Hyperglycemic responses to cold shock in the freshwater giant prawn, Macrobrachium rosenbergii

Crustacean hyperglycemic hormone (CHH), a neurohormone synthesized and released from the x-organ sinus gland complex, is primarily involved in carbohydrate metabolism; biogenic amines and peptidergic neuroregulators are known to modulate the release of CHH. Marked elevations of hemolymph glucose titers, which peaked within 2 h, were observed in both intact and bilaterally eyestalk-ablated prawns, Macrobrachium rosenbergii, when they were transferred directly from their optimal temperature of 28 °C to lower temperatures close to their lethal limit. Hyperglycemia can therefore be considered a characteristic response in this species under cold shock. Involvement of biogenic amines in the hyperglycemic response was also demonstrated. Hyperglycemic effects of epinephrine, dopamine and serotonin were mediated through CHH at the eyestalk level, but the response under cold shock was not exclusively mediated through CHH. It is suggested that factor(s) other than CHH are involved in the hyperglycemic response, possibly norepinephrine or/and octopamine. Accepted: 24 October 1998     

The role of biogenic amines in the control of blood glucose level in the decapod crustacean,Carcinus maenas L.

1. Administration of biogenic amines into intact Carcinus maenas induces dose- and timedependent elevation of hemolymph glucose level.2. Removal of the neurosecretory centre containing the crustacean hyperglycemic hormone (CHH) by ablation of the eyestalks did not induce hypoglycemia.3. Injection of dopamine (DA) into eyestalkless crabs showed no hyperglycemic effect, while serotonin (5-HT), epinephrine (E), norepinephrine (NE), and octopamine (OA) elevated glucose levels.4. The dopaminergic effect was significantly reduced by administration of trifluoperazine (TFP).5. 5-HT and OA were found to be strong elevators of glucose levels, while the other biogenic amines had moderate effects only.6. The results indicate, that DA exerts its hyperglycemic effect by stimulating the release of CHH from the eyestalk neurosecretory centre. Elevation of hemolymph glucose level by 5-HT, OA, E, and NE, occurs independently of CHH.     

On the mode of action of methionine enkephalin, FK 33-824 and naloxone in regulating the hemolymph glucose level in the fresh water field crab Oziotelphusa senex senex

The possible involvement of opioid system in the regulation of hemolymph glucose level in the fresh water crab Oziotelphusa senex senex Fabricius, was investigated. Opioid agonist and antagonist was also used in addition to methionine-enkephalin itself. Injection of the opioid, methionine-enkephalin and FK 33-824 significantly elevated hemolymph glucose level. In contrast, injection of naloxone in to crab resulted in decrease in hemolymph glucose level. Injection of naloxone prior to injection of methionine-enkephalin blocked the hyperglycemic action of methionine-enkephalin. Injection of methionine-enkephalin, FK 33824 and naloxone produced no significant effect on hemolymph glucose level in eyestalk-less crab. The alterations in the intact crab hemolymph glucose level hypothesised to be due to stimulation of release of hyperglycemic hormone during methionine-enkephalin and FK 33824 treatment and blocking of release of hyperglycemic hormone during naloxone treatment from the eyestalks of crab Oziotelphusa senex senex.     

Preparation of two recombinant crustacean hyperglycemic hormones from the giant freshwater prawn, Macrobrachium rosenbergii, and their hyperglycemic activities

Crustacean hyperglycemic hormone (CHH) is released from the X-organ/sinus gland complex located in the eyestalks, and regulates glucose levels in the hemolymph. In the giant freshwater prawn (Macrobrachium rosenbergii), two cDNAs encoding different CHH molecules were previously cloned by other workers. One of these (Mar-CHH-2) was expressed only in the eyestalks, whereas the other (Mar-CHH-L) was expressed in the heart, gills, antennal gland, and thoracic ganglion, but not in the eyestalks. However, their biological activities had not yet been characterized. Therefore, in this study, recombinant Mar-CHH-2 (rMar-CHH-2) and Mar-CHH-L (rMar-CHH-L) were produced using an E. coli expression system, by expression in bacterial cells and recovery in the insoluble fraction. Thereafter, rMar-CHH-2 and rMar-CHH-L were subjected to refolding and were subsequently purified by reversed-phase HPLC. The rMar-CHH-2 and rMar-CHH-L thus obtained exhibited the same disulfide bond arrangements as those of other CHHs reported previously, indicative of natural conformation. In in vivo bioassay, rMar-CHH-2 showed significant hyperglycemic activity, whereas rMar-CHH-L had no effect. These results indicate that Mar-CHH-L does not function as a CHH, but may have some other, unknown function.     

Dopaminergic regulation of crustacean hyperglycemic hormone and glucose levels in the hemolymph of the crayfish Procambarus clarkii

The effects of dopamine on crustacean hyperglycemic hormone (CHH) release and hemolymph glucose levels in the crayfish Procambarus clarkii were investigated. A quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using antibodies specific for Prc CHH was developed and characterized. The sensitivity of the ELISA was about 1 fmol/well. Specific measurement of CHH in hemolymph samples by the ELISA was demonstrated by the parallelism between CHH standard curve and sample (hemolymph) titration curve. Moreover, thermally stressed P. clarkii exhibited a characteristic change of hemolymph CHH levels as revealed by the ELISA. CHH and glucose levels increased significantly within 30 min of dopamine injection, peaked at 1 h, and returned to the basal levels at 4 h. Dose-dependent effects of dopamine on CHH and glucose levels were observed between 10(-8) to 10(-6) mol/animal. Dopamine-induced increases in CHH and glucose levels were absent in eyestalk-ablated animals. Finally, dopamine significantly stimulated the release of CHH from in vitro incubated eyestalk ganglia. These results suggest that dopamine enhances release of CHH into hemolymph that in turn evokes hyperglycemic responses and that the predominant site of dopamine-induced CHH release is the X-organ-sinus gland complex located within the eyestalk.     

Reproductive tract and pancreatic norepinephrine levels in pre- and overt-diabetic C57BL/KsJ mice: relationship to body weight, blood glucose, serum insulin, and reproductive dysfunction

Diabetes-associated changes in tissue norepinephrine (NE) levels were determined in pre- and overt-diabetic C57BL/KsJ (db/db) mice relative to age-matched controls (+/?). At 4 weeks of age, both pre- and overt-diabetic mice exhibited obesity and hyperinsulinemia relative to controls. Prediabetic mice demonstrated normoglycemia and tissue (i.e., pancreatic, ovarian, and uterine) NE levels which were comparable to control levels. However, the hyperglycemic condition that characterized the overt-diabetic mice was found to be associated with significant elevations in tissue NE levels relative to both control and prediabetic values. These data demonstrate that the Type II diabetes-obesity syndrome in this mutant murine model is accompanied by a dramatic imbalance in the adrenergic influence on reproductive tissue glucose homeostasis. The hyperglycemic component of the diabetes-obesity syndrome is temporally linked to the noradrenergic disturbances in this model, which occur independently of insulin- or obesity-related changes that also accompany the expression of the genomic mutation and reproductive tract involution.     

Effect of melatonin on hemolymph glucose and lactate levels in the fiddler crab Uca pugilator

Melatonin was injected into intact and eyestalk-ablated fiddler crabs (Uca pugilator), and its effects on hemolymph glucose and lactate levels were studied. In intact crabs, glucose and lactate levels cycled simultaneously, with peaks occurring during early and late photophase. Melatonin caused a shift in the glucose and lactate cycles, with only one peak occurring closer to mid-photophase. In eyestalk-ablated animals, the glucose rhythmicity was lost; lactate cycled, but levels were significantly lower than in intact animals. Melatonin caused a delayed hyperglycemia in eyestalk-ablated animals, with concurrent but much lower increases in lactate. Overall, melatonin demonstrated delayed hyperglycemic effects that do not appear to be mediated solely via eyestalk factors such as crustacean hyperglycemic hormone (CHH), though involvement of the eyestalks cannot be ruled out. An influence on extra-eyestalk CHH secretion is a potential mechanism of melatonin activity.     

Changes in free amino acids in the hemolymph of giant freshwater prawn Macrobrachium rosenbergii exposed to varying salinities: relationship to osmoregulatory ability

Changes in free amino acids (FAA) in the hemolymph of the giant freshwater prawn, Macrobrachium rosenbergii, were examined in individuals exposed to varying salinities for up to 1 week. In freshwater and under conditions of low salinity, total FAA concentrations were maintained between approximately 0.85 and 1 mM and did not exhibit changes in response to salinity exposure. Under high salinities, total FAA concentrations increased dramatically, reaching up to 2.1 mM depending on treatment. Examination of individual amino acid concentrations revealed that these increases were based on specific changes in glycine, arginine, alanine, proline and lysine. Among these, alanine showed the greatest increases, resulting in levels six-fold higher under high salinity than in freshwater and under low salinity. The other amino acid species showed increases of 2.5-fold compared to original values. These five FAAs in freshwater and under low salinity together occupied approximately 45% of total FAA contents and under high salinity comprised more than 70% of total FAA contents. These results suggest that specific hemolymph FAAs are involved in mediating response to salinity exposure in freshwater prawns.     

Morphological response of the red palm weevil,Rhynchophorus ferrugineus,to a transient low temperature analyzed by computer tomography and holographic microscopy

The red palm weevil (RPW), Rhynchophorus ferrugineus, is one of the worst palm pests worldwide. Our study aims to assess its internal and external morphological response to a sudden but transient decrease in the environmental temperature. Wild pre-pupae were subjected for 7 days to either low (5.0 ± 0.5 °C) or ambient temperature (23 ± 1 °C). Such conditions mimic a thermal anomaly happening in the larval stage most exposed to environmental factors. We quantified the changes undergone at: 1) the internal morphology, by X-Ray Computer Tomography (CT); 2) the 3-D integument’ architecture, by Digital Holographic Microscopy (DHM); and 3) the glucose in hemolymph as a potential endogenous cryoprotectant. From X-ray CT we found that both pre-pupae subjected to cold and those remaining at ambient temperature follow a development where their fat body content decreases while a thick and dense cuticle is formed. There was no difference between both groups in the rate of change of fat body/dense tissues. Nevertheless, the cold group presents a slight developmental delay at the level of hemolymph content. Through DHM we again obtained that pre-pupae subjected to cold have not experienced a stop in their development. However, a more obvious developmental delay is now observed in this group at the level of the integumental roughness. Finally, regarding glucose, we found similar levels in control and ambient temperature larvae, while it was clearly increased in 51,7% of those subjected to cold. Our whole results provide morphological and biochemical evidence showing that the larval-pupal transition of the RPW continues almost undisturbed even during the quiescent state induced by a sudden and severe cold event. Nevertheless, a certain developmental delay is observed in both internal and external morphology. Additionally, the increased glucose level only found in the cold group suggests that glucose is part of the RPW cold tolerance strategy.     

A decreased hyperglycemic response to adrenaline after prolonged, exhausting exercise in dogs.

The aim of the investigations carried out on dogs was to elucidate possible causes of the decreased hyperglycemic response to adrenaline after exhausting exercise. The k-values of i.v. glucose tolerance tests were calculated and the dose-response relationship to infused adrenaline was analyzed under control, resting conditions, and after exhausting exercise. K-values (KG-exc.) were significantly higher (p less than 0.01) after exercise than in control experiments, whereas hyperglycemic responses to the applied doses of adrenaline: 0.25, 0.5, 1.0, 2.0 and 3.0 mug-kg-1min-1 were considerably lower. In both situations a linear relationship was found between the hyperglycemic response and the dose of adrenaline, although the slope of the regression line, expressing this relationship, was significantly lower after exercise than that under control conditions. It is suggested that the decreased hyperglycemic response to adrenaline after prolonged, exhausting exercise may be attributed to both the increased rate of glucose elimination from blood, and a decreased responsiveness of the liver to adrenaline.     

Molecular cloning and characterization of the crustacean hyperglycemic hormone cDNA from Litopenaeus schmitti. Functional analysis by double-stranded RNA interference technique

The crustacean hyperglycemic hormone (CHH) plays an important role in the regulation of hemolymph glucose levels, but it is also involved in other functions such as growth, molting and reproduction. In the present study we describe the first CHH family gene isolated from the Atlantic Ocean shrimp Litopenaeus schmitti. Sequence analysis of the amplified cDNA fragment revealed a high nucleotide sequence identity with other CHHs. Northern blot analysis showed that the isolated CHH mRNA from L. schmitti is present in the eyestalk but not in muscle or stomach. We also investigated the ability of dsRNA to inhibit the CHH function in shrimps in vivo. Injection of CHH dsRNA into the abdominal hemolymph sinuses resulted in undetectable CHH mRNA levels within 24 h and a corresponding decrease in hemolymph glucose levels, suggesting that functional gene silencing had occurred. These findings are the first evidence that dsRNA technique is operative in adult shrimps in vivo.     

Serotonergic regulation of blood glucose levels in the crayfish, Procambarus clarkii: site of action and receptor characterization

The present study investigated the site of action of 5-hydroxytryptamine (5-HT) and pharmacologically characterized the receptors involved in regulating blood glucose levels in the crayfish, Procambarus clarkii. Injection of 5-HT into intact animals increased glucose levels in a dose-dependent manner. In contrast, 5-HT failed to elicit a hyperglycemic response in eyestalk-ablated animals. Effects of several 5-HT receptor agonists and antagonists were examined. 5-CT, oxymetazoline (both 5-HT(1) receptor agonists) and alpha-methyl-5-HT (a 5-HT(2) receptor agonist), but not 1-phenylbiguanide, m-CPBG (both 5-HT(3) receptor agonists), or RS 67333 (a 5-HT(4) receptor agonist), induced hyperglycemic responses in a dose-dependent manner. In addition, 8-OH-DPAT (a 5-HT(1A) receptor agonist), L-694,247 (a 5-HT(1B/1D) receptor agonist), and DOI (a 5-HT(2A) receptor agonist) were effective in significantly increasing the glucose levels, whereas both BW 723C86 (a 5-HT(2B) receptor agonist) and m-CPP (a 5-HT(2C) receptor agonist) were ineffective. Finally, ketanserin (a 5-HT(2A) receptor antagonist), but not p-MPPF (a 5-HT(1A) receptor antagonist), GR 55562 (a 5-HT(1B/1D) receptor antagonist), SB 206553 (a 5-HT(2B/2C) receptor antagonist), or tropisetron (a 5-HT(3) receptor antagonist), was able to block 5-HT-induced hyperglycemia. The combined results support the hypothesis that 5-HT exerts its hyperglycemic effect by enhancing the release of hyperglycemic factor(s) from the eyestalks, and suggest that 5 HT-induced hyperglycemia is mediated by 5-HT(1)- and 5-HT(2)-like receptors.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) co-localizes with activity-dependent neuroprotective protein (ADNP) in the mouse brains

The crustacean hyperglycemic hormone is the most abundant neuropeptide present in the eyestalk of Crustacea and its main role is to control the glucose level in the hemolymph. Our study was aimed at assessing the importance of C-terminal amidation for its biological activity. Two recombinant peptides were produced, Asl-rcHH-Gly with a free carboxyl terminus and Asl-rcHH-amide with an amidated C-terminus. Homologous bioassays performed on the astacid crayfish Astacus leptodactylus showed that the amidated peptide had a stronger hyperglycemic effect compared to the non-amidated peptide. To assess the relevance of amidation also in other decapods and how much the differences in the cHH amino acid sequence can affect the functionality of the peptides, we carried out heterologous bioassays on the cambarid Procambarus clarkii and palaemonid Palaemon elegans. The Asl-rcHH-amide elicited a good response in P. clarkii and in P. elegans. The injection of Asl-rcHH-Gly evoked a weak response in both species. These results prove the importance of C-terminal amidation for the biological activity of cHH in crayfish as well as the role of the peptide primary sequence for the species-specificity hormone-receptor recognition.     

Hyperglycemic and osmotic effects of dopamine and recombinant hormone CHH-B1 in the Pacific white shrimp Litopenaeus vannamei

The effects of dopamine (DA) on crustacean hyperglycemic hormone (CHH) release and osmoregulation were investigated in the white shrimp Litopenaeus vannamei. Application of 2 μg of the recombinant CHH-B1_His hormone or 2?×?10^?6 mol L^?1 of DA to intact shrimp caused an increase in the hemolymph glucose levels 1 h post-injection, suggesting that DA might stimulate hyperglycemia through CHH release from the sinus glands. This assumption was supported by similar experiments using bilaterally eyestalk-ablated shrimp. Additionally, rCHH-B1_His restored the osmoregulatory capacity (OC) of shrimp under hyperosmotic conditions to basal values 2 h post-injection, whereas DA led to an OC decrease in shrimp at all sampling times. These neuroendocrine factors may be involved in the control of metabolism and osmoregulation in L. vannamei and could be important for its adaptation to different environmental conditions.     

Species-specific effects on hemolymph glucose control by serotonin, dopamine, and L-enkephalin and their inhibitors in Squilla mantis and Astacus leptodactylus (crustacea)

Hemolymph glucose level is controlled by crustacean Hyperglycemic Hormone (cHH) released from the eyestalk neuroendocrine centers under conditions of both physiological and environmental stress. Biogenic amines and enkephalin have been found to mediate the release of several neurohormones from crustacean neuroendocrine tissue. We investigated the effect of serotonin, dopamine, and Leucine-enkephalin in vivo--injected into the stomatopod Squilla mantis and the decapod Astacus leptodactylus--whether increasing or depressing glycemia. Serotonin had a marked effect in elevating glucose level compared with initial values in both species. 5-HT1-like receptors are more involved in mediating serotonin action as co-injected cyproheptadine was a more effective antagonist than ketanserin (5-HT2-like receptor inhibitor). Dopamine injection in intact animals produced a decrease below initial levels of hemolymph glucose. This effect was significantly antagonized by domperidone. No significant effect of both amines occurred in eyestalkless animals. L-enkephalin shows a differential effect: in S. mantis it induced hypoglycemia while in A. leptodactylus it caused an increase of glucose level. Co-injected antagonist naloxone affected the direction of the response. Serotonin appears to provide a major control on glucose mobilization, whereas dopamine and L-enkephalin act as modulators whose plasticity in use or action varies among species.     

Use of the glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol as a probe to investigate hypertrehalosemic hormone-mediated change in carbohydrate and lipid metabolism in the cockroach, Periplaneta americana

The glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DIA) was used to investigate the action of hypertrehalosemic hormone (HTH) on carbohydrate, neutral lipid, and phospholipid metabolism in the hemolymph and fat body of the cockroach, Periplaneta americana. DIA blocked the hypertrehalosemic and hyperglycemic effects of HTH, as well as the decrease in hemolymph neutral lipid and phospholipid normally induced by HTH. DIA diminished the accumulation of neutral lipid in the fat body formed under the influence of HTH and partly blocked the decrease in fat body phospholipid evoked by HTH. The increased incorporation of (14)C-glucose into fat body triacylglycerol in the presence of HTH was decreased by more than two-thirds when DIA was coinjected with the hormone. The results suggest that glucose derived from hemolymph trehalose is an important contributor to the formation of the glycerol backbone of newly formed triacylglycerol in the fat body.     

Reference ranges for hemolymph chemistries from Elliptio complanata of North Carolina

Hemolymph chemistries may be useful nonlethal measures of bivalve health. The prognostic value of hemolymph, however, depends on a comparison of chemistry results to reference ranges from healthy individuals. Currently, knowledge of expected hemolymph values in healthy and unhealthy freshwater mussels is extremely limited. The purpose of this study was to develop a set of reference ranges for clinical evaluation of hemolymph from a freshwater mussel species common to southeastern USA. We collected hemolymph from 380 Elliptio complanata from 19 apparently healthy populations from northwest of Raleigh, North Carolina, during May through July 2001. We present reference ranges for hemolymph parameters ammonia, glucose, calcium, magnesium, phosphorus, aspartate aminotransferase (AST), bicarbonate, protein and cell count, and for tissue glycogen. We compare the subpopulations of mussels from regions with an agricultural riparian buffer to those surrounded predominantly by forested lands. We further present correlations noted between hemolymph chemistries and physical or physiologic parameters. The only statistically significant differences between populations contiguous to agricultural and forested lands were in hemolymph calcium and glucose concentrations. Other statistically significant correlations identified were between gravidity and hemolymph protein concentration and tissue glycogen content, as well as between gravidity and parasite burden, and between shell length and hemolymph glucose, AST, calcium and bicarbonate concentrations. The results of this study will aid the interpretation of health measures from populations of E. complanata of similar geographic and seasonal origin.     

The effects of exposure to critical thermal maxima on the physiological,metabolic, and immunological responses in adult white shrimp Litopenaeus vannamei (Boone)

Exposure to thermal stress was shown to have a significant effect on the osmotic pressure of the hemolymph, glucose levels, total count of hemocyte (TCH), and proPO activity in adult white shrimp Litopenaeus vannamei. Exposure of the shrimp to CTMax significantly increased the osmotic pressure of the hemolymph relative to the control group. In organisms reaching CTMax, temperature elicited a secondary stress response that included an increase in hemolymph glucose of 31?mg?mL^?1. Metabolites in hemolymph such as cholesterol, acylglycerides, and total protein were not significantly affected by exposure to CTMax. CTMax exposure affected several immunological parameters causing decreases in TCH and proPO activity. We suggested that biomarkers such as osmolality, glucose levels, TCH, and proPO activity could be used as sensitive predictors of exposure to CTMax in white shrimp.     

An invertebrate hyperglycemic model for the identification of anti-diabetic drugs

The number of individuals diagnosed with type 2 diabetes mellitus, which is caused by insulin resistance and/or abnormal insulin secretion, is increasing worldwide, creating a strong demand for the development of more effective anti-diabetic drugs. However, animal-based screening for anti-diabetic compounds requires sacrifice of a large number of diabetic animals, which presents issues in terms of animal welfare. Here, we established a method for evaluating the anti-diabetic effects of compounds using an invertebrate animal, the silkworm, Bombyx mori. Sugar levels in silkworm hemolymph increased immediately after feeding silkworms a high glucose-containing diet, resulting in impaired growth. Human insulin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, decreased the hemolymph sugar levels of the hyperglycemic silkworms and restored growth. Treatment of the isolated fat body with human insulin in an in vitro culture system increased total sugar in the fat body and stimulated Akt phosphorylation. These responses were inhibited by wortmannin, an inhibitor of phosphoinositide 3 kinase. Moreover, AICAR stimulated AMPK phosphorylation in the silkworm fat body. Administration of aminoguanidine, a Maillard reaction inhibitor, repressed the accumulation of Maillard reaction products (advanced glycation end-products; AGEs) in the hyperglycemic silkworms and restored growth, suggesting that the growth defect of hyperglycemic silkworms is caused by AGE accumulation in the hemolymph. Furthermore, we identified galactose as a hypoglycemic compound in jiou, an herbal medicine for diabetes, by monitoring its hypoglycemic activity in hyperglycemic silkworms. These results suggest that the hyperglycemic silkworm model is useful for identifying anti-diabetic drugs that show therapeutic effects in mammals.     

Arginine-vasopressin mediates central and peripheral glucose regulation in response to carotid body receptor stimulation with Na-cyanide.

Hypoxic stimulation of the carotid body receptors (CBR) results in a rapid hyperglycemia with an increase in brain glucose retention. Previous work indicates that neurohypophysectomy inhibits this hyperglycemic response. Here, we show that systemic arginine vasopressin (AVP) induced a transient, but significant, increase in blood glucose levels and increased brain glucose retention, a response similar to that observed after CBR stimulation. Comparable results were obtained after intracerebral infusion of AVP. Systemic AVP-induced changes were maintained in hypophysectomized rats but were not observed after adrenalectomy. Glycemic changes after CBR stimulation were inhibited by pharmacological blockage of AVP V1a receptors with a V1a-selective receptor antagonist ([beta-Mercapto-beta,beta-cyclopentamethylenepropionyl1,O-me-Tyr2, Arg8]-vasopressin). Importantly, local application of micro-doses of this antagonist to the liver was sufficient to abolish the hyperglycemic response after CBR stimulation. These results suggest that AVP is a mediator of the hyperglycemic reflex and cerebral glucose retention following CBR stimulation. We propose that hepatic activation of AVP V1a receptors is essential for this hyperglycemic response.