A signaling role of glutamine in insulin secretion

Children with hypoglycemia due to recessive loss of function mutations of the beta-cell ATP-sensitive potassium (K(ATP)) channel can develop hypoglycemia in response to protein feeding. We hypothesized that amino acids might stimulate insulin secretion by unknown mechanisms, because the K(ATP) channel-dependent pathway of insulin secretion is defective. We therefore investigated the effects of amino acids on insulin secretion and intracellular calcium in islets from normal and sulfonylurea receptor 1 knockout (SUR1-/-) mice. Even though SUR1-/- mice are euglycemic, their islets are considered a suitable model for studies of the human genetic defect. SUR1-/- islets, but not normal islets, released insulin in response to an amino acid mixture ramp. This response to amino acids was decreased by 60% when glutamine was omitted. Insulin release by SUR1-/- islets was also stimulated by a ramp of glutamine alone. Glutamine was more potent than leucine or dimethyl glutamate. Basal intracellular calcium was elevated in SUR1-/- islets and was increased further by glutamine. In normal islets, methionine sulfoximine, a glutamine synthetase inhibitor, suppressed insulin release in response to a glucose ramp. This inhibition was reversed by glutamine or by 6-diazo-5-oxo-l-norleucine, a non-metabolizable glutamine analogue. High glucose doubled glutamine levels of islets. Methionine sulfoximine inhibition of glucose stimulated insulin secretion was associated with accumulation of glutamate and aspartate. We hypothesize that glutamine plays a critical role as a signaling molecule in amino acid- and glucose-stimulated insulin secretion, and that beta-cell depolarization and subsequent intracellular calcium elevation are required for this glutamine effect to occur.     

Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities

The SCF ubiquitin ligase complex regulates diverse cellular functions by ubiquitinating numerous protein substrates. Cand1, a 120 kDa HEAT repeat protein, forms a tight complex with the Cul1-Roc1 SCF catalytic core, inhibiting the assembly of the multisubunit E3 complex. The crystal structure of the Cand1-Cul1-Roc1 complex shows that Cand1 adopts a highly sinuous superhelical structure, clamping around the elongated SCF scaffold protein Cul1. At one end, a Cand1 beta hairpin protrusion partially occupies the adaptor binding site on Cul1, inhibiting its interactions with the Skp1 adaptor and the substrate-recruiting F box protein subunits. At the other end, two Cand1 HEAT repeats pack against a conserved Cul1 surface cleft and bury a Cul1 lysine residue, whose modification by the ubiquitin-like protein, Nedd8, is able to block Cand1-Cul1 association. Together with biochemical evidence, these structural results elucidate the mechanisms by which Cand1 and Nedd8 regulate the assembly-disassembly cycles of SCF and other cullin-dependent E3 complexes.     

Activation of the p70 S6 kinase and phosphorylation of the 4E-BP1 repressor of mRNA translation by type I interferons

The Type I IFN receptor-generated signals required for initiation of mRNA translation and, ultimately, induction of protein products that mediate IFN responses, remain unknown. We have previously shown that IFNalpha and IFNbeta induce phosphorylation of insulin receptor substrate proteins and downstream engagement of the phosphatidylinositol (PI) 3'-kinase pathway. In the present study we provide evidence for the existence of a Type I IFN-dependent signaling cascade activated downstream of PI 3'-kinase, involving p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated on threonine 421 and serine 424 and is activated during treatment of cells with IFNalpha or IFNbeta. Such activation of p70 S6K is blocked by pharmacological inhibitors of the PI 3'-kinase or the FKBP 12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR). Consistent with this, the Type I IFN-dependent phosphorylation/activation of p70 S6K is defective in embryonic fibroblasts from mice with targeted disruption of the p85alpha and p85beta subunits of the PI 3'-kinase (p85alpha-/-beta-/-). Treatment of sensitive cell lines with IFNalpha or IFNbeta also results in phosphorylation/inactivation of the 4E-BP-1 repressor of mRNA translation. Such 4E-BP1 phosphorylation is also PI3'-kinase-dependent and rapamycin-sensitive, indicating that the Type I IFN-inducible activation of PI3'-kinase and FRAP/mTOR results in dissociation of 4E-BP1 from the eukaryotic initiation factor-4E (eIF4E) complex. Altogether, our data establish that the Type I IFN receptor-activated PI 3'-kinase pathway mediates activation of the p70 S6 kinase and inactivation of 4E-BP1, to regulate mRNA translation and induction of Type I IFN responses.     

Single chain antibodies specific for fatty acids derived from a semi-synthetic phage display library

The biological activities of many acylated molecules are lipid dependent. Lipids, however, are poorly immunogenic or non-immunogenic. We employed a phage display semi-synthetic human antibody library to isolate anti-lipid antibodies. Selection was done against methyl palmitate, a 16 carbon aliphatic chain, and a major component of bacterial glycolipids and lipoproteins in animal cells. The selected single chain variable fragment (scFv) bound specifically to a 16 carbon aliphatic chain and to a lesser extent to a 14 or 18 carbon aliphatic chain and poorly to either 12, 22 or 8 carbon aliphatic chains. Furthermore, the scFv prevented micelle formation of lipoteichoic acid from Gram-positive bacteria; inhibited lipopolysaccharide-induced tumor necrosis factor alpha release in mononuclear cells; bound to hydrophobic bacterial surfaces, especially those of Gram-positive bacteria, and bound to Lck, a mammalian palmitated lipoprotein. Our data suggest that the phage antibody library can be successfully employed to obtain human anti-aliphatic scFv human antibody fragment with potential therapeutic applications in neutralizing the deleterious effects of bacterial toxins as well as in structure--function analysis of lipoproteins in animal cells.     

Regulation of urea synthesis by agmatine in the perfused liver: studies with 15N

Administration of arginine or a high-protein diet increases the hepatic content of N-acetylglutamate (NAG) and the synthesis of urea. However, the underlying mechanism is unknown. We have explored the hypothesis that agmatine, a metabolite of arginine, may stimulate NAG synthesis and, thereby, urea synthesis. We tested this hypothesis in a liver perfusion system to determine 1) the metabolism of l-[guanidino-15N2]arginine to either agmatine, nitric oxide (NO), and/or urea; 2) hepatic uptake of perfusate agmatine and its action on hepatic N metabolism; and 3) the role of arginine, agmatine, or NO in regulating NAG synthesis and ureagenesis in livers perfused with 15N-labeled glutamine and unlabeled ammonia or 15NH4Cl and unlabeled glutamine. Our principal findings are 1) [guanidino-15N2]agmatine is formed in the liver from perfusate l-[guanidino-15N2]arginine ( approximately 90% of hepatic agmatine is derived from perfusate arginine); 2) perfusions with agmatine significantly stimulated the synthesis of 15N-labeled NAG and [15N]urea from 15N-labeled ammonia or glutamine; and 3) the increased levels of hepatic agmatine are strongly correlated with increased levels and synthesis of 15N-labeled NAG and [15N]urea. These data suggest a possible therapeutic strategy encompassing the use of agmatine for the treatment of disturbed ureagenesis, whether secondary to inborn errors of metabolism or to liver disease.     

Generation and epitope mapping of high-affinity scFv to eukaryotic elongation factor 1A by dual application of phage display.

To generate specific tools for, in particular, localization studies of the eukaryotic elongation factor 1A (eEF1A), we have applied phage display in various formats to affinity-improve and map epitopes of two previously isolated, low-affinity single-chain Fv (scFv) G3 and D1. The scFv differ in their reactivity toward the eEF1A isoforms, eEF1A-1 and eEF1A-2. By PCR-based randomization of six residues within the variable light chain CDR3 (LCDR3), and subsequent phage-based affinity-selection, two 'families' of affinity-improved scFv were obtained. The scFv of highest affinity, A8, has a Kd of 9 nM to eEF1A-1. Interestingly, two affinity-improved scFvs have abnormally short LCDR3 consisting of two and four residues compared to 11 in the parental scFv. Hence, the LCDR3 of the parental clones may play a modulating rather than a direct role in antigen-binding. Despite different preferences for the eEF1A isoforms, both families of scFv recognize antigenic determinant(s), which was mapped to residues 413-450 of eEF1A-1/2 by Western blot analysis of recombinant human eEF1A (hEF1A) fragments. Prior to the Western blotting analysis, the epitope location had been suggested using a novel approach where phage-antibody repertoire derived scFv were used to select phage-displayed peptides. Hereby, peptides containing a SFXD motif, matching the SFSD(414-418) sequence found in hEF1A-1 were isolated. The structure of eukaryotic EF1A from yeast indicates a discontinuous nature of the epitope with distal functional elements juxtaposed by the protein fold. Finally, the scFv A8 was applied for immunofluorescence studies of transformed human amnion cells and MCF-7 fibroblasts. In both cases a perinuclear localization of hEF1A was observed. No evidence for the reported nuclear localization of hEF1A was obtained.     

Effects of Ketone Bodies on Astrocyte Amino Acid Metabolism

Abstract: The effects of acetoacetate and 3-hydroxybutyrate on glial amino acid metabolism were studied in primary cultures of astrocytes. The exchange of nitrogen among amino acids was measured with ¹⁵N as a metabolic probe and gas chromatography-mass spectrometry as a tool with which to quantify isotope abundance. Addition of either acetoacetate or 3-hydroxybutyrate (5 m M ) to the incubation medium did not alter the initial rate of appearance of [¹⁵N]glutamate in the glia, but it did inhibit transamination of glutamate to [¹⁵N]aspartate. Addition of acetoacetate also inhibited formation of [2-¹⁵N]glutamine, but 3-hydroxybutyrate had a stimulatory effect. The presence in the medium of sodium acetate (5 m M ) was also associated with diminished production of [¹⁵N]aspartate and [2-¹⁵N]glutamine with [¹⁵N]glutamate as precursor. Studies with [2-¹⁵N]glutamine as precursor indicated that treatment of the astrocytes with ketone bodies did not alter flux through the glutaminase pathway. Nor did the presence of the ketone bodies reduce significantly the flux of nitrogen from [¹⁵N]GABA to [2-¹⁵N]glutamine when the former species served as a metabolic tracer. The concentration of internal citrate increased in the presence of acetoacetate, 3-hydroxybutyrate, and acetate. Studies with purified sheep brain glutamine synthetase showed that citrate inhibited this enzyme. These findings are considered in terms of the known anticonvulsant effect of a ketogenic diet.     

Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.