Toward the active conformation of insulin: stereospecific modulation of a structural switch in the B chain

How insulin binds to the insulin receptor has long been a subject of speculation. Although the structure of the free hormone has been extensively characterized, a variety of evidence suggests that a conformational change occurs upon receptor binding. Here, we employ chiral mutagenesis, comparison of corresponding d and l amino acid substitutions, to investigate a possible switch in the B-chain. To investigate the interrelation of structure, function, and stability, isomeric analogs have been synthesized in which an invariant glycine in a beta-turn (Gly(B8)) is replaced by d- or l-Ser. The d substitution enhances stability (DeltaDeltaG(u) 0.9 kcal/mol) but impairs receptor binding by 100-fold; by contrast, the l substitution markedly impairs stability (DeltaDeltaG(u) -3.0 kcal/mol) with only 2-fold reduction in receptor binding. Although the isomeric structures each retain a native-like overall fold, the l-Ser(B8) analog exhibits fewer helix-related and long range nuclear Overhauser effects than does the d-Ser(B8) analog or native monomer. Evidence for enhanced conformational fluctuations in the unstable analog is provided by its attenuated CD spectrum. The inverse relationship between stereospecific stabilization and receptor binding strongly suggests that the B7-B10 beta-turn changes conformation on receptor binding.     

Chiral mutagenesis of insulin. Contribution of the B20-B23 beta-turn to activity and stability

Insulin contains a beta-turn (residues B20-B23) interposed between two receptor-binding elements, the central alpha-helix of the B chain (B9-B19) and its C-terminal beta-strand (B24-B28). The turn contains conserved glycines at B20 and B23. Although insulin exhibits marked conformational variability among crystal forms, these glycines consistently maintain positive phi dihedral angles within a classic type-I beta-turn. Because the Ramachandran conformations of GlyB20 and GlyB23 are ordinarily forbidden to L-amino acids, turn architecture may contribute to structure or function. Here, we employ "chiral mutagenesis," comparison of corresponding D- and L-Ala substitutions, to investigate this turn. Control substitutions are introduced at GluB21, a neighboring residue exhibiting a conventional (negative) phi angle. The D- and L-Ala substitutions at B23 are associated with a marked stereospecific difference in activity. Whereas the D-AlaB23 analog retains native activity, the L analog exhibits a 20-fold decrease in receptor binding. By contrast, D- and L-AlaB20 analogs each exhibit high activity. Stereospecific differences between the thermodynamic stabilities of the analogs are nonetheless more pronounced at B20 (delta deltaG(u) 2.0 kcal/mole) than at B23 (delta deltaG(u) 0.7 kcal/mole). Control substitutions at B21 are well tolerated without significant stereospecificity. Chiral mutagenesis thus defines the complementary contributions of these conserved glycines to protein stability (GlyB20) or receptor recognition (GlyB23).     

Vasodilator effects of L-arginine are stereospecific and augmented by insulin in humans

The amino acid l-arginine, the precursor of nitric oxide (NO) synthesis, induces vasodilation in vivo, but the mechanism behind this effect is unclear. There is, however, some evidence to assume that the l-arginine membrane transport capacity is dependent on insulin plasma levels. We hypothesized that vasodilator effects of l-arginine may be dependent on insulin plasma levels. Accordingly, we performed two randomized, double-blind crossover studies in healthy male subjects. In protocol 1 (n = 15), subjects received an infusion of insulin (6 mU x kg(-1) x min(-1) for 120 min) or placebo and, during the last 30 min, l-arginine or d-arginine (1 g/min for 30 min) x In protocol 2 (n = 8), subjects received l-arginine in stepwise increasing doses in the presence (1.5 mU x kg(-1) x min(-1)) or absence of insulin. Renal plasma flow and glomerular filtration rate were assessed by the para-aminohippurate and inulin plasma clearance methods, respectively. Pulsatile choroidal blood flow was assessed with laser interferometric measurement of fundus pulsation, and mean flow velocity in the ophthalmic artery was measured with Doppler sonography. l-arginine, but not d-arginine, significantly increased renal and ocular hemodynamic parameters. Coinfusion of l-arginine with insulin caused a dose-dependent leftward shift of the vasodilator effect of l-arginine. This stereospecific renal and ocular vasodilator potency of l-arginine is enhanced by insulin, which may result from facilitated l-arginine membrane transport, enhanced intracellular NO formation, or increased NO bioavailability.     

ACC synthase and its cognate E3 ligase are inversely regulated by light

1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the key enzyme in ethylene biosynthesis, catalyzing the conversion of S-adenosylmethionine (AdoMet) to ACC, which is the immediate precursor of ethylene. The regulation of ACS protein stability plays an important role in controlling ethylene biosynthesis. We have recently shown that 14-3-3 positively regulates ACS protein stability by both a direct effect and via downregulation of the stability of the E3 ligases regulating its turnover, Ethylene Overproducer1 (ETO1)/ETO1-like (EOL). Here, we report that treatment of etiolated Arabidopsis seedlings with light rapidly increases the stability of ACS5 protein. In contrast, light destabilizes the ETO1/EOLs proteins, suggesting that light acts to increase ethylene biosynthesis in part through a decrease in the level of the ETO1/EOL proteins. This demonstrates that the ETO1/EOLs are regulated in response to at least one environmental cue and that their regulated degradation may represent a novel input controlling ethylene biosynthesis.     

Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid.

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases, which include protein phosphatase-2A (PP2A) and protein phosphatase-1 (PP1) with roles in regulating apoptosis and cell growth. Thus far, in vitro studies on ceramide-activated protein phosphatases have been restricted to the use of short chain ceramides, limiting the extent of mechanistic insight. In this study, we show that the long chain D-erythro-C18-ceramide activated PP2A (AB'C trimer), PP2Ac (catalytic subunit of PP2A), and PP1gammac and -alphac (catalytic subunits of PP1gamma and -1alpha isoforms, respectively) 2-6-fold in the presence of dodecane, a lipid-solubilizing agent, with 50% maximal activation achieved at approximately 10 microM D-erythro-C18-ceramide. The diastereoisomers of D-erythroC18-ceramide, D-threo-, and L-threo-C18-ceramide, as well as the enantiomeric L-erythro-C18-ceramide, did not activate PP1 or PP2A, but they inhibited PP1 and PP2A activity. The addition of phosphatidic acid decreased the basal activity of PP1c but also increased the stimulation by D-erythro-C18-ceramide from 1.8- to 2. 8-fold and decreased the EC50 of D-erythro-C18-ceramide to 4.45 microM. The addition of 150 mM KCl decreased the basal activity of PP1 and the dose of D-erythro-C18-ceramide necessary to activate PP1c (EC50 = 6.25 microM) and increased the ceramide responsiveness up to 10-17-fold. These studies disclose stereospecific activation of PP1 and PP2A by long chain natural ceramides under near physiologic ionic strengths in vitro. The implications of these studies for mechanisms of ceramide action are discussed.     

Bipolar spindle frequency and genome content are inversely regulated by the activity of two N-type kinesins in Entamoeba histolytica

Bipolar microtubular spindles are seen infrequently in Entamoeba histolytica trophozoites while monopolar or radial microtubular assemblies are common. Additionally, heterogeneity in nuclear DNA content and multi-nucleation is found in amoeba cells growing in axenic culture. Taken together these observations indicate that genome segregation is irregular in these cells. In order to identify proteins involved in regulating genome segregation, we have focused on studying E. histolytica homologues of kinesin motor proteins that are known to affect stability of bipolar mitotic spindles. We have demonstrated earlier that increased levels of the kinesin – Eh Klp5 – led to increased frequency of bipolar spindles accompanied with a reduction in the heterogeneity of genome content, showing that bipolar spindle frequency was inversely linked to genome content in E. histolytica . In this study, we have investigated the role of E. histolytica kinesins (Eh KlpA1, 2–4) in regulating bipolar spindle frequency and genome content. While downregulation of Eh Klp3, 4 and A1 showed no effect, downregulation of Eh Klp2 led to increased frequency of bipolar spindles and homogenization of genome content, similar to the effect of increased expression of Eh Klp5. In addition to microtubules, Eh Klp2–4 associated with F-actin in the cytoplasm, suggesting that these kinesins are multi-functional.     

Antagonism of insulin action on muscle by the albumin-bound B chain of insulin

1. The presence of a substance associated with human albumin that exerts anti-insulin activity on the isolated rat diaphragm has been confirmed. This factor has been removed from albumin, thereby providing a source of non-antagonistic carrier protein. 2. Derivatives of the polypeptide B chain of insulin obtained by chemical scission of the interchain disulphide bonds have been separated by conventional techniques. In the presence of non-antagonistic albumin, the reduced and sulpho-B chain preparations inhibited insulin action on muscle. 3. The B chain resulting from reductive cleavage of insulin by bovine-liver extracts, in association with human albumin, exhibited a comparable anti-insulin effect. 4. It is postulated that the B chain interacts with albumin to enable solubilization of the chain and that inhibition of insulin action on muscle may occur as a result of competition for cellular receptor sites by the B chain. 5. The implication of these findings in relation to a circulating insulin antagonist is discussed.     

Limited proteolysis patterns of the B chain of insulin.

The oxidized B chain of insulin was used as a simple model for further consideration of limited proteolysis with low substrate:enzyme ratios. With low B chain:trypsin ratios, the ordinarily slower cleavage rate of the -Lys₂₉-Ala₃₀ bond essentially equaled the cleavage saturation rate of the -Arg₂₂-Gly₂₃ bond. This led to the disappearance of octapeptide which ordinarily forms most rapidly. Heptapeptide and alanine, formed mainly by cleavage of the octapeptide, decreased somewhat at high enzyme relative levels. Trypsin added to B chain formed a single chromatographic peak.     

Immunogenicity of B chain in insulin responder and nonresponder mice.

The potential immunogenicity of insulin B chain in beef insulin low-responder H-2k,a and high-responder H-2b,d mice was examined using lymph node proliferation assays. Oxidized B chain was immunogenic in H-2k,a, but not H-2b,d, mice. The T cell population recognized a determinant in OX-B chain associated with I-Ak. These cells did not respond to intact insulin, suggesting that the B chain determinant was not available to I-Ak during immunologic processing of insulin. Responses were observed in H-2k and H-2d, but not H-2b, after immunization with reduced and carboxyamidomethylated-insulin which contains equimolar A chain and B chain. These responses were I-A-restricted and heterogeneous, with reactivity to A chain and B chain determinants. In each case, little or no cross-reactivity was observed between RCAM-insulin and intact insulin. Furthermore, T cell populations induced in H-2k mice selectively recognized OX-B chain or RCAM-B chain, which differ in chemical modification of the thiols of Cys B7 and Cys B19. Similarly, RCAM-BINS-immune T cells from H-2d did not react to OX-B chain. These results indicate that derivatization of the cysteine thiols, through disulfide bonds, oxidation, or carboxyamidomethylation, radically affects T cell recognition of insulin B chain.     

Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner.

The activities of the individual hepatitis B virus (HBV) promoters and the effects of the HBV enhancer on these promoters in several human cell types have been compared by measuring the activity and RNA levels of the linked reporter function chloramphenicol acetyltransferase. The relative promoter activities in the human HepG2 (liver), HeLa, and HS27 (fibroblast) cell lines are in the order precore greater than X greater than preS2 greater than preS1; thus, the promoters of the gene producing the largest quantity of viral proteins have relatively low activity. The juxtaposition of the HBV enhancer in either orientation increased the promoter activities only modestly (2- to 5-fold) in the nonliver cell lines, whereas it dramatically increased (20- to 100-fold) the promoter activities in the liver cell line. Thus, the HBV enhancer is especially active in liver cells. This may be one of the causes of hepatotrophicity of the virus.     

Action of human liver cathepsin B on the oxidized insulin B chain.

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.     

Temporally and spatially regulated somatic mutagenesis in mice.

In mice transgenesis through oocyte injection or DNA recombination in embryonal stem (ES) cells allows mutations to be introduced into the germline. However, the earliest phenotype of the introduced mutation can eclipse later effects. We show in mice that site-specific genomic recombination can be induced in a selected cell type, B lymphocytes, at a chosen time. This precision of somatic mutagenesis was accomplished by limiting expression of a Cre recombinase-estrogen receptor fusion protein to B lymphocytes by use of tissue-specific elements in the promoter of the transgene employed. The expressed fusion protein remained inactive until derepressed by systemic administration of an exogenous ligand for the estrogen receptor, 4-OH-tamoxifen. Upon derepression the Cre recombinase enzyme deleted specific DNA segments, flanked by loxP sites, in B lymphocytes only. The efficiency of recombination in cells expressing the fusion protein could be varied from low levels to >80%, depending on the dose of ligand administered. Our work presents a paradigm applicable to other uses of site-specific recombination in somatic mutagenesis where both temporal and spatial regulation are desired.     

Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.     

B cell response pathways regulated by IL-5 and IL-2. Secretory microH chain-mRNA and J chain mRNA expression are separately controlled events

We have examined the effect of IL-5 and/or IL-2 on the expression of the secretory form of microH chain (microsecond) and J chain mRNA in a homogeneous neoplastic B cell clone, in which proliferation, IL-2R up-regulation and entry into the IgM secretory state are separately controlled events. The IL-5 signal triggers a partial induction of CL-3 cells into the IgM secretory state, characterized by a striking increase of microsecond mRNA expression and an increase in the ratio of the secretory to membrane forms of microH chain mRNA, with a modest increase of J chain mRNA. In contrast, amplification of J chain mRNA is accomplished by the late-acting B cell differentiation stimulus, IL-2, acting on IL-5-pretreated CL-3 cells or acting simultaneously with IL-5 on CL-3 cells. Such dually stimulated cells now are fully induced into IgM secreting cells. These results define the relative roles of IL-5 and IL-2 in B cell differentiation by showing important regulatory effects at the mRNA level. In addition, these results substantiate that appearance of mRNA for J chain, a molecule key to the formation of pentameric IgM, is a limiting factor for high level IgM secretion. The separate control of microsecond and J chain mRNA found in CL-3 cells stimulated with IL-5 and IL-2 elucidates a molecular mechanism by which these two lymphokines synergize in the development of CL-3 cells into IgM secreting cells.     

Single chain des-(B30) insulin. Intramolecular crosslinking of insulin by trypsin catalyzed transpeptidation

Single chain des-(B30) insulin (SCI) has been synthesized from porcine insulin by trypsin in a medium with a low content of water. Trypsin catalyzes an intramolecular transpeptidation reaction in which the glycineA1 residue substitutes the alanineB30 residue, rendering a LysB29 -GlyA1 peptide link between the A- and B-chains of insulin. The insulin derivative has been purified by column chromatography and appears to be homogeneous in HPLC and disc electrophoresis. The structure was proven to be B(1-29)-A(1-21) insulin by proteolysis with Armilliaria mellea protease followed by a few steps of Edman degradation. The electrophoretic mobility indicates that SCI has a more condensed structure than that of insulin. Perfect rhombohedral crystals were obtained under conditions resembling those under which insulin crystallizes in the same form. SCI was devoid of effect in the blood sugar lowering assay in mice, the estimated potency being less than 0.1% of that of insulin.     

How membrane chain melting properties are regulated by the polar surface of the lipid bilayer

The principle of regulation of various membrane properties by the hydrocarbon membrane interior is now well understood. The mechanism by which the interfacial membrane region including aqueous solution affects the state of the lipid bilayer matrix, however, is as yet unclear, despite its great biological and physiological significance. Data and analysis presented in this paper show that apart from the lipid chain type, length, and degree of unsaturation the main factors determining the characteristics of lipid membranes are surface polarity and interfacial hydration. These incorporate the effects of head group dipole and multipole moments as well as the head group ability for hydrogen bonding and can account for most of the changes in the physicochemical membrane state caused by the lipid head group structure, bulk pH value, salt content, solute adsorption, etc. The effects of membrane potential are much less, only 10-30% of the former. Variations in hydration thus not only govern the short- and medium-range intermolecular and intermembrane interactions but also provide a fast and energetically inexpensive regulatory mechanism for lipid membranes to adapt their characteristics, at least locally or transiently, to new requirements.     

Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats

Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P < 0.05); refeeding reversed these changes (P < 0.05). Consistent with these effects being regulated by S6K1, activation of this kinase was suppressed by FD (-91%, P < 0.05) but was increased by refeeding. Gavaging rats subjected to FD with a mixture of amino acids partially restored muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P < 0.0001). Thus feeding stimulates fractional protein synthesis in skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.     

Endosomal proteolysis of internalized insulin at the C-terminal region of the B chain by cathepsin D.

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.     

Circadian rhythms of gastrointestinal function are regulated by both central and peripheral oscillators

Circadian clocks are responsible for daily rhythms in a wide array of processes, including gastrointestinal (GI) function. These are vital for normal digestive rhythms and overall health. Previous studies demonstrated circadian clocks within the cells of GI tissue. The present study examines the roles played by the suprachiasmatic nuclei (SCN), master circadian pacemaker for overt circadian rhythms, and the sympathetic nervous system in regulation of circadian GI rhythms in the mouse Mus musculus. Surgical ablation of the SCN abolishes circadian locomotor, feeding, and stool output rhythms when animals are presented with food ad libitum, while restricted feeding reestablishes these rhythms temporarily. In intact mice, chemical sympathectomy with 6-hydroxydopamine has no effect on feeding and locomotor rhythmicity in light-dark cycles or constant darkness but attenuates stool weight and stool number rhythms. Again, however, restricted feeding reestablishes rhythms in locomotor activity, feeding, and stool output rhythms. Ex vivo, intestinal tissue from PER2::LUC transgenic mice expresses circadian rhythms of luciferase bioluminescence. Chemical sympathectomy has little effect on these rhythms, but timed administration of the β-adrenergic agonist isoproterenol causes a phase-dependent shift in PERIOD2 expression rhythms. Collectively, the data suggest that the SCN are required to maintain feeding, locomotor, and stool output rhythms during ad libitum conditions, acting at least in part through daily activation of sympathetic activity. Even so, this input is not necessary for entrainment to timed feeding, which may be the province of oscillators within the intestines themselves or other components of the GI system.