Characterization of Acetylcholine-induced Increases in Cytosolic Free Calcium Concentration in Individual Rat Pancreatic β-cells

The intracellular free Ca²⁺ ion concentration ([Ca²⁺]i) was measured using fura-2 microspec-trofluorimetry in individual rat pancreatic β-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The mean basal concentration of [Ca²⁺]i in β-cells in the presence of 4.4 mM glucose and 1.8 mM Ca²⁺ was 112±1.6 nM (n=207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in [Ca²⁺]i spikes of increasing amplitude and duration in some, but not all of the β-cells. In addition, the β-cells demonstrated variable sensitivity to ACh. The increases in [Ca²⁺]i were rapid, transient and were blocked by atropine at 10^-6M. A brief exposure to 50 mM K⁺ resulted in a transient increase in [Ca²⁺]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100μL 10^-4M or 10^-3M) induced [Ca²⁺]i oscillations in 11 out of 57 β-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca²⁺ free medium, the source of the increase in [Ca²⁺]i was deduced to be from extracellular spaces. Changing the temperature from 22 to 37°C did not affect the action of ACh on [Ca²⁺]i. These data strongly suggest that ACh exerted a direct action on [Ca²⁺]i in normal rat pancreatic β-cells and support a role for Ca²⁺ as a second messenger in the action of ACh.     

Selective Expression of Huntingtin-associated Protein 1 in ��-Cells of the Rat Pancreatic Islets

Huntingtin-associated protein-1 (HAP1) was initially identified as a binding partner of huntingtin, the Huntington''s disease protein. Based on its preferred distribution among neurons and endocrine cells, HAP1 has been suggested to play roles in vesicular transportation in neurons and hormonal secretion of endocrine cells. Given that HAP1 is selectively expressed in the islets of rat pancreas, in this study, we analyzed the expression pattern of HAP1 in the islets. In rats injected intraperitoneally with streptozotocin, which can selectively destroy β-cells of the pancreatic islets, the number of HAP1 immunoreactive cells was dramatically decreased and was accompanied by a parallel decrease in the number of insulin-immunoreactive cells. Immunofluorescent double staining of pancreas sections showed that, in rat islets, HAP1 is selectively expressed in the insulin-immunoreactive β-cells but not in the glucagon-immunoreactive α-cells and somatostatin immunoreactive δ-cells. In isolated rat pancreatic islets, ∼80% of cells expressed both HAP1 and insulin. Expression of HAP1 in the INS-1 rat insulinoma cell line was also demonstrated by immunofluorescent staining. Western blotting further revealed that HAP1 in both the isolated rat pancreatic islets and the INS-1 cells also has two isoforms, HAP1A and HAP1B, which are the same as those in the hypothalamus. These results demonstrated that HAP1 is selectively expressed in β-cells of rat pancreatic islets, suggesting the involvement of HAP1 in the regulation of cellular trafficking and secretion of insulin. (J Histochem Cytochem 58:255–263, 2010)     

Identification of Markers for Newly Formed ��-Cells in the Perinatal Period: A Time of Recognized ��-Cell Immaturity

Markers of β-cell maturity would be useful in staging the differentiation of stem/progenitor cells to β-cells whether in vivo or in vitro. We previously identified markers for newly formed β-cells in regenerating rat pancreases after 90% partial pancreatectomy. To test the generality of these markers of newly formed β-cells, we examined their expression during the perinatal period, a time of recognized β-cell immaturity. We show by semiquantitative RT-PCR and immunostaining over the time course from embryonic day 18/20 to birth, 1 day, 2 days, 3 days, 7 days, and adult that MMP-2, CK-19, and SPD are truly markers of new and immature β-cells and that their expression transiently peaks in the perinatal period and is not entirely synchronous. The shared expression of these markers among fetal, newborn, and newly regenerated β-cells, but not adult, strongly supports their use as potential markers for new β-cells in the assessment of both the maturity of stem cell–derived insulin-producing cells and the presence of newly formed islets (neogenesis) in the adult pancreas. (J Histochem Cytochem 58:369–376, 2010)     

Nutrient–secretion coupling in the pancreatic islet β-cell: recent advances

Insulin secretion from pancreatic islet β-cells is a tightly regulated process, under the close control of blood glucose concentrations, and several hormones and neurotransmitters. Defects in glucose-triggered insulin secretion are ultimately responsible for the development of type II diabetes, a condition in which the total β-cell mass is essentially unaltered, but β-cells become progressively “glucose blind” and unable to meet the enhanced demand for insulin resulting for peripheral insulin resistance. At present, the mechanisms by which glucose (and other nutrients including certain amino acids) trigger insulin secretion in healthy individuals are understood only in part. It is clear, however, that the metabolism of nutrients, and the generation of intracellular signalling molecules including the products of mitochondrial metabolism, probably play a central role. Closure of ATP-sensitive K⁺(K_ATP) channels in the plasma membrane, cell depolarisation, and influx of intracellular Ca²⁺, then prompt the “first phase” on insulin release. However, recent data indicate that glucose also enhances insulin secretion through mechanisms which do not involve a change in K_ATP channel activity, and seem likely to underlie the second, sustained phase of glucose-stimulated insulin secretion. In this review, I will discuss recent advances in our understanding of each of these signalling processes.     

Evidence for modulation of cell-to-cell electrical coupling by cAMP in mouse islets of Langerhans

The effects of forskolin on electrical coupling among pancreatic β-cells were studied. Two microelectrodes were used to measure membrane potentials simultaneously in pairs of islet β-cells. Intracellular injection of a current pulse (ΔI) elicited a membrane response ΔV₁ in the injected cell and also a response ΔV₂ in a nearby β-cell confirming the existence of cell-to-cell electrical coupling among islet β-cells. In the presence of glucose (7 mM), application of forskolin evoked a transient depolarization of the membrane and electrical activity suggesting that the drug induced a partial inhibition of the β-cell membrane K⁺ conductance. Concomitant with this depolarization of the membrane there was a marked decrease in β-cell input resistance (ΔV₂/ΔI) suggesting that exposure to forskolin enhanced intercellular coupling. Direct measurements of the coupling ratio ΔV₂/ΔV₁ provided further support to the idea that forskolin enhances electrical coupling among islet cells. Indeed, application of forskolin reversibly increased the coupling ratio. These results suggest that cAMP might be involved in the modulation of electrical coupling among islet β-cells.     

The Production of γ-Decalactone by Fermentation of Castor Oil

A microbial process for the production of optically-active γ-decalactone from the ricinoleic acid present as triglycerides in castor oil has been developed, γ-decalactone (γDL) is a component of some fruit flavours, being an important organoleptic component of peach flavours. Screening showed two red yeast microorganisms, Rhodotorula glutinis and Sporobolomyces odortts to be especially suitable for this biotransformation. The process involves lipase-mediated hydrolysis of the castor oil to give free ricinoleic acid, uptake of the acid by the cells and aerobic fermentation to achieve abbreviated β-oxidation of the ricinoleic acid (12-hydroxyoleic acid) into 4-hydroxydecanoic acid (4HDA), lactonisation of the acid into γ-DL, followed by solvent extraction and distillation. γ-DL broth concentrations of 0.5-1.2g · 1^-t were obtained after 3-5 days from fermentation media containing 10 g · 1^-1 castor oil, representing an 8.3-20.0% theoretical yield. Intermediates detected were consistent with the operation of the β-oxidation pathway. Appreciable amounts of novel metabolites identified as cis and trans isomers of a tetrahydrofuran (C₁₀) were also produced. Their formation from 4HDA appeared to be non-enzymic and was favoured by anaerobic conditions. Yields of γ-DL were inversely proportional to the concentration of castor oil present in the medium, indicating that substrate inhibition takes place. The highest yields of γ-DL were obtained when castor oil was present from the beginning of the fermentation, rather than when added once the fermentation had become established, demonstrating that the β-oxidation pathway and/or transport system require continual induction. Significant amounts of γ-DL were not produced from other fatty acids, including ricinelaidic acid, the trans isomer of ricinoleic acid. γ-DL formation was dramatically inhibited by antibiotic inhibitors of oxidative phosphorylation, indicating the importance of intact β-oxidation pathways, whereas inhibitors of protein synthesis and cell-wall synthesis had much less marked effects. Selective extraction of 4HDA from the fermentation broths, and of γDL from broth lactonised by heating at low pH, could be achieved by adsorption to Amberlite XAD-1 and XAD-7 resins respectively. Some product could be recovered from the exit gases of the fermenter by passing through propylene glycol traps. This pathway is unusual in that it is a rare example of the truncated β-oxidation of a fatty acid by microorganisms. This effect probably occurs because of partial inhibition of one or more enzymes of the β-oxidation pathway by the C₁₀ hydroxylated fatty acid intermediate(s) allowing intracellular accumulation of the 4HDA, followed by leakage out of the cell; although further metabolism of this C₁₀ intermediate does take place slowly.     

Unsaturated fatty acids as cytoprotective agents in the pancreatic β-cell

It is widely accepted that, in type 2 diabetes, elevated levels of free fatty acids and glucose contribute to a state of glucolipotoxicity in which β-cell function declines and, ultimately, cell viability is compromised. This suggests that β-cells do not readily tolerate chronic elevations in fatty acid levels. In vitro studies suggest, however, that β-cells respond differentially to long chain fatty acids, such that saturated species are lipotoxic whereas long chain mono-unsaturated fatty acids can provide cytoprotection. This difference does not appear to be mediated by a mutual metabolic antagonism between saturated and unsaturated species (although differential alterations in neutral lipid disposition may occur in response to these fatty acids) and the mechanisms remain unclear. This review summaries the current understanding of the actions of mono-unsaturated fatty acids in β-cells and highlights areas of controversy as well as key unresolved issues which require to be addressed.     

Beta(1)/beta(2)/beta(3)-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting

Catecholamines are viewed as major stimulants of diet- and cold-induced thermogenesis and of fasting-induced lipolysis, through the β-adrenoceptors (β₁/β₂/β₃). To test this hypothesis, we generated β₁/β₂/β₃-adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold-intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein-1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, β-adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.     

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).     

Central and peripheral administration of amylin induces energy expenditure in anesthetized rats

Amylin is a peptide hormone that is co-released with insulin from pancreatic β-cells following a meal. Intracerebroventricular (icv) administration of amylin (1–100 pmol), or an amylin agonist, salmon calcitonin, elicited dose-dependent thermogenic, tachycardic, and hyperthermic responses in urethane-anesthetized rats. Intravenous (iv) administration of higher doses of amylin (100 pmol–20 nmol) also induced similar responses, although the amplitudes of these responses were significantly smaller than those elicited by icv administration, suggesting the primary action of amylin to be in the brain. However, the iv administration of amylin induced the responses slightly faster than the icv injection, the former responses occurring <4 min and the latter, at 8–10 min, after the administration. The iv but not the icv injection of amylin increased the respiratory exchange ratio transiently (<20 min), though the thermogenic response lasted for a longer period after both injections, indicating a shift from mixed fuel to predominantly carbohydrate utilization in the initial phase of thermogenesis induced by the iv injection of amylin. The differences in substrate utilization and latency of the responses suggest that the actions of amylin include partly different targets when administered centrally and peripherally. Moreover, pretreatment with a β-adrenergic blocker, propranolol (5 mg kg⁻¹, iv), blocked all responses elicited by either icv or iv administration of amylin, whereas ablation of the area postrema in the hindbrain did not influence the effects of icv-administered amylin. These results suggest the involvement of amylin in postprandial energy expenditure, mediated by peripheral β-adrenoceptors.     

Fourier transform infrared spectroscopy used to evidence the prevention of beta-sheet formation of amyloid beta(1-40) peptide by a short amyloid fragment

Reflectance Fourier transform infrared (FT-IR) microspectroscopy was applied to study the prevention of β-sheet formation of amyloid β (Aβ)(1–40) peptide by co-incubation with a hexapeptide containing a KLVFF sequence (Aβ(15–20) fragment). Second-derivative spectral analysis was used to locate the position of the overlapping components of the amide I band of Aβ peptide and assigned them to different secondary components. The result indicates that each intact sample of Aβ(15–20) fragment or Aβ(1–40) peptide previously incubated in distilled water at 37 °C transformed their secondary structure from 1649 (1651) or 1653 cm⁻¹ to 1624 cm⁻¹, suggesting the transformation from -helix and/or random coil structures to β-sheet structure. By co-incubating both samples with different molar ratio in distilled water at 37 °C, the structural transformation was not found for Aβ(1–40) peptide after 24 h-incubation. But the β-sheet formation of Aβ(1–40) peptide after 48 h-incubation was evidenced from the appearance of the IR peak at 1626 cm⁻¹ by adding a little amount of Aβ(15–20) fragment. There was no β-sheet formation of Aβ(1–40) peptide after addition with much amount of Aβ(15–20) fragment, however, suggesting the higher amount of Aβ(15–20) fragment used might inhibit the β-sheet formation of Aβ(1–40) peptide. The more Aβ(15–20) fragment used made the more stable structure of Aβ(1–40) peptide and the less β-sheet formation of Aβ(1–40) peptide. The study indicates that the reflectance FT-IR microspectroscopy can easily evidence the prevention of β-sheet formation of Aβ(1–40) peptide by a short amyloid fragment.     

The relationship between mitochondrial activation and toxicity of some substituted carboxylic acids

The activation of 4-bromocrotonic acid, 4-bromo-2-octenoic acid, valproic acid, and 3-methylglycidic acid by conversion to their CoA thioesters and the effects of these carboxylic acids on palmitoylcarnitine-supported respiration were studied with rat liver and rat heart mitochondria. 4-Bromocrotonic acid was activated by both liver and heart mitochondria, whereas 4-bromo-2-octenoic acid and valproic acid were only activated by liver mitochondria. 3-Methylglycidic acid was not a substrate of mitochondrial activation. All of the carboxylic acids that were activated also inhibited palmitoylcarnitine-supported respiration. 3-Methylglycidoyl-CoA was found to irreversibly inhibit 3-ketoacyl-CoA thiolase in a concentration-dependent and time-dependent manner. Together, these results lead to the conclusion that substituted medium-chain carboxylic acids, which enter mitochondria directly, may inhibit β-oxidation as long as they are activated and perhaps further metabolized in the mitochondrial matrix to compounds that sequester CoA and/or inhibit β-oxidation enzymes. Liver is more susceptible to inhibition by such xenobiotic carboxylic acids due to the broader substrate specificity of its mitochondrial medium-chain acyl-CoA synthetase (EC 6.2.1.2).     

Crystallinity and polysaccharide chains of beta-glucan in white sorghum, SK(5912).

The polysaccharide chains and the crystallinity of β-glucan in a white sorghum variety, SK₅₉₁₂ were investigated using chemical and enzymic studies. Mild periodate oxidation and methylation, coupled to descending paper chromatography of products revealed the presence of unresolved non-carbohydrate moiety, 2, 4-and 2, 3-di-O-methyl -glucose residues (molar ratio; 18:3) and 2, 4, 6-and 2, 3, 6-tri-O-methyl -glucose residues (molar ratio; 1:14). Paper chromatography of the total acid hydrolysate also revealed a non-carbohydrate spot, identified as protein on the basis of positive Biuret and ninhydrin tests. The O-methyl -glucose residues suggest two polysaccharide chains designated X and Y. Chain X is formed through linking of β- -glucopyranosyl residues by (1→3) linkages with 85–86% (1→6) bonds at branch points and constitute about 6–7% of the β-glucan sample. Chain Y, which is 93–94% of the β-glucan polysaccharide chains, constitutes β- -glucopyranosyl residues in (1→4) linkages and 4–5% (1→6) bonds at branch points. Of the 18 branch points on the X-chains in a given β-glucan sample, about 15 are the Y chains interlinked to the X-chains through their (Y-chains) reducing ends. Both acid and enzyme hydrolyses of the β-glucan suggest two structural organizations, a crystalline and less crystalline granules, based on two first order kinetics. This was correlated by the progress curves obtained during hydrolysis with two purified isoforms of β-glucanases from the sorghum malt. The short and highly branched polysaccharide chains, and longer but less branched polysaccharide chains found in this β-glucan are reminiscent of the structures of amylopectin and amylose, respectively. The K_ms of 0.30–0.32 and 0.42–0.50 mg β-glucan/ml for the β-glucanase isoforms also lay credence to both the crystalline forms and the highly polymerised nature of the β-glucan in white sorghum.     

Study of the biosynthesis of phleic acids, polyunsaturated fatty acids synthesised by Mycobacterium phlei (author's transl)]

Because of their structures, phleic acids (general formula: CH3-(CH2)m-(CH=CH-CH2-CH2)n-CO2H; main component: m = 14, n = 5) cannot be synthesized by the same kinds of enzymatic systems as other natural polyunsaturated fatty acids. By using specifically labelled 14C compounds, we have tested the ability of different molecules to be incorporated in the phleate skeletons by Mycobacterium phlei. The localisation of radioactive carbon atoms has been studied by chemical degradation of labelled phleates, isolation and purification of the degradation products, and determination of their specific radioactivity. When M. phlei cells are incubated with labelled acetate, the unsaturated and saturated parts of the molecules of phleic acids are unequally labelled. The radioactivity of succinate monoester on the one hand and fatty acids (mixture of myristic and palmitic acids) on the other hand, measured after oxidative degradation of phleate esters, shows a constant ratio under definite conditions. Whether [1-14C]acetate or [2-14C]acetate is used for incubation, the same ratio is observed. Therefore acetate is the precursor of the unsaturated part as well as of the saturated part of the phleate molecules. By using labelled fatty acid esters, it has been found that palmitic acid is the precursor of phleates with m = 14, while myristic acid is the precursor of phleates with m = 12. Stearic and eicosanoic acids are not incorporated without degradation. The hypothesis of a condensation of a saturated fatty acid with a preformed polyunsaturated molecule was examined. Search for such a molecule in the lipids of M. phlei gives negative results. Pentaunsaturated phleate arising from palmitate is more abundant than pentaunsaturated phleate arising from myristate, while the reverse is true for hexaunsaturated phleates. These observations make very unlikely such an hypothesis. An elongation process fits well with the observed facts provided that this process involves elongation by two acetate units simultaneously, making elongation by four carbon atoms at a time. Such a requirement would be easily satisfied if two molecules of acetate are condensed together before their utilization in the elongation process. In such a hypothetical process, crotonate would be the most probable substrate of the elongation reaction.     

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.     

Beta-ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single β-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-β-hydroxybutyrate (PHB) synthesis, and in β-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953–963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial β-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to β-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to β-hydroxyacyl-CoA and β-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second β-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on β-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on β-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced β-ketothiolase activity and PHB accumulation, showing that this is the β-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under β-oxidation conditions and to possess β-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for β-ketothiolases in A. vinelandii.     

Kinetics of Photobleaching of β-Carotene in Chloroform and Formation of Transient Carotenoid Species Absorbing in the Near Infrared

Upon laser flash photolysis of β-carotene in chloroform instantaneous bleaching of β-carotene and concomitant formation of near infrared absorbing species are observed. One species, absorbing with maximum at 920 nm, is formed during the laser pulse (10 ns) and is practically gone in one millisecond, the decay showing a bi-exponential behaviour. The second species, absorbing with maximum at 1000 nm, is formed from the species absorbing at 920 nm by first order kinetics with a rate constant of 4.9·10⁴ s^-1 at 20°C. This second species decays by second order kinetics and is gone within a few milliseconds. An additional slow bleaching of β-carotene and formation of the species absorbing at 920 nm is observed. This slow bleaching/formation of transient absorption is probably due to processes involving free radicals generated during the instantaneous bleaching. The species absorbing at 920 nm is suggested to be either (i) a free radical adduct formed from β-carotene and chloroform or (ii) β-carotene after abstraction of a hydrogen atom. The species absorbing at 1000 nm is most likely the radical cation. Formation and decay of the near infrared absorbing species and bleaching of β-carotene are independent of whether oxygen is present or absent in the solutions.