKA) in regulation of the human ether-a-go-go-related gene (HERG) potassium channel activation. HERG clones with single mutations destroying one of four consensus PKA phosphorylation sites (S283A, S890A, T895A, S1137A), as well as one clone carrying all mutations with no PKA phosphorylation sites (HERG 4M) were constructed. These clones were expressed heterologously in Xenopus oocytes, and HERG potassium currents were measured with the two microelectrode voltage clamp technique. Application of the cAMP-specific phosphodiesterase (PDE IV) inhibitor Ro-20-1724 (100 microM), which results in an increased cAMP level and PKA stimulation, induced a reduction of HERG wild type outward currents by 19.1% due to a shift in the activation curve of 12.4 mV. When 100 microM Ro-20-1724 was applied to the HERG 4M channel, missing all PKA sites, there was no significant shift in the activation curve, and the current amplitude was not reduced. Furthermore, the adenylate cyclase activator forskolin that leads to PKA activation (400 microM, 60 min), shifted HERG wild type channel activation by 14.1 mV and reduced currents by 39.9%, whereas HERG 4M channels showed only a small shift of 4.3 mV and a weaker current reduction of 22.3%. We conclude that PKA regulates HERG channel activation, and direct phosphorylation of the HERG channel protein has a functional role that may be important in regulation of cardiac repolarization.     

Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

Retinal progenitor cells （RPCs） are neural stem cells able to differentiate into any normal adult retinal cell type, except for pigment epithelial cells. Retinoic acid （RA） is a powerful growth/differentiation factor that generally causes growth inhibition, differentiation and/or apoptosis. In this study, we demonstrate that RA not only affects mouse RPC differentiation but also improves cell survival by reducing spontaneous apoptotic rate without affecting RPC proliferation. The enhanced cell survival was accompanied by a significant upregulation of the expression of protein kinase A （PKA） and several protein kinase C （PKC） isoforms. Treatment of cells grown in RA-free media with 8-bromoadenosine3＇,5＇-cyclic monophosphate, a known activator of PKA, resulted in an anti-apoptotic effect similar to that caused by RA; whereas the PKA inhibitor N-[2-（p-bromocinnamylamino）ethyl]-5-isoquinolinesul- fonamide dihydrochloride led to a significant （-32%） increase in apoptosis. In contrast, treatment of RPCs with any of two PKC selective inhibitors, 2,2＇,3,3＇,4,4＇-hexahydroxy-1,1 ＇-biphenyl-6,6＇-dimethanol dimethyl ether and bisindolylmaleimide XI, led to diminished apoptosis; while a PKC activator, phorbol 12-myristate 13-acetate, increased apoptosis. These and other data suggest that the effect of RA on RPC survival is mostly due to the increased anti-apoptotic activity elicited by PKA, which might in turn be antagonized by PKC. Such a mechanism is a new example of tight regulation of important biological processes triggered by RA. Although the detailed mechanisms remain to be elucidated, we provide evidence that the pro-survival effect of RA on RPCs is not mediated by changed expression of p53 or bcl-2, and appears to be independent of 15-amyloid, Fas ligand, TNF-α, ganglioside GM1 and ceramide C 16-induced apoptotic pathways.     

A new pathway for glucose-dependent insulinotropic polypeptide (GIP) receptor signaling: evidence for the involvement of phospholipase A2 in GIP-stimulated insulin secretion

The hormone glucose-dependent insulinotropic polypeptide (GIP) is an important regulator of insulin secretion. GIP has been shown to increase adenylyl cyclase activity, elevate intracellular Ca(2+) levels, and stimulate a mitogen-activated protein kinase pathway in the pancreatic beta-cell. In the current study we demonstrate a role for arachidonic acid in GIP-mediated signal transduction. Static incubations revealed that both GIP (100 nm) and ATP (5 microm) significantly increased [(3)H]arachidonic acid ([(3)H]AA) efflux from transfected Chinese hamster ovary K1 cells expressing the GIP receptor (basal, 128 +/- 11 cpm/well; GIP, 212 +/- 32 cpm/well; ATP, 263 +/- 35 cpm/well; n = 4; p < 0.05). In addition, GIP receptors were shown for the first time to be capable of functionally coupling to AA production through Gbetagamma dimers in Chinese hamster ovary K1 cells. In a beta-cell model (betaTC-3), GIP was found to elicit [(3)H]AA release, independent of glucose, in a concentration-dependent manner (EC(50) value of 1.4 +/- 0.62 nm; n = 3). Although GIP did not potentiate insulin release under extracellular Ca(2+)-free conditions, it was still capable of elevating intracellular cAMP and stimulating [(3)H]AA release. Our data suggest that cAMP is the proximal signaling intermediate responsible for GIP-stimulated AA release. Finally, stimulation of GIP-mediated AA production was shown to be mediated via a Ca(2+)-independent phospholipase A(2). Arachidonic acid is therefore a new component of GIP-mediated signal transduction in the beta-cell.     

Novel mechanism for regulation of epidermal growth factor receptor endocytosis revealed by protein kinase A inhibition

Current models put forward that the epidermal growth factor receptor (EGFR) is efficiently internalized via clathrin-coated pits only in response to ligand-induced activation of its intrinsic tyrosine kinase and is subsequently directed into a lysosomal-proteasomal degradation pathway by mechanisms that include receptor tyrosine phosphorylation and ubiquitylation. Herein, we report a novel mechanism of EGFR internalization that does not require ligand binding, receptor kinase activity, or ubiquitylation and does not direct the receptor into a degradative pathway. Inhibition of basal protein kinase A (PKA) activity by H89 and the cell-permeable substrate peptide Myr-PKI induced internalization of 40-60% unoccupied, inactive EGFR, and its accumulation into early endosomes without affecting endocytosis of transferrin and mu-opioid receptors. This effect was abrogated by interfering with clathrin function. Thus, the predominant distribution of inactive EGFR at the plasma membrane is not simply by default but involves a PKA-dependent restrictive condition resulting in receptor avoidance of endocytosis until it is stimulated by ligand. Furthermore, PKA inhibition may contribute to ligand-induced EGFR endocytosis because epidermal growth factor inhibited 26% of PKA basal activity. On the other hand, H89 did not alter ligand-induced internalization of EGFR but doubled its half-time of down-regulation by retarding its segregation into degradative compartments, seemingly due to a delay in the receptor tyrosine phosphorylation and ubiquitylation. Our results reveal that PKA basal activity controls EGFR function at two levels: 1) residence time of inactive EGFR at the cell surface by a process of "endocytic evasion," modulating the accessibility of receptors to stimuli; and 2) sorting events leading to the down-regulation pathway of ligand-activated EGFR, determining the length of its intracellular signaling. They add a new dimension to the fine-tuning of EGFR function in response to cellular demands and cross talk with other signaling receptors.     

Sphingosine activates protein kinase A type II by a novel cAMP-independent mechanism

Protein kinase A (PKA) has long been recognized as playing a major role in many regulatory processes in cells through its activation by the ubiquitous second messenger cAMP. We show here a novel mode of activation of PKA type II that is independent of cAMP and is, instead, dependent on sphingosine. PKA type II is specifically activated by sphingosine and its analog, dimethylsphingosine, but not by sphingosine-1-phosphate or other lipids. Like cAMP, sphingosine activates PKA holoenzyme but not the catalytic subunit alone, suggesting that the activation is mediated by the regulatory subunits. However, sphingosine-activated PKA, but not cAMP-activated PKA, is inhibited by phosphatidylserine, suggesting a distinct mechanism of activation. Furthermore, unlike cAMP, sphingosine does not induce the dissociation of PKA holoenzyme into catalytic and regulatory subunits. Modulation of sphingosine levels in vivo results in alteration in basal membrane-associated PKA activity consistent with a direct effect of membrane sphingosine on PKA type II. Importantly, sphingosine-dependent but not cAMP-dependent activation of PKA specifically phosphorylates Ser58 of the multifunctional adapter protein 14-3-3zeta, promoting the conversion of dimeric 14-3-3 to a monomeric state, thus potentially modulating several biological functions. These results define a new mode of PKA activation that is sphingosine-dependent and mechanistically different from the classical cAMP-dependent activation of PKA. Furthermore, they suggest that stimuli that induce sphingosine accumulation and modulate phospholipid content at the cell membrane have the potential to activate PKA, thereby inducing the phosphorylation of distinct substrates and biological activities.     

Diets influence the diabetic phenotype of transgenic mice expressing a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn)

Transgenic mice overexpressing a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPR(dn)) have recently been shown to develop diabetes mellitus due to disturbed postnatal development of the endocrine pancreas. In this study, the effects of feeding a high fibre/low calorie diet on the diabetic phenotype of GIPR(dn) transgenic mice were examined. Transgenic and control animals received either a conventional breeding diet (BD) or a high fibre diet (HF). Both fasting and postprandial blood glucose levels and HbA1C levels were largely elevated in transgenic mice vs. controls (p<0.05), irrespective of the diet fed. Food and water intake and the daily urine volume of GIPR(dn) transgenic mice were higher than that of control mice (p<0.05). Transgenic animals receiving the HF diet showed significantly lower blood glucose and HbA1C levels as well as less food and water intake than transgenic mice fed BD. The 365-day survival of transgenic mice was significantly lower than that of control mice. Transgenic animals fed the HF diet lived significantly longer than their counterparts receiving BD. GIPR(dn) transgenic mice develop a severe diabetic phenotype which can be ameliorated by a HF diet, thereby resembling some aspects of the pathophysiology of human type 2 diabetes mellitus.     

Molecular cloning and characterization of LKv1, a novel voltage-gated potassium channel in leech

We have cloned a novel voltage-gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage-gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I(K). Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage-gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker-related K channels in jellyfish. Thus, this analysis indicates that this group of voltage-gated K channels contains several evolutionarily divergent lineages.     

Phosphorylation of the A-kinase-anchoring protein Yotiao contributes to protein kinase A regulation of a heart potassium channel

Regulation of the heart by the sympathetic nervous system, fundamental to the physiological response to stress and exercise, requires coordinated phosphorylation of multiple downstream molecular targets, including the I(Ks) (slowly activating potassium current) channel. Sympathetic nervous system stimulation increases intracellular cAMP for which targeted regulation is directed in large part by distinct scaffold or anchoring proteins. Yotiao is an A-kinase-anchoring protein (AKAP) that recruits the cyclic AMP-dependent protein kinase (protein kinase A (PKA)) and protein phosphatase 1 to the carboxyl terminus of the I(Ks) channel to form a molecular complex and control its phosphorylation state, crucial to the cardiac cellular response to sympathetic nervous system stimulation. Here we report that Yotiao itself is a substrate for PKA phosphorylation, and we identify a Yotiao amino-terminal (N-T) residue (Ser-43) that is PKA-phosphorylated in response to beta-adrenergic receptor stimulation. The replacement of Ser-43 by Ala ablates the PKA phosphorylation of N-T Yotiao and markedly diminishes the functional response of the wild type and pseudo-phosphorylated I(Ks) channel to cAMP but neither prevents the PKA phosphorylation of KCNQ1 nor its binding to Yotiao. These results suggest, for the first time, a critical role for the PKA phosphorylation of an AKAP in the functional regulation of an ion channel protein and postphosphorylation allosteric modulation of the I(Ks) channel by Yotiao.