Increase In Trehalase Activity Research Articles

Involvement of 20-hydroxyecdysone on feeding behavior and carbohydrate metabolizing enzymes in the adult stage of the red flour beetle, Tribolium castaneum

Since the red flour beetle, Tribolium castaneum, feeds on starch, α-amylase is an important enzyme for hydrolyzing carbohydrates into sugar to meet energy demands. It has been reported that 20-hydroxyecdysone (20E) involve in the regulation of α-amylase in the red flour beetle. Injection of 20E decreased α-amylase activity in dose- and time-dependent manner. α-Amylase activity in males and females showed different patterns responding to 20E. Adults injected with high concentrations of 20E (300–500 ng/insect) had low levels of food consumption leading to a decrease in body mass. Interestingly, glucose levels in treated adults increased significantly in parallel with the increase in trehalase activity in both males and females. 20E down-regulated Amylase (TcAmy), while Trehalase-1 and -2 (TcTre-1 and -2, respectively) genes were up-regulated. Taken together, 20E caused a decrease in α-amylase activity directly at the level of gene expression and indirectly by repressing food consumption and causing low ingestion levels. However, understanding the mechanism(s) by which feeding behavior suppression is caused by 20E in the red flour beetle needs to be examined in the future.

Free Trehalose Accumulation in Dormant Mycobacterium smegmatis Cells and Its Breakdown in Early Resuscitation Phase.

Under gradual acidification of growth medium resulting in the formation of dormant Mycobacterium smegmatis, a significant accumulation of free trehalose in dormant cells was observed. According to 1H- and 13C-NMR spectroscopy up to 64% of total organic substances in the dormant cell extract was represented by trehalose whilst the trehalose content in an extract of active cells taken from early stationary phase was not more than 15%. Trehalose biosynthesis during transition to the dormant state is provided by activation of genes involved in the OtsA-OtsB and TreY-TreZ pathways (according to RT-PCR). Varying the concentration of free trehalose in dormant cells by expression of MSMEG_4535 coding for trehalase we found that cell viability depends on trehalose level: cells with a high amount of trehalose survive much better than cells with a low amount. Upon resuscitation of dormant M. smegmatis, a decrease of free trehalose and an increase in glucose concentration occurred in the early period of resuscitation (after 2 h). Evidently, breakdown of trehalose by trehalase takes place at this time as a transient increase in trehalase activity was observed between 1 and 3 h of resuscitation. Activation of trehalase was not due to de novo biosynthesis but because of self-activation of the enzyme from the inactive state in dormant cells. Because, even a low concentration of ATP (2 mM) prevents self-activation of trehalase in vitro and after activation the enzyme is still sensitive to ATP we suggest that the transient character of trehalase activation in cells is due to variation in intracellular ATP concentration found in the early resuscitation period. The negative influence of the trehalase inhibitor validamycin A on the resuscitation of dormant cells proves the importance of trehalase for resuscitation. These experiments demonstrate the significance of free trehalose accumulation for the maintenance of dormant mycobacterial viability and the involvement of trehalose breakdown in early events leading to cell reactivation similar to yeast and fungal spores.

Trehalase as a possible marker of intestinal ischemia--reperfusion injury.

Different pathological affections of the small intestine cause corresponding morphological and functional changes. The present study was aimed to assess intestinal trehalase activities during ischemia and following reperfusion, correlate them with the pathological changes and determine whether trehalase could be used as a biochemical marker of the intestinal ischemia, ischemia - reperfusion injury. Wistar rats, randomly divided into 5 experimental groups (IR) (each n=15), were subjected to one hour mesenteric ischemia followed by 0, 1, 4, 12 and 24 hours of reperfusion. As a control group sham operated animals were used (n=15). The activity of trehalase was determined using an adapted Dahlqwist method. The range of intestinal injury was determined using histological (histopathological injury index and goblet cell quantification) and immunohistochemical (Ki67, InSitu TUNEL) methods. The highest activities of trehalase were recorded in the control group (C=4.42 ± 0.373 μmol/mg/h). The most altered intestinal histology detected in group IR1 was accompanied by the lowest trehalase activity (IR1=0.97 ± 0.209 μmol/mg/h; p < 0.001 C vs. IR1). Improved histological structure in the remaining reperfusion periods correlated with increase in trehalase activity. Almost normal mucosal histological architecture and 72% of the enzymatic activity were restored after 24 hours of reperfusion (IR24=3.20 ± 0.266 μmol/mg/h; p < 0.01 IR1 vs. IR24). The correlation between intestinal histology and trehalase activities during intestinal injury has been shown. Trehalase activity is closely associated with the status of the histological architecture of the small intestine.

Dual control of midgut trehalase activity by 20-hydroxyecdysone and an inhibitory factor in the bamboo borer Omphisa fuscidentalis Hampson

During larval diapause lasting 9 months from September to May, trehalase activity in the midgut of the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae) was low from December to April, followed by a fourfold increase in May that remained high during the pupal stage in July. An application of juvenile hormone analog (JHA) produced increases in the ecdysteroid titer, while trehalase activity was increased by both JHA and 20-hydroxyecdysone (20E) injection. The trehalase activity in the midgut of diapausing larvae was doubled by incubating the midgut with 20E for 48h. During diapause as well as after JHA application, expression of two ecdysone receptor isoform genes (EcR-A and EcR-B1) in the midgut increased simultaneously with the increase in hemolymph ecdysteroid titer, followed by an increase in trehalase activity. The hemolymph of diapausing larvae contained a trehalase inhibitor and inhibitory activity was high during diapause. After 20E injection, trehalase inhibition decreased as midgut trehalase activity increased. Taken together, at least two factors may participate in the change in midgut trehalase activity: increase in trehalase activity and decrease in trehalase inhibitor activity, both of which may be induced by 20E.

Physiological implications of trehalose in the ectomycorrhizal fungus Pisolithus sp. under thermal stress

The trehalose cycle in filamentous fungi has been suggested to be an important mechanism of tolerance against adverse stress conditions, particularly in thermal stress. Here, we demonstrate that trehalose and trehalase activity can be involved as an additional mechanism in development of thermotolerance in Pisolithus sp. In response to heat shock at 42 °C, an accumulation of intracellular trehalose and an increase in trehalase activity in the mycelial mass of the fungus was observed, when compared to the control treatment (28 °C). In vitro, assays showed that trehalose had a protective effect on β-glucosidase activity under thermal stress. Therefore, trehalose production may be an important mechanism of protection in ectomycorrhizal fungi, and this capacity could be used in the selection of isolates with greater capacity for adaptation to environmental stress.

Shifts in glycogen metabolism in hemolymph and fat body of the silkworm, Bombyx mori (Lepidoptera: Bombycidae) in response to organophosphorus insecticides toxicity

The toxic potential of widely used commercial-grade organophosphorus insecticides namely, fenitrothion and ethion, as a function of gycogen metabolism of hemolymph and fat body on days 1, 3, 5, and 7 of the fifth-instar silkworm, Bombyx mori were studied. Results showed that both lethal and sublethal doses of fenitrothion and ethion registered significant depletion in fat body glycogen reserves followed by concomitant increases in fat body phosphorylase and trehalase activities. Increase in glycogen phosphorylase activity and decrease in glycogen content indicated increased glycogenolysis at tissue level. Increase in trehalase activity may be attributed to the failure of homeostatic mechanism in silkworms on exposure to insecticides. In contrast, both insecticides produced a significant hyper and hypo-trehalosemia, hyper and hypo-glycaemia in hemolymph, and fat body, respectively, suggesting mobilization of trehalose and glucose from fat body to hemolymph to counteract the induced insecticides toxicity. The magnitude of these changes increased with time on exposure (1<3<5<7). These changes clearly indicated that there was a breakdown of homeostatic mechanism coupled with glycogenolysis to meet the required energy demands of the silkworms under insecticides toxicity. Food consumption and faeces defecated during the fifth-instar decreased significantly at lethal dose, while it increased marginally at sublethal doses of fenitrothion and ethion. The potential effects of fenitrothion and ethion on glycogen metabolism of hemolymph and fat body of the silkworms were dose dependent. These effects were more pronounced on exposure to lethal dose than sublethal dose of both insecticides and in general, the changes induced by ethion were more striking than those caused by fenitrothion.

Enhancement of Neutral Trehalase Activity by Oxidative Stress in the Fission YeastSchizosaccharomyces pombe

Addition of hydrogen peroxide, menadione, or plumbagin to growing cultures of the fission yeastSchizosaccharomyces pombeincreased trehalase activity. The effect was inhibited only slightly in the presence of cycloheximide, indicating that the stimulation of trehalase triggered by oxidative stress is mostly due to posttranscriptional activation. Northern blot analysis of trehalase mRNA level revealed that oxidative stress also induces a moderate rise in transcription of trehalase. Mutants disrupted in genes encoding elements of the mitogen-activated protein kinase (MAPK) cascade showed a reduced increase in trehalase activity upon oxidative challenge, which was coincident with a block in transcription of trehalase. Taken together, the results support the idea that the enhancement of trehalase by oxidative stress is due to enzyme activation (via the Pka1/Sck1 phosphorylation pathway) and induction of trehalase mRNA (via the MAPK signaling pathway). In spite of the trehalase increase, a net accumulation of trehalose was noticed during the oxidative stress.

Protein kinase Sck1 is involved in trehalase activation by glucose and nitrogen source in the fission yeast Schizosaccharomyces pombe.

Trehalase activity is markedly enhanced upon addition of glucose and a nitrogen source to cells of the fission yeast Schizosaccharomyces pombe. This increase corresponds to a post-translational activation of the enzyme, which is controlled by cAMP-dependent and cAMP-independent pathways. Recent work has shown that overexpression of SCK1 in Schiz. pombe is able to suppress mutations that result in reduced Pka1 (cAMP-dependent protein kinase A activity, suggesting that Sck1 (suppressor of loss of cAMP-dependent protein kinase) might be a functional analogue of Pka1 in the fission yeast. Here, an analysis of the possible role of Sck1 in the activation of trehalase triggered by glucose and a nitrogen source is reported in cells that were deficient in either Pka1, Sck1 or both protein kinases. The results showed that, except in repressed cells, Sck1 probably mediates a cAMP-independent activation of trehalase following the signal(s) triggered by glucose and the nitrogen source. The absence of functional Sck1 in depressed cells renders trehalase insensitive to activation by glucose and the nitrogen source even in the presence of Pka1, indicating that the Sck1-dependent, cAMP-independent pathway is the main signalling pathway controlling trehalase activation under derepression conditions. It is proposed that, during the activation of trehalase induced by glucose or a nitrogen source, the cAMP-Pka1 activation pathway previously characterized is to some extent parallel to this newly described one which includes Sck1 as phosphorylating enzyme. Neither of these two pathways, however, plays a key role in the heat-induced increase in trehalase activity.

Activation of cytoplasmic trehalase by cyclic-AMP-dependent and cyclic-AMP-independent signalling pathways in the yeast Candida utilis.

Derepressed cells of Candida utilis suspended in buffer exhibited both a transient cAMP-mediated signal and a marked activation of cytoplasmic trehalase when supplemented with glucose. Nitrogen sources or protein synthesis inhibitors, as well as protonophores or uncouplers, were also able to cause trehalase stimulation in derepressed cells even in the absence of the sugar. The increase in trehalase activity caused by nitrogen sources or protein synthesis inhibitors was not accompanied by changes in cAMP levels. Moreover, acridine orange inhibited both the cAMP signal and the glucose-induced activation of trehalase without affecting the increase in trehalase activity caused by nitrogen sources or protein synthesis inhibitors. These results suggest that cAMP is not involved as second messenger in the signal for trehalase stimulation induced by the latter compounds. By contrast, the addition of glucose to repressed cells suspended in buffer failed to cause the cAMP-mediated glucose signal and sugar-induced trehalase activation. No significant changes in either trehalase activity or cAMP concentration were observed upon addition to these cells of asparagine, cycloheximide, anisomycin or other agents, including protonophores and uncouplers. However, heat treatment of repressed cultures resulted in a moderate increase in trehalase activity with negligible change in cAMP levels, whereas such an effect was not observed in derepressed cultures. The thermally induced increase in trehalase activity was dependent on de novo protein synthesis and required the presence of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the biochemical mechanisms of anorexia caused by formamidine pesticides in the American cockroach, Periplaneta americana L

Chlordimeform (CDM), a formamidine pesticide, causes intense anorexia in the American cockroach, Periplaneta americana L. at doses as low as 1 to 5 μg/insect. We have studied the effect of this compound on the levels of mono- and disaccharide sugars in the hemolymph and found that such a phenomenon is accompanied by an increase in glucose and a simultaneous decrease in trehalose level. The combined level of glucose and trehalose remained relatively constant during the test period of 5 hr. An in vitro study has shown that trehalase activity in the thoracic muscles increased as a result of in vivo administration of CDM. The same phenomenon could be reproduced in vitro using N-demethylchlordimeform (DCDM) in place of CDM. These phenomena are very similar to those produced by octopamine (OA) both in vivo and in vitro. Phentolamine (PA) antagonized such actions of OA and DCDM in the thoracic muscles. Furthermore, both DCDM and OA could elevate cAMP levels in vitro in muscle homogenate preparations. These results support the notion that CDM, after metabolic activation to the N-demethyl and N,N-didemethyl forms in vivo, acts on the OA receptor causing activation of the cAMP-mediated response system. The result of this activation is an increase in trehalase activity in the thoracic muscles which leads to the conversion of trehalose to glucose. The resulting increase in the hemolymph glucose is the likely biochemical cause of anorexia in this species.

20-Hydroxyecdysone acts in the male pupa to commit accessory glands toward trehalase production in the adult mealworm beetle ( Tenebrio molitor)

During postecdysial adult maturation, the bean-shaped accessory reproductive glands (BAGs) of adult male mealworm beetles produce increasing amounts of trehalase. In order to determine when the BAGs become competent to produce trehalase, we transplanted pupal BAGs into 0-day female adults. After 8 days, trehalase activity had increased in BAGs from 4- and 5-day pupae (at the time of the pupal ecdysteroid peak) but not in those from 1-and 2-day pupae (before the ecdysteroid peak). BAGs from 0- and 2-day pupae were exposed to 20-hydroxyecdysone in vitro before implantation into 0-day female adults. Increase in trehalase activity was dose dependent. Both dose (ED 50, 5 × 10 −6 M) and exposure time (>6 hr) of hormone required are greater for commitment than for acceleration of pupal cell cycling ( T. Yaginuma, H. Kai, and G. M. Happ, 1988, Dev. Biol. 126, 173–181). Since trehalase activity increased markedly in isolated adult male abdomens, factors from the cephalic and thoracic centers are not required to sustain trehalase production in the adult BAGs.

Properties of a germination mutant of Phycomyces blakesleeanus

Spores of the Phycomyces blakesleeanus strain S440 germinated only for some 4 to 7% when activated with a heat treatment or with ammonium acetate. Contrary to wild type spores, they showed no increase in trehalase activity during or after the activating treatment. This was not due to a variant trehalase or a defective protein kinase but rather to the absence of an increase in cellular cyclic AMP which normally occurs in the wild type. Phosphodiesterase activity in the mutant was comparable to wild type activity and in both strains phosphodiesterase was inactivated by a heat treatment. The phosphodiesterase inhibitor 1-isobutyl, 3-methyl xanthine caused germination and trehalase activation in the wild type but not in the mutant. The results corroborate the importance of cyclic AMP in the breaking of dormancy and the activation of trehalase in this fungus.

Trehalase activity and cyclic AMP content during early development of Mucor rouxii spores.

Incubation of Mucor rouxii sporangiospores in complex medium under aerobic conditions resulted in a transient 20-fold increase in trehalase activity. Maximum activity was reached after 15 min. Simultaneously, the cyclic AMP (cAMP) content increased approximately eightfold, reaching a maximum within 10 min. Increases in trehalase activity and cAMP content were also observed under anaerobic conditions (CO2). The extent of trehalase activation and the changes in cAMP content, during both aerobic and anaerobic incubation, varied with the medium used. Trehalase was activated in vitro by a cAMP- and ATP-dependent process. An even faster activation was obtained when cAMP was replaced by the catalytic subunit of beef heart protein kinase. The coincidence of, and the correlation between, increased cAMP contents and trehalase activities support the involvement of a cAMP-dependent phosphorylation in the in vivo regulation of trehalase activity.

Activation of trehalase by membrane-depolarizing agents in yeast vegetative cells and ascospores.

The membrane-depolarizing agents 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazone, and nystatin are known to cause a rapid increase in the cyclic AMP level in fungal cells. Addition of these proton ionophores to yeast stationary-phase cells or ascospores causes an immediate 10-fold increase in trehalase activity. This observation is in agreement with a role for cyclic AMP-induced phosphorylation in the activation process of trehalase. It also provides an explanation for previous results on the induction of trehalose breakdown by 2,4-dinitrophenol in resting yeast cells.

Cyclic AMP, Trehalase and Germination of Phycomyces blakesleeanus Spores

When heat activated Phycomyces blakesleeanus spores were incubated at room temperature a rapid and transient increase in cyclic AMP content was observed. A similar increase was observed during the heat treatment itself but the cyclic AMP content had returned to the value of dormant spores at the end of the 5 min treatment. The cyclic AMP content increased only after treatments at temperatures which also induced spore germination. A more gradual rise in cyclic AMP was also found during activation at room temperature with acetate and after activation of the spores with pyrosulphite. The increase in cyclic AMP content is the earliest event known to occur after activation of the spores. It clearly preceded the increase in trehalase activity observed during and/or after all the activating treatments, suggesting that, as shown for in vitro activation, the in vivo activation of trehalase is due to a cyclic AMP-dependent phosphorylation. An increase in cyclic AMP was a common feature of all activation methods investigated and appears to be the factor triggering spore germination.

Glucose-induced Trehalase Activation and Trehalose Mobilization during Early Germination of Phycomyces blakesleeanus Spores

SUMMARY: When dormant spores of Phycomyces blakesleeanus were activated without concomitant activation of trehalase, breakdown of storage trehalose during early germination was not prevented. Measurement of trehalase activity during early germination of spores activated in this way indicated a subsequent rapid activation of trehalase upon incubation of the spores in germination medium. Trehalase activity reached a maximum after about 10 min of germination; thereafter it declined to values somewhat higher than those found in dormant spores. The same was observed when the activation of trehalase which normally occurs during heat activation of the spores was suppressed by adding long-chain alcohols to the activation medium. These results argue against previous speculation that trehalase is a ‘luxury’ molecule in the spores and that its activation has no significant role in the induction of germination. They point, on the other hand, to an important role for trehalase in the induction of germination. The main factor in the germination medium responsible for the activation of trehalase was found to be glucose. When spores were incubated under conditions in which they reverted to the dormant state, this subsequent trehalase activation was not seen. The increase in trehalase activity was not dependent on protein synthesis. A less pronounced increase was seen with glucose analogues. In the presence of azide the activation was only retarded, whereas in the presence of azide and salicylhydroxamic acid strong inhibition of trehalase activation was observed.

Changes in the activity and properties of trehalase during early germination of yeast ascospores: correlation with trehalose breakdown as studied by in vivo 13C NMR.

The regulation of trehalose breakdown during dormancy and the induction of germination in yeast ascospores was studied both by in vivo high-resolution NMR spectroscopy and in vitro assays of trehalase activity. Natural-abundance (13)C NMR spectra taken during the induction of germination with glucose and phosphate showed a rapid breakdown of part of the trehalose content. The presence of both glucose and phosphate was important for maximal trehalose breakdown. The (13)C NMR spectra showed that the externally added glucose and the internal trehalose were metabolized mainly to glycerol and ethanol. Under these conditions of nitrogen deprivation, full germination is not possible and trehalose breakdown stopped after approximately 1 hr. At this moment resynthesis of trehalose occurred while glycerol and ethanol production from the exogenous glucose continued. In complex media where full spore germination can occur, trehalose breakdown was more pronounced. Measurements of trehalase activity in spore extracts made after addition of varying amounts of glucose and phosphate to the spores revealed a sudden 10-fold increase in the activity of trehalase, within the first minutes of spore germination. The activation was transient: after reaching a maximum between 5 and 10 min, the activity declined back to low values during the next hours. The increase in trehalase activity was not inhibited by cycloheximide or by anaerobic conditions. The decline in trehalase activity that occurred after the initial activation could be correlated with the extent of trehalose breakdown as measured by (13)C NMR. In addition to the increase in trehalase activity, differences in the control properties were found between the enzymes from dormant and germinating spores. Trehalase from dormant spores was strongly inhibited by ATP at a concentration of approximately 0.5 mM, which corresponds with the ATP concentration found in dormant spores. On the other hand, trehalase from germinating spores was not inhibited by ATP up to the much higher ATP concentrations that are found in germinating spores. It is suggested that the low activity and the stringent ATP feedback inhibition of trehalase from dormant spores are responsible for the very slow mobilization of the huge amount of trehalose in dormant spores. Therefore, dormancy seems to be caused primarily by extreme curtailment of the energy production within the spore at one selective and primary point. The switch towards high activity and low ATP inhibition upon induction of germination is suggested to be responsible for the breaking of dormancy and for the rapid breakdown of trehalose that occurs during the initial phase of germination.

Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.

Metabolism and chemical activation of Phycomyces blakesleeanus spores

Respiratory and other data indicated that acetate was quickly metabolized by Phycomyces spores. Azide prevented metabolism of acetate although it did not inhibit oxygen uptake by dormant spores. Azide also inhibited activation of dormant spores by acetate, suggesting that acetate metabolism was necessary for spore activation. Pyruvate uptake by the spores was very limited in culture medium but could be increased substantially by lowering the pH to 3. However, pyruvate failed to activate dormant spores event at low pH values. Therefore, the lack of pyruvate metabolism could be involved in maintaining spore dormancy. Ammonium salts at pH 9 largely activated the spores. Under such conditions no increase in trehalase activity was found in the spores. Incubations with acetate in the presence of azide yielded a stimulation of trehalase activity without triggering spore activation. Therefore, spore activation and stimulation of trehalase activity seem to be two independent phenomena in Phycomyces spores.

The effect of gamma radiation on breaking of dormancy in Phycomyces spores.

Dormant sporangiospores of Phycomyces blakesleeanus were activated by Cs-137 gamma rays. After a dose of about 300 krad, between 80 and 90% of the spores germinated normally. However, further development of the mycelium was inhibited. Even with dry spores a partial activation was obtained. Activation by gamma rays was not accompanied by an increase in trehalase activity.