Structural Mapping and Functional Characterization of Zebrafish Class B G-Protein Coupled Receptor (GPCR) with Dual Ligand Selectivity towards GLP-1 and Glucagon.

GLP-1 and glucagon regulate glucose metabolism through a network of metabolic pathways initiated upon binding to their specific receptors that belong to class B G-protein coupled receptors (GPCRs). The therapeutic potential of glucagon is currently being evaluated, while GLP-1 is already used in the treatment of type 2 diabetes and obesity. Development of a second generation of GLP-1 based therapeutics depends on a molecular and structural understanding of the interactions between the GLP-1 receptor (GLP-1R) and its ligand GLP-1.

Proglucagons in vertebrates: Expression and processing of multiple genes in a bony fish.

In contrast to mammals, where a single proglucagon (PG) gene encodes three peptides: glucagon, glucagon-like peptide 1 and glucagon-like peptide 2 (GLP-1; GLP-2), many non-mammalian vertebrates carry multiple PG genes. Here, we investigate proglucagon mRNA sequences, their tissue expression and processing in a diploid bony fish. Copper rockfish (Sebastes caurinus) express two independent genes coding for distinct proglucagon sequences (PG I, PG II), with PG II lacking the GLP-2 sequence.

Concentration-response relationships and temporal patterns in hepatic gene expression of Chinook salmon (Oncorhynchus tshawytscha) exposed to sewage.

Changes in liver gene expression were examined in juvenile Chinook salmon (Oncorhynchus tshawytscha) exposed in vivo for 8d to seawater (control) or one of 5 concentrations of sewage (environmentally-relevant dilutions of 0.05%, 0.1%, and 0.

Glucosensing and glucose homeostasis: from fish to mammals.

This review is focused on two topics related to glucose in vertebrates. In a first section devoted to glucose homeostasis we describe how glucose levels fluctuate and are regulated in different classes of vertebrates. The detection of these fluctuations is essential for homeostasis and for other physiological processes such as regulation of food intake.

Growth hormone transgenesis influences carbohydrate, lipid and protein metabolism capacity for energy production in coho salmon (Oncorhynchus kisutch).

Growth hormone (GH) transgenesis results in increased growth, feed intake and consequent metabolic rates in fish, and alters the utilization of dietary and stored carbohydrates, lipid and protein. However, the manner in which GH transgenesis differentially alters these energy sources in fish has not been well explored. We examined the effects of GH transgenesis and dietary carbohydrate, lipid and protein levels on metabolic enzyme activity in coho salmon (Oncorhynchus kisutch).

Is the alkaline tide a signal to activate metabolic or ionoregulatory enzymes in the dogfish shark (Squalus acanthias)?

Experimental metabolic alkalosis is known to stimulate whole-animal urea production and active ion secretion by the rectal gland in the dogfish shark. Furthermore, recent evidence indicates that a marked alkaline tide (systemic metabolic alkalosis) follows feeding in this species and that the activities of the enzymes of the ornithine-urea cycle (OUC) for urea synthesis in skeletal muscle and liver and of energy metabolism and ion transport in the rectal gland are increased at this time. We therefore evaluated whether alkalosis and/or NaCl/volume loading (which also occurs with feeding) could serve as a signal for activation of these enzymes independent of nutrient loading.

Gene expression pattern in the liver during recovery from an acute stressor in rainbow trout.

The physiological response to stressors, including hormonal profiles and associated tissue responsiveness, has been extensively studied with salmonid fish, but less is known about the molecular basis of this adaptive response. As liver is the major target organ for metabolic adjustments, we exploited a selective transcriptomics approach to address molecular response in this tissue during acute stress adaptation in rainbow trout. The stressor consisted of a standardized 3 min handling disturbance of trout, and plasma and liver samples were collected either prior to or 1 and 24 h after stressor exposure.

Metabolic organization and effects of feeding on enzyme activities of the dogfish shark (Squalus acanthias) rectal gland.

In order to investigate the metabolic poise of the elasmobranch rectal gland, we conducted two lines of experimentation. First, we examined the effects of feeding on plasma metabolites and enzyme activities from several metabolic pathways in several tissues of the dogfish shark, Squalus acanthias, after starvation and at 6, 20, 30 and 48 h post-feeding. We found a rapid and sustained ten-fold decrease in plasma beta-hydroxybutyrate at 6 h and beyond compared with starved dogfish, suggesting an upregulation in the use of this substrate, a decrease in production, or both.

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding.

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues.

Alkaline tide and nitrogen conservation after feeding in an elasmobranch (Squalus acanthias).

We investigated the consequences of feeding for acid-base balance, nitrogen excretion, blood metabolites and osmoregulation in the Pacific spiny dogfish. Sharks that had been starved for 7 days were surgically fitted with indwelling stomach tubes for gastric feeding and blood catheters for repetitive blood sampling and were confined in chambers, allowing measurement of ammonia-N and urea-N fluxes. The experimental meal infused via the stomach tube consisted of flatfish muscle (2% of body mass) suspended in saline (4% of body mass total volume).

Salmon spawning migration and muscle protein metabolism: the August Krogh principle at work.

The August Krogh principle, stating that for any particular question in biology, nature holds an ideal study system, was applied by choosing the anorexic, long-distance migration of salmon as a model to analyze protein degradation and amino acid metabolism. Reexamining an original study done over 20 years ago on migrating sockeye salmon (Oncorhynchus nerka), data on fish migration and starvation are reviewed and a general model is developed on how fish deal with muscle proteolysis. It is shown that lysosomal activation and degradation of muscle protein by lysosomal cathepsins, especially cathepsin D and sometimes cathepsin L, are responsible for the degradation of muscle protein during fish migration, maturation and starvation.

Cell volume affects glycogen phosphorylase activity in fish hepatocytes.

The activity of glycogen phosphorylase (GPase) in the active a-form (GPase a) is dependent on the hydration state of hepatocytes. We establish that GPase a catalysis in catfish (Ameiurus nebulosus) hepatocytes is a function of medium osmolarity and that a linear relationship exists between GPase a activity and osmolarity between 254 mosmol l(-1) and 478 mosmol l(-1). Exposure of isolated hepatocytes to hyperosmotic media increases enzyme activity up to 7-fold, indicative of covalent phosphorylation.

Glutamine synthetase in tilapia gastrointestinal tract: zonation, cDNA and induction by cortisol.

Glutamine synthetase, an enzyme generally associated with ammonia detoxication in the vertebrate brain and with hepatic nitrogen turnover in mammals, shows substantial activities in the gastrointestinal tract of teleostean fishes. Enzyme activity is highest in the central area of the stomach and reveals a distinct distribution pattern in stomach and along the intestine of tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss) and copper rockfish (Sebastes caurinus). In all three species, intestinal activity peaks in the distal region of the intestine.

Metabolic zonation in teleost gastrointestinal tract. Effects of fasting and cortisol in tilapia.

Activities of several metabolic enzymes show distinct patterns of zonation along the intestinal tract of tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss) and copper rockfish (Sebastes caurinus). Zonation is species and enzyme specific, with different metabolic activities concentrated in specific areas, and few generalizations can be made. The rockfish show the smallest degree of zonation, with highest activities in the third quarter of the intestine, and shallow gradients to either side, and a general upswing in activity towards the distal end.

A second glutamine synthetase gene with expression in the gills of the gulf toadfish (Opsanus beta).

We characterized the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.

Evolution of glutamine synthetase in vertebrates: multiple glutamine synthetase genes expressed in rainbow trout (Oncorhynchus mykiss).

Glutamine synthetase (GSase) is a key enzyme in nitrogen metabolism and encoded by a single gene in mammals. Using PCR cloning techniques, including RT-PCR from total RNA and PCR from a cDNA library, we find evidence of four expressed GSase mRNAs for the tetraploid rainbow trout. For two of these mRNAs (Onmy-GS01, -GS02) we characterize the full-length coding regions, and for two others (Onmy-GS03, -GS04), we describe partial sequences.

Novel role for prostaglandin E2 in fish hepatocytes: regulation of glucose metabolism.

Prostaglandin E(2) (PGE(2)) potently activated glycogenolysis and gluconeogenesis in isolated rockfish (Sebastes caurinus) hepatocytes. The average degree of activation for glycogenolysis was 6.4+/-0.

Estradiol impairs hyposmoregulatory capacity in the euryhaline tilapia, Oreochromis mossambicus.

Freshwater (FW)-adapted tilapia (Oreochromis mossambicus) were treated with estradiol (E(2)) for 4 days to stimulate protein synthesis and sampled at 0, 4, and 24 h after exposure to 50% seawater (SW). E(2) increased circulating vitellogenin (VTG) levels in large amounts, indicative of unusually high rates of hepatic protein synthesis. E(2) treatment prevented the recovery of plasma osmolality in 50% SW that was evident in the sham group.