Overexpression of lactate dehydrogenase A attenuates glucose-induced insulin secretion in stable MIN-6 β-cell lines

Since islet β-cells express little

Full-size image

-lactate dehydrogenase (LDH) activity, we have examined the effects on these cells of LDH overexpression. In mock-transfected MIN6 β-cells, LDH activity was 38 nmol/min/mg protein, and 30 mM glucose stimulated secretion 10.4-fold. In two MIN6 cell clones stably overexpressing human LDH-A cDNA, insulin secretion was stimulated only 2.7- and 2.1-fold by high glucose. K⁺-stimulated insulin secretion was unaffected, and leucine stimulation enhanced, by LDH-A overexpression. LDH-A-overexpressing clones displayed unaltered activities of hexokinase, glucokinase, and malate dehydrogenase, slightly elevated plasma membrane lactate transport activity, and lowered insulin content. Low LDH activity would therefore appear important in β-cell glucose sensing.     

Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic β-cells

We have studied the effects of cholinegic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca²⁺ ([Ca²⁺]_i) in the β-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 β-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increased in DAG. ACh caused a transient rise in [Ca²⁺]_i which was abolished by omission of Ca²⁺ or by addition of devapamil. Thus, cholinergic stimulation of β-cell insulin release is associated with changes in both [Ca²⁺]_i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca²⁺ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca²⁺]_i and DAG had returned to control values suggesting that additional mechanisms may be involved.     

Islet neogenesis-associated protein pentadecapeptide (INGAP-PP): Mechanisms involved in its effect upon β-cell mass and function

The effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) administration to normal male hamsters upon serum glucose and triglyceride levels, β-cell mass and function was studied. INGAP-PP (500 μg) or saline was injected twice daily during 10 days. Both groups showed comparable body weight, serum glucose and triglyceride levels. INGAP-PP treated animals had significantly higher HOMA-IR and HOMA-β and their islets released more insulin in response to glucose; they had lower islet DNA content, significantly increased number of islets/unit area, β-cell replication rate and mass, cells co-expressing Pdx-1/INGAP and islets in contact with ducts, and decreased β-cell apoptosis rate. The percentage of cells expressing Pdx-1 alone or together with INGAP or insulin increased significantly in ducts. These animals also showed a significantly higher concentration of Pdx-1 and Ngn-3 mRNA and a lower number of INGAP-positive cells. In conclusion, INGAP-PP promoted a controlled and functionally active increase of β-cell mass; our data demonstrate for the first time the mechanism responsible for such changes; that Ngn-3 would be involved in INGAP-PP-induced neogenesis; and the existence of a negative feedback loop with endogenous INGAP-producing cells. Accordingly, INGAP-PP could be used to induce these effects in people with or at risk of developing diabetes.     

Kinectin participates in microtubule-dependent hormone secretion in pancreatic islet beta-cells

Kinectin (KNT) is a candidate membrane receptor for kinesin in the movement of intracellular organelles along microtubules. Isoforms of KNT exist containing different combinations of six small (residues 23-33) variable domains (vd) vd1-6 within the C-terminus. Here we investigate a role for KNT and its isoform KNTvd4(-) in the transport of amylin and insulin-containing secretory vesicles in the pancreatic islet beta-cell line RINm5F. KNTvd4(-) lacks vd4 that forms the kinesin-binding domain, and hence its role in the cell is an enigma. We report that amylin-containing vesicles also contained insulin, and exhibited microtubule, and small G-protein-dependent secretion. Knockdown of KNT by small interference RNA (siRNA) inhibited amylin expression and secretion. In contrast, recombinant KNTvd4(-) overexpressed in RINm5F cells associated with amylin-containing vesicles and inhibited amylin secretion, but had no discernible affect on amylin expression. The data suggests that both KNT and KNTvd4(-) participate in microtubule-dependent secretion of amylin in islet beta-cells.     

Fungus β-glycosidases: immobilization and use in alkyl-β-glycoside synthesis

Production of β-glycosidases: β-xylosidase and β-glucosidase by the fungus Sclerotinia sclerotiorum was optimized in the presence of different carbon sources. Immobilization supports with different physico-chemical characteristics were evaluated for use in continuous reactors. Immobilization and activity yields were calculated. Among the adsorption on Duolite, Amberlite, Celite and DEAE-sepharose, and entrapment in polyacrylamide gel or reticulation using glutaraldehyde, highest yields were obtained when β-xylosidase was adsorbed on Duolite A 7 and when β-glucosidase was adsorbed on DEAE-sepharose.

Enzyme preparations from S. sclerotiorum cultures were used in a biphasic (alcohol/aqueous) medium for the synthesis of alkyl-glycosides by trans-glycosylation of sugars and long-chain alcohols. The synthesis was studied under different conditions with primary and secondary alcohols as substrates, in the presence of free or immobilized enzyme. Xylan and cellobiose were used for the synthesis of alkyl-xylosides and alkyl-glucosides, respectively. The majority of the immobilized preparations were unable to catalyze the synthesis of alkyl-glycosides.

Highest yields were obtained when using xylan and C₄–C₆-alcohols. The reaction produced alkyl-β-xyloside and alkyl-β-xylobioside, as confirmed by MS/MS. Up to 22 mM iso-amyl-xyloside and 14 mM iso-amyl-xylobioside were produced from iso-amyl alcohol and xylan.     

Proteolytic modification of membrane-associated phospholipase C-β by μ-calpain enhances its activation by G-protein βγ subunits in human platelets

Membrane-associated phosphoinositide-phospholipase C (PI-PLC)-β (150 kDa) and its truncated forms (100 kDa and 45 kDa) were purified from human platelets. The 100 kDa PI-PLC-β was found to be activated to a greater extent by brain G-protein βγ subunits compared to the intact 150 kDa enzyme. Furthermore, treatment with μ-calpain of the intact PI-PLC-β (150 kDa) caused a marked augmentation of its activation by βγ subunits. This enhanced PLC activation by βγ subunits was due to truncation by μ-calpain, producing a 100 kDa PI-PLC, but not by another protease,thrombin.     

Enzymic Preparation of Benzyl β-D-Glucopyranoside

Benzyl β-D-glucopyranoside was prepared by an enzyme-catalysed direct reaction between D-glucose, or better cellobiose, and benzyl alcohol in the presence of a minimum amount of water. The enzyme β-glucosidase was used in the immobilized form (adsorbed onto macroporous polyethylene terephthalate or covalently bound on polyglycidyl methacrylate), enabling multiple application.     

Leptin modulates pancreatic β-cell membrane potential through Src kinase–mediated phosphorylation of NMDA receptors

The adipocyte-derived hormone leptin increases trafficking of K_ATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca²⁺ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.     

Biotransformations of α,β-Epoxyalcohols Catalyzed by Epoxide Hydrolases

Microsomal and cytosolic fractions of mammalian livers were screened for their capacity to resolve racemic mixtures of trans -2,3-epoxy-l-alkanols. The epoxide hydrolase activities showed some specificity for the 2S, 3S enantiomers which were attacked at the proximate carbon atom. The best resolutions were observed with guinea pig liver microsomal enzymes.     

Temporal Changes of Acid Phosphatase, Arylsulphatase, β-Galactosidase and β-N-ACETYL-d-Glucosaminidase Activities in Subcellular Fractions of Rat Liver

In this paper circadian changes in the liver enzyme activities of rat housed under highly standardized conditions with 12:12 hour light-dark cycle are shown. Activities of acid phosphatase, arylsulphatase, β-galactosidase and β-N-acetyl-d-glucosaminidase in microsomal and lysosomal fractions and crude homogenate were estimated every 4 hr during one 24-hr period. The enzyme activities were related to 1 mg of protein, 1 mg of DNA and 1 g fresh tissue. Daily changes of enzyme activities were found. In case of activity calculated per 1 mg DNA two maxima at 0500 and at 2100 hr were observed, while activity calculated per 1 mg protein showed one maximum at 0500 hr. Activity calculated per 1 g fresh tissue showed the maximum at 0500 hr for each enzyme only in microsomal fraction. As far as acrophase table is concerned for all enzymes and fractions the acrophase occurred during the night. The obtained results are discussed in relation to lysosomal enzymes synthesis process as well as different reference values.     

Biotransformation of β-amino nitriles: the role of the N-protecting group

N-Tolylsulfonyl- and N-butyloxycarbonyl-protected β-amino nitriles were prepared to study the effect of the N-protecting group on the biotransformation of the β-amino nitriles to the corresponding β-amino amides and acids. The bioconversions were carried out by using whole cells of Rhodococcus sp. R312 and Rhodococcus erythropolis NCIMB 11540. The bioconversion products of five-membered carbocyclic nitriles were mainly the respective acids whereas the carbocyclic six-membered nitriles were accumulated at the stage of the amide. Benefits of the enzymatic compared with the chemical hydrolysis of β-amino nitriles are the mild reaction conditions for the transformation of the nitrile group in the presence of acid or base labile N-protecting groups. In the present work we concentrated on this chemoselectivity of the biotransformation rather than its potential enantioselectivity, which will be subject of future investigations. Thus, some new compounds were prepared: (±)-(2-cyano-cyclohexyl) carbamic acid tert-butyl ester (4a), (±)-(2-carbamoyl-cyclopentyl) carbamic acid tert-butyl ester (3b) and (±)-(2-carbamoyl-cyclohexyl) carbamic acid tert-butyl ester (4b).     

Whole-cell bioconversion of β-sitosterol in aqueous–organic two-phase systems

The selective cleavage of the β-sitosterol side-chain by free Mycobacterium sp. NRRL B-3805 cells was used as a model system for the study of solvent effects in a whole-cell bioconversion in two phase aqueous–organic media. This multi-step degradation pathway leads to the production of 4-androstene-4,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) as a minor product. In an attempt to correlate the substrate and cell partition effects and solvent hydrophobicity (log P) with biocatalytic activity, 15 carboxylic acid esters with log P values between 3 and 10 were screened. The results indicated that the toxicity of the tested solvents in this system could not be correlated to their log P, but seemed to depend on their ability to accumulate in the cells, as these showed a strong affinity towards the organic phase. Different solvent/aqueous ratios and hydrodynamic conditions were further tested in the solvent systems (phthalates) showing significant biodegradation activity. The bioconversion rate was generally not much affected by the stirring speed in the employed range (150–300 rpm) but was strongly influenced by the aqueous/organic phase ratio. Results suggest that the bioconversion takes place at the interphase, its rate being possibly limited by mass transport inside the organic phase.     

Matrix alkalinization: a novel mitochondrial signal for sustained pancreatic β‐cell activation

Nutrient secretagogues activate mitochondria of the pancreatic β‐cell through the provision of substrate, hyperpolarisation of the inner mitochondrial membrane and mitochondrial calcium rises. We report that mitochondrial matrix pH, a parameter not previously studied in the β‐cell, also exerts an important control function in mitochondrial metabolism. During nutrient stimulation matrix pH alkalinises, monitored by the mitochondrial targeted fluorescent pH‐sensitive protein mtAlpHi or ³¹P‐NMR inorganic phosphate chemical shifts following saturation transfer. Compared with other cell types, the resting mitochondrial pH was surprisingly low, rising from pH 7.25 to 7.7 during nutrient stimulation of rat β‐cells. As cytosolic alkalinisation to the nutrient was of much smaller amplitude, the matrix alkalinisation was accompanied by a pronounced increase of the ΔpH across the inner mitochondrial membrane. Furthermore, matrix alkalinisation closely correlates with the cytosolic ATP net increase, which is also associated with elevated ATP synthesis rates in mitochondria. Preventing ΔpH increases in permeabilised cells abrogated substrate‐driven ATP synthesis. We propose that the mitochondrial pH and ΔpH are key determinants of mitochondrial energy metabolism and metabolite transport important for cell activation.     

N-Nitroso-β-lactams as β-lactamase inhibitor

N-Nitroso-β-phenyl-β-lactam has been found to be a specific inhibitor of β-lactamase. N-Nitroso--phenyl-β-lactam, by contrast, was virtually ineffective although a transient inhibition of short duration was observed. The acyl enzyme derived from the β-phenyl isomer is presumably involved in a cross-linking reaction, whereas that from the -phenyl isomer was quenched by spontaneous hydrolysis without formation of a covalent bond. No inhibitory effect of the β-phenyl isomer on chymotrypsin has been observed.     

Synthetic β-endorphin-like peptide immunorphin binds to non-opioid receptors for β-endorphin on T lymphocytes

The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364–373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [¹²⁵I]β-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (K_i = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (K_i = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (K_i = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K_i = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K_i = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K_i = 1.0 nM) possessed the ability to inhibit specific binding of [¹²⁵I]β-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. K_d values characterizing the specific binding of ¹²⁵I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4 nM and 36.3 nM, respectively.     

Inhibition of protein synthesis by the β-subunit of spectrin

The 220 kDa β-subunit of erythroid cell spectrin is a potent inhibitor of protein synthesis in lysates from rabbit reticulocytes. On the basis of weight of protein added to a lysate reaction mixture, it has about half the inhibitory activity of highly purified heme-regulated eIF-2 kinase. Inhibition appears to be at the level of peptide initiation but does not involve a kinase that phosphorylates eIF-2 on its -subunit.     

Baker's yeast mediated reduction of β-keto esters and β-keto amides in an organic solvent system

The baker's yeast mediated reduction of four β-keto esters in petroleum ether indicated that the size of the group attached to the keto carbon affected their reactivity. Ethyl 3-phenyl-3-oxopropanoate (1), which has a phenyl group directly attached to the keto carbon, is incompletely reduced using 20 g yeast/mmol substrate, ethyl 4-phenyl-3-oxobutanoate (2), which has one methylene group between the phenyl and keto carbon, was also incompletely reduced using 20 g yeast/mmol, although the extent of reduction was about double that of (1), ethyl 5-phenyl-3-oxopentanoate (3), which has two methylene groups between the phenyl and keto carbon, is completely reduced using 10 g yeast/mmol and ethyl 3-oxobutanoate (4), which has a methyl group attached to the keto carbon shows complete reduction using only 1 g yeast/mmol. The corresponding β-keto amides are considerably less reactive than the corresponding β-keto esters with only the amides derived from ethyl 3-oxobutanoate indicating any significant reduction using 20 g yeast/mmol.     

Analysis of the α4β1 Integrin–Osteopontin Interaction

The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN.