Effects of stanniocalcin 1 hormone on lactate metabolism in rat kidney under fed and fasted conditions.

To test the hypothesis of STC-1 participation in maintenance of glucose homeostasis in fed and fasting (48 h) rats, we investigated that this hormone may be implicated in the regulation of renal gluconeogenesis pathway from lactate and lactate oxidation in renal cortex and medulla. Our results demonstrate the hSTC-1 role on lactate metabolism in the renal cortex and medulla from fed and fasting rats. hSTC-1 increased the gluconeogenesis activity in fed state in renal cortex, and this increase was induced by raise in Pck1 gene expression. In fasting animals hSTC-1 increase the renal medulla gluconeogenesis activity, but Pck1 gene expression was not alter. The stimulatory effect of hSTC-1 on 14C-lactate oxidation occurred only in the renal cortex from fed rats. These findings show the hSTC-1 contribution to lactate homeostasis and supplies glucose to other tissues. This response may represent a strategy of action of STC-1 in response to fasting stress as postulated by different authors. On the other hand, hSTC-1 acts downstream of adenylcyclase pathway, decreasing the gluconeogenesis activity induced by cAMP intracellular increase or stimulating the phosphodiesterase activity in the renal cortex. However, no hSTC-1 effect on 14C-lactate oxidation was found after increase in the intracellular cAMP. The findings also revealed that the renal cortex and medulla respond differently to hSTC-1, possibly due to the higher level of STC-1 gene expression in inner renal medulla than in renal cortex.

Diet composition and long-term starvation do not affect crustacean hyperglycemic hormone (CHH) transcription in the burrowing crab Neohelice granulata (Dana, 1851).

The burrowing crab Neohelice granulata is a key omnivorous species in intertidal areas along the southwestern Atlantic from southern Brazil to northern Argentinean Patagonia. This crab is adapted to starvation and can endure natural periods of food deprivation. The metabolic adjustments during starvation depend on the type of diet the crabs were fed previously. Since eyestalk-crustacean hyperglycemic hormone (CHH) is the principal regulator of glucose homeostasis in decapods, we investigated whether CHH transcription was affected by diet composition and starvation. Crabs were maintained in the laboratory for two weeks and subsequently divided in two groups. One received a high carbohydrate (HC) diet, and the other was fed a high protein (HP) diet. After this period, they were starved for four weeks. The full-length cDNA sequence of N. granulata CHH was determined and aligned with CHH sequences of other crabs. Levels of circulating glucose and glycogen were higher in the hepatopancreas and muscle of the HC-fed group and decreased after starvation. Glucose and glycogen concentrations were not altered by starvation in the HP group. Triglyceride levels within the hemolymph were not altered by diet or starvation. However, triglycerides concentration was higher in the hepatopancreas of HC compared to HP-fed group. Long-term starvation and diet composition did not affect CHH transcription.

Metabolic effects of epinephrine on the crab Neohelice granulata.

Although widely known for their involvement in the control of carbohydrate and lipid metabolism of vertebrates, the participation of catecholamines (CAs) in the metabolism of invertebrates is less understood. This study was designed to identify the physiological role of Epinephrine (E) in the intermediary metabolism of the burrowing crab Neohelice granulata and how E regulates the metabolism in crabs fed with a high-carbohydrate (HC) or a high-protein (HP) diet. To answer these questions, we evaluated in vivo the effects of E injections on glucose and triglycerides in the hemolymph and tissue glycogen levels of crabs fed with HC or HP diet. An in vitro investigation was carried out to assess the direct effects of E on glycogenolysis, lipolysis and glycolysis pathways in the hepatopancreas, mandibular muscle and anterior and posterior gills of this crab. E injections increased glucose and did not affect triglycerides levels in the hemolymph of either group of crabs, and E decreased glycogen in the hepatopancreas and mandibular muscle only in HP crabs, suggesting that these effects may be mediated by the crustacean hyperglycemic hormone (CHH). When the tissues were incubated with different concentrations of E, the concentration of glucose released to the medium decreased in the hepatopancreas and posterior gills, while glucose oxidation increased in the posterior gills of HP crabs. Incubation with E did not alter any parameter in tissues of HC crabs. These effects suggest that E may be involved in the metabolic response to osmotic stress.

Insulin-like receptors and carbohydrate metabolism in gills of the euryhaline crab Neohelice granulata: Effects of osmotic stress.

The present study determined the effect of osmotic stress on the insulin-like receptor binding characteristics and on glucose metabolism in the anterior (AG) and posterior (PG) gills of the crab Neohelice granulata. Bovine insulin increased the capacity of the PG cell membrane to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and the glucose uptake in the control crab group. The crabs were submitted to three periods of hyperosmotic (HR) and hyposmotic (HO) stress, for 24, 72 and 144 h, to investigate the insulin-like receptor phosphorylation capacity of gills. Acclimation to HO for 24 h or HR for 144 h of stress inhibited the effects of insulin in the PG, decreasing the capacity of insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and decreasing the glucose uptake. Hyperosmotic stress for the same period of 144 h significantly affected 125I-insulin binding in the AG and PG. However, HO stress for 24 h significantly reduced 125I-insulin-specific uptake only in the PG. Therefore, osmotic stress induces alterations in the gill insulin-like receptors that decrease insulin binding in the PG. These findings indicate that osmotic stress induced a pattern of insulin resistance in the PG. The free-glucose concentration in the PG decreased during acclimation to 144 h of HR stress and 24 h of HO stress. This decrease in the cell free-glucose concentration was not accompanied by a significant change in hemolymph glucose levels. In AG from the control group, neither the capacity of bovine insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) nor the glucose uptake changed; however, genistein decreased tyrosine-kinase activity, confirming that this receptor belongs to the tyrosine-kinase family. Acclimation to HO (24 h) or HR (144 h) stress decreased tyrosine-kinase activity in the AG. This study provided new information on the mechanisms involved in the osmoregulation process in crustaceans, demonstrating for the first time in an estuarine crab that osmotic challenge inhibited insulin-like signaling and the effect of insulin on glucose uptake in the PG.

Serotonin effects in the crab Neohelice granulata: Possible involvement of two types of receptors in peripheral tissues.

In crustaceans, serotonin (5-HT) controls various physiological processes, such as hormonal secretion, color changes, reproduction, and metabolism. Since 5-HT injections cause hyperglycemia, this study was designed to further investigate this action of 5-HT in the crab Neohelice granulate, fed with a high-carbohydrate (HC) or a high-protein (HP) diet. The effects of pre-treatment with mammalian 5-HT receptor antagonists, cyproheptadine and methiothepin, were also investigated. A series of in vivo experiments with (3)H-5-HT was carried out in order to investigate the presence of putative receptors in peripheral tissues. Since gills were the tissue with the highest labeling in in vivo experiments, in vitro studies with isolated anterior and posterior gills were also conducted. Cyproheptadine blocked the hyperglycemic effect of 5-HT in HP-fed crabs. Methiothepin reduced glycogen levels in the anterior gills of HP crabs and partially blocked the 5-HT-like posture. The injection of (3)H-5-HT identified specific binding sites in all the tissues studied and revealed that the binding can be influenced by the type of diet administered to the crabs. Incubation of the anterior and posterior gills with (3)H-5-HT and 5-HT confirmed the specificity of the binding sites. Both antagonists inhibited (3)H-5-HT binding. In conclusion, this study highlights the importance of serotonin in the control of glucose homeostasis in crustaceans and provides evidences of at least two types of 5-HT binding sites in peripheral tissues. Further studies are necessary to identify the structure of these receptors and their signaling pathways.

Effect of starvation and refeeding on amino acid metabolism in muscle of crab Neohelice granulata previously fed protein- or carbohydrate-rich diets.

The present study assesses the effects of starvation and refeeding on 1-[(14)C]-methyl aminoisobutyric acid ((14)C-MeAIB) uptake, (14)C-total lipids, (14)CO(2) production from (14)C-glycine, (14)C-protein synthesis from (14)C-leucine and Na(+)-K(+)-ATPase activity in jaw muscle of Neohelice granulata previously maintained on a carbohydrate-rich (HC) or high-protein (HP) diet. In N. granulata the metabolic adjustments during starvation and refeeding use different pathways according to the composition of the diet previously offered to the crabs. During starvation, (14)CO(2) production from (14)C-glycine, and (14)C-protein synthesis from (14)C-leucine were reduced in HC-fed crabs. In crabs maintained on the HP or HC diet, (14)C-total lipid synthesis increased after 15 days of starvation. In crabs fed HP diet, (14)C-MeAIB uptake and Na(+)-K(+)-ATPase activity decreased in refeeding state. In crabs refeeding HC diet, (14)C-MeAIB uptake and (14)CO(2) production decreased during the refeeding. In contrast, the (14)C-protein synthesis increased after 120h of refeeding. In both dietary groups, (14)C-total lipid synthesis increased during refeeding. Changes in the carbon amino acid flux between different metabolic pathways in muscle are among the strategies used by this crab to face starvation and refeeding. Protein or carbohydrate levels in the diet administered to this crab modulate the carbon flux between the different metabolic pathways.

Control of carbohydrate metabolism in an anoxia-tolerant nervous system.

Anoxia-tolerant animal models are crucial to understand protective mechanisms during low oxygen excursions. As glycogen is the main fermentable fuel supporting energy production during oxygen tension reduction, understanding glycogen metabolism can provide important insights about processes involved in anoxia survival. In this report we studied carbohydrate metabolism regulation in the central nervous system (CNS) of an anoxia-tolerant land snail during experimental anoxia exposure and subsequent reoxygenation. Glucose uptake, glycogen synthesis from glucose, and the key enzymes of glycogen metabolism, glycogen synthase (GS) and glycogen phosphorylase (GP), were analyzed. When exposed to anoxia, the nervous ganglia of the snail achieved a sustained glucose uptake and glycogen synthesis levels, which seems important to maintain neural homeostasis. However, the activities of GS and GP were reduced, indicating a possible metabolic depression in the CNS. During the aerobic recovery period, the enzyme activities returned to basal values. The possible strategies used by Megalobulimus abbreviatus CNS to survive anoxia are discussed.

Seasonal variation in glucose and neutral amino acid uptake in the estuarine crab Neohelice granulata.

This study investigates the mechanisms of glucose and amino acid transport in gills and jaw muscle of N. granulata collected from an estuarine natural population. The physicochemical parameters of the estuarine environment and of this crustacean's hemolymph were measured during different seasons of the year. In summer, the lagoon water osmolality increased (5-6 times), and hemolymph osmolality decreased. In fall, water pH increased, whereas hemolymph pH decreased markedly. In all seasons, 2-deoxi glucose (DG) uptake in gills was significantly higher than 3-O methyl-glucose (MG) uptake. Phloretin reduced DG uptake in gills; phloretin and phlorizin did not affect MG uptake in this organ. DG and MG uptake was highest in gills during spring and summer. In jaw muscle, MG uptake in winter and spring was higher than DG uptake. In fall, gill methyl aminoisobutyric acid (MeAIB) uptake increased. In jaw muscle, MeAIB uptake was higher in spring. The observed changes in glucose uptake and in the type of glucose and amino acid transporter used in gills and muscle appear to be strategies used by N. granulata to minimize seasonal oscillations in the environmental parameters of their estuarine habitat.

Lactate metabolism in the muscle of the crab Chasmagnathus granulatus during hypoxia and post-hypoxia recovery.

The present study showed that the lactate/glucose ratio in the hemolymph of Chasmagnathus granulatus maintained in normoxia (controls) was 4.9, suggesting that lactate is an important substrate for this crab. Periods of hypoxia are part of the biological cycle of this crab, and lactate is the main end product of anaerobiosis in this crab. Our hypothesis was that this lactate would be, therefore, used by gluconeogenic pathway or can be oxidized or excreted to the aquatic medium during hypoxia and post-hypoxia periods in C. granulatus. The concentrations of hemolymphatic lactate in animals in normoxia are high, and are used as an energy substrate. In hypoxia, muscle gluconeogenesis and excretion of lactate to the aquatic medium would contribute significantly in regulating the concentration of circulating lactate. Utilization of these pathways would serve the objective of maintaining the acid-base equilibrium of the organism. Muscle gluconeogenesis participates, during the recovery process, in metabolizing the lactate produced during the period of hypoxia. Lactate excretion to the external medium, was one of the strategies used to decrease the higher hemolymphatic lactate levels. However, oxidation of lactate in the muscle is not a main strategy used by this crab to metabolize lactate in the recovery periods.