Species-specific low plasma glucose in fish is associated with relatively high tissue glucose content and is inversely correlated with cardiac glycogen content.

The relationship between plasma glucose concentration and intracellular glucose (liver, heart, brain, gill, gonad, intestine, kidney, spleen, white muscle) was determined in fish species with a range in plasma glucose (Atlantic cod, 5.06 mM; cunner, 3.8 mM; rainbow trout, 3.7 mM; lumpfish, 0.9 mM; short-horned sculpin, 0.6 mM; and winter flounder, 0.6 mM). The ratio of intracellular glucose/plasma glucose was always higher than one in liver for all species consistent with a diffusion gradient from the tissue to the plasma. In all other tissues in Atlantic cod, cunner, and rainbow trout the diffusion gradient was from the plasma to the intracellular space. In short-horned sculpin, the mean ratio in heart and white muscle exceeded one and in winter flounder the ratio was significantly greater than one at 5.97 and 2.92 for heart and muscle, respectively. The presence of an active glucose 6-phosphatase in white muscle could account for elevated amounts of free glucose. The white muscle of all species displayed phosphoenolpyruvate carboxykinase and in winter flounder the activity was as high in white muscle as in liver suggesting that gluconeogenesis may be associated with a relatively high-muscle glucose content. The glycogen content was highest in liver followed by heart with lower amounts in all other tissues. There was an inverse correlation between heart glycogen content and plasma glucose concentration when all species were included. It is contended that in species with low plasma glucose, heart glycogen is accumulated at a slow rate under normoxia, to be called upon under hypoxic conditions when the gradient for inward diffusion is unfavourable for high rates of glucose metabolism.

Low levels of extracellular glucose limit cardiac anaerobic metabolism in some species of fish.

There is a wide interspecific range in plasma glucose levels in teleosts from less than 0.5 to greater than 10 mmol l-1 Here we assessed how glucose availability influences glucose metabolism in hearts of Atlantic cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), lumpfish (Cyclopterus lumpus) and short-horned sculpin (Myoxocephalus scorpius) under normoxic and hypoxic conditions. These species had plasma glucose levels of 5.1, 4.8, 0.9 and 0.5 mmol l-1, respectively. Rates of glucose metabolism and lactate production were determined in isolated hearts perfused with medium containing physiological levels of glucose. Under normoxic conditions there was no significant difference in rates of either glucose metabolism (average 15 nmol g-1 min-1) or lactate production (average 30 nmol g-1 min-1) across species. Under hypoxia (12% of air saturation) there were significant increases in rates of glucose metabolism and lactate production in hearts from Atlantic cod (glucose-130; lactate-663 nmol g-1 min-1) and rainbow trout (glucose-103; lactate-774 nmol g-1 min-1); however, there was no change in rate of glucose metabolism in hearts from either lumpfish or short-horned sculpin and only increases in lactate production to rates much lower than the other species. Furthermore, Atlantic cod hearts perfused with medium containing low non-physiological levels of glucose (0.5 mmol l-1) had the same rates of glucose metabolism under normoxic and hypoxic treatments. Anaerobic metabolism supported by extracellular glucose is compromised in fish with low levels of plasma glucose, which in turn may decrease performance under oxygen-limiting conditions at the whole-animal level.

High rates of glucose utilization in the gas gland of Atlantic cod (Gadus morhua) are supported by GLUT1 and HK1b.

The gas gland of physoclistous fish utilizes glucose to generate lactic acid that leads to the off-loading of oxygen from haemoglobin. This study addresses characteristics of the first two steps in glucose utilization in the gas gland of Atlantic cod (Gadus morhua). Glucose metabolism by isolated gas gland cells was 12- and 170-fold higher, respectively, than that in heart and red blood cells (RBCs) as determined by the production of (3)H2O from [2-(3)H]glucose. In the gas gland, essentially all of the glucose consumed was converted to lactate. Glucose uptake in the gas gland shows a very high dependence upon facilitated transport as evidenced by saturation of uptake of 2-deoxyglucose at a low extracellular concentration and a requirement for high levels of cytochalasin B for uptake inhibition despite the high efficacy of this treatment in heart and RBCs. Glucose transport is via glucose transporter 1 (GLUT1), which is localized to the glandular cells. GLUT1 western blot analysis from whole-tissue lysates displayed a band with a relative molecular mass of 52 kDa, consistent with the deduced amino acid sequence. Levels of 52 kDa GLUT1 in the gas gland were 2.3- and 33-fold higher, respectively, than those in heart and RBCs, respectively. Glucose phosphorylation is catalysed by hexokinase Ib (HKIb), a paralogue that cannot bind to the outer mitochondrial membrane. Transcript levels of HKIb in the gas gland were 52- and 57-fold more abundant, respectively, than those in heart and RBCs. It appears that high levels of GLUT1 protein and an unusual isoform of HKI are both critical for the high rates of glycolysis in gas gland cells.

Extracellular glucose supports lactate production but not aerobic metabolism in cardiomyocytes from both normoglycemic Atlantic cod and low glycemic short-horned sculpin.

Fish exhibit a wide range of species-specific blood glucose levels. How this relates to glucose utilization is yet to be fully realized. Here, we assessed glucose transport and metabolism in myocytes isolated from Atlantic cod (Gadus morhua) and short-horned sculpin (Myoxocephalus scorpius), species with blood glucose levels of 3.7 and 0.57 mmol l(-1), respectively. Glucose metabolism was assessed by the production of (3)H2O from [2-(3)H]glucose. Glucose metabolism was 3.5- to 6-fold higher by myocytes from Atlantic cod than by those from short-horned sculpin at the same level of extracellular glucose. In Atlantic cod myocytes, glucose metabolism displayed what appears to be a saturable component with respect to extracellular glucose, and cytochalasin B inhibited glucose metabolism. These features revealed a facilitated glucose diffusion mechanism that accounts for between 30% and 55% of glucose entry at physiological levels of extracellular glucose. Facilitated glucose diffusion appears to be minimal in myocytes for short-horned sculpin. Glucose entry by simple diffusion occurs in both cell types with the same linear relationship between glucose metabolism and extracellular glucose concentration, presumably due to similarities in membrane composition. Oxygen consumption by myocytes incubated in medium containing physiological levels of extracellular glucose (Atlantic cod 5 mmol l(-1), short-horned sculpin 0.5 mmol l(-1)) was similar in the two species and was not decreased by cytochalasin B, suggesting that these cells have the capability of oxidizing alternative on-board metabolic fuels. Cells produced lactate at low rates but glycogen levels did not change during the incubation period. In cells from both species, glucose utilization assessed by both simple chemical analysis of glucose disappearance from the medium and (3)H2O production was half the rate of lactate production and as such extracellular glucose was not available for oxidative metabolism. Overall, extracellular glucose makes only a minor contribution to ATP production but a sustained glycolysis may be necessary to support Ca(2+) transport mechanisms at either the sarcoplasmic reticulum or the sarcolemmal membrane.

Extracellular glucose can fuel metabolism in red blood cells from high glycemic Atlantic cod (Gadus morhua) but not low glycemic short-horned sculpin (Myoxocephalus scorpius).

Energy metabolism was assessed in red blood cells (RBCs) from Atlantic cod and short-horned sculpin, two species that have markedly different levels of blood glucose. The objective was to determine whether the level of extracellular glucose has an impact on rates of glucose metabolism. The blood glucose level was 2.5 mmol l(-1) in Atlantic cod and 0.2 mmol l(-1) in short-horned sculpin, respectively. Oxygen consumption, lactate production and glucose utilization were measured in whole blood and related to grams of RBCs. Glucose utilization was assessed by measuring both glucose disappearance and the production of (3)H2O from [2-(3)H]-glucose. RBCs from both species have an aerobic-based metabolism. In Atlantic cod, extracellular glucose is sufficient to provide the sum of glucosyl equivalents to support both oxidative metabolism and lactate production. In contrast, extracellular glucose can account for only 10% of the metabolic rate in short-horned sculpin RBCs. In both species, about 70% of glucose enters the RBCs via facilitated transport. The difference in rates of extracellular glucose utilization is related to the extremely low levels of blood glucose in short-horned sculpin. In this species energy metabolism by RBCs must be supported by alternative fuels.

Transcript levels of class I GLUTs within individual tissues and the direct relationship between GLUT1 expression and glucose metabolism in Atlantic cod (Gadus morhua).

GLUTs 1-4 are sodium-independent facilitated glucose transporters and are considered to play a major role in glucose trafficking. The relative transcript levels of GLUTs 1-4 were determined in tissues of Atlantic cod (Gadus morhua). The distribution profile of GLUTs normalized to RNA is similar to mammals and with a few exceptions other fish. GLUT1 is ubiquitous, GLUT2 is relatively abundant in tissues that release glucose, GLUT3 expression is relatively strong in brain, and GLUT4 is relatively high in heart and muscle. The functionally significant level of transcript is presumably the level in the cell. Normalization of relative GLUT levels to tissue mass reveals there are extremely high levels of GLUT1 transcript in gas gland consistent with the high lactate production rates, GLUT3 is dominant in gill and head kidney as well as brain, and GLUT4 expression in gill is elevated relative to other tissues. Consideration of GLUTs within tissues reveals that GLUT1 is the dominant transcript in a group of tissues including gas gland, heart, white muscle, and RBCs. Brain, gill, and spleen display a co-dominance of GLUTs 1 and 3. There are relatively low levels of GLUT4 in most tissues, the highest being found in white muscle where GLUT4 accounts for only 12 % of the total transcript level. The apparent low level of GLUT4 transcript may reflect two tissues that were not included in the current study, red muscle and adipose tissue, due to their low abundance in Atlantic cod. The rate of glucose metabolism in isolated cells prepared from gas gland, heart, and RBCs was determined by tracking the rate of (3)H2O production from [2-(3)H]-glucose. The steady-state rate of basal glycolysis in these three tissues correlates with relative transcript levels of GLUT1.

Glucose uptake and metabolism by red blood cells from fish with different extracellular glucose levels.

The aim of the present study was to assess whether mechanisms of glucose trafficking by red blood cells (RBCs) relate to species-specific extracellular glucose levels. Atlantic cod (Gadus morhua), Atlantic salmon (Salmo salar), cunner (Tautogolabrus adspersus) and short-horned sculpin (Myoxocephalus scorpius) had plasma glucose levels of 4, 4.1, 1.95 and 0.73 mmol l(-1), respectively. Glucose uptake by isolated RBCs was measured by the initial incorporation of [6-(14)C]-glucose and steady-state glucose metabolism was determined by the production of (3)H(2)O from [2-(3)H]-glucose. Saturation kinetics of glucose uptake and inhibition of both glucose uptake and metabolism by cytochalasin B and phloretin revealed that Atlantic cod, cunner and sculpin RBCs all had a facilitated transport component to glucose trafficking. RBCs from Atlantic salmon showed a linear relationship between glucose uptake and extracellular glucose level, but exhibited clear inhibition of glucose metabolism by cytochalasin B and phloretin, suggesting a component of facilitated glucose transport that is more elusive to detect. The production of (3)H(2)O was linear for at least 6 h and as such presents a rigorous approach to measuring glycolytic rate. Steady-state rates of glucose metabolism were achieved at extracellular levels of approximately 1 mmol l(-1) glucose for RBCs from all species, showing that within-species normal extracellular glucose level is not a primary determinant of the basal level of glycolysis. At physiological levels of extracellular glucose, the ratio of initial glucose uptake to glucose metabolism was 1.5 to 4 for all RBCs, suggesting that there is scope to increase metabolic rate without alteration of the basal glucose uptake capacity.

Expression analysis of glycerol synthesis-related liver transcripts in rainbow smelt (Osmerus mordax) exposed to a controlled decrease in temperature.

Rainbow smelt (Osmerus mordax) accumulate high glycerol levels to avoid freezing at subzero temperatures. Glyceroneogenesis is activated by low temperature and occurs in liver via a branch in glycolysis and gluconeogenesis. In this study, carbohydrate and liver transcript levels of 21 genes potentially associated with glycerol production were assessed during a controlled warm to cold transition. Smelt were held at 8°C (warm smelt; non-glycerol accumulating) or subjected to a controlled decrease in water temperature from 8° to 0°C (cold smelt; glycerol accumulating) and sampled at the end of the temperature decrease and 1 mo later. In cold smelt compared with warm smelt, liver glycogen levels were lower and phosphoglucomutase transcript levels were higher. Plasma glycerol levels were higher and increased over time in cold smelt; in cold smelt, liver phosphofructokinase and pyruvate dehydrogenase kinase transcript levels increased over time. These findings imply that glycerol production is being fueled by glycogen degradation and inhibition of pyruvate oxidation serves to channel metabolic flux toward glycerol as opposed to complete glycolysis. Plasma glucose and liver glucose-6-phosphatase transcript levels were higher. Lipoprotein lipase transcript levels were higher, suggesting enhanced lipid breakdown to fuel energy metabolism. Glutamine synthetase transcript levels were higher, perhaps to store nitrogen for biosynthesis in spring.

Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax).

PURPOSE: The rainbow smelt (Osmerus mordax), is a teleost fish, which avoids freezing by becoming virtually isosmotic with seawater. The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known. New knowledge about the osmotic adaptation of the rainbow smelt eye is highly relevant to the adaptation and survival of this species and to its ability to feed as a visual predator in the face of environmental pressures. Moreover, the molecular physiologic response of the smelt to osmotic stress might provide valuable insights into understanding and managing mammalian pathological hyperosmolarity conditions, such as diabetes. We undertook the present study to provide an initial assessment of gene expression in ocular vasculature during osmotic adaptation in rainbow smelt. METHODS: Immunohistochemistry with species cross reactive antibodies was used to assess blood vessel protein expression in paraffin sections. Western blotting was used to further verify antibody specificity for orthologs of mammalian blood vessel proteins in rainbow smelt. Thermal hysteresis and the analysis of glycerol concentrations in vitreous fluid were used to assess the physiologic adaptive properties of cold stressed eyes. RESULTS: Glycerol levels and osmotic pressure were significantly increased in the vitreal fluid of smelt maintained at <0.5 °C versus those maintained at 8-10 °C. Compared to the 8-10 °C adapted specimens, the rete mirabile blood vessels and connecting regions of the endothelial linings of the choroidal vessels of the <0.5 °C adapted specimens showed a higher expression level of Tubedown (Tbdn) protein, a marker of the endothelial transcellular permeability pathway. Expression of the zonula occludens protein ZO-1, a marker of the endothelial paracellular permeability pathway showed a reciprocal expression pattern and was downregulated in rete mirabile blood vessels and connecting regions in the endothelial linings of choroidal vessels in <0.5 °C adapted specimens. Smelt orthologs of the mammalian Tbdn and zoluna occludens protein 1 (ZO-1) proteins were also detected by western blotting using anti-mammalian antibodies raised against the same epitopes as those used for immunohistochemistry. CONCLUSIONS: This work provides the first evidence that molecules known to play a role in ocular vascular homeostasis are expressed and may be differentially regulated during anti-freezing cold adaptation in smelt eyes. We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space. This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

Glycerol loss to water exceeds glycerol catabolism via glycerol kinase in freeze-resistant rainbow smelt (Osmerus mordax).

Rainbow smelt accumulate high amounts of glycerol in winter. In smelt, there is a predictable profile of plasma glycerol levels that starts to increase in November (<5 µmol/ml), peaks in mid-February (>200 µmol/ml), and thereafter decreases to reach the initial levels in the beginning of May. The aim of this study was to investigate the respective role of the two main mechanisms that might be involved in glycerol clearance from mid-February: 1) breakdown of glycerol to glycerol-3-phosphate through the action of the glycerol kinase (GK) and 2) direct loss toward the environment. Over the entire glycerol cycle, loss to water represents a daily loss of ∼10% of the total glycerol content of fish. GK activities were very low in all tissues investigated and likely have a minor quantitative role in the glycerol cycle. These results suggest that glycerol levels are dictated by the rate of glycerol synthesis (accelerated and deactivated during the accumulation and decrease stages, respectively). Although not important in glycerol clearance, GK in liver might have an important metabolic function for other purposes, such as gluconeogenesis, as evidenced by the significant increase of activity at the end of the cycle.

Seasonal changes in hepatic gene expression reveal modulation of multiple processes in rainbow smelt (Osmerus mordax).

Rainbow smelt (Osmerus mordax) are freeze-resistant fish that accumulate glycerol and produce an antifreeze protein during winter. Quantitative reverse transcription PCR (qPCR) and subtractive hybridization studies have previously revealed five genes in rainbow smelt liver to be differentially regulated in winter in comparison with the fall when water temperatures are warmer. In order to further define the suite of processes that are regulated seasonally, we undertook a large-scale analysis of gene expression by hybridization of smelt cDNA to the salmonid 16K cGRASP microarray. In total, 69 genes were identified as up-regulated and 14 genes as down-regulated under winter conditions. A subset of these genes was examined for differential regulation by qPCR in the individual cDNA samples that were pooled for microarray analysis. Ten of the 15 genes tested showed significant change in the same direction as microarray results, whereas one showed significant change in the opposite direction. Fructose-bisphosphate aldolase B and the cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase were among the most highly up-regulated genes, a result supporting a metabolic focus on glycerol synthesis during winter. Modulation of other processes, including endoplasmic reticulum stress, lipid metabolism and transport, and protein synthesis, was also suggested by the qPCR analysis of array-identified genes. The 15 genes were subsequently examined by qPCR for seasonal variation in expression over five sampling times between October and March, and ten showed significant variation in expression over the sampling period. Taken together, these results provide new understanding of the biochemical adaptations of vertebrates to an extremely low seasonal temperature.

Seasonal expressed sequence tags of rainbow smelt (Osmerus mordax) revealed by subtractive hybridization and the identification of two genes up-regulated during winter.

The rainbow smelt (Osmerus mordax) is freeze-resistant and maintains swimming and feeding activity during winter. In order to identify genes differentially expressed in smelt liver response to winter water temperatures, a large-scale analysis of gene expression using suppression subtractive hybridization was carried out using samples obtained in fall and winter. Forward and reverse subtractions were performed, subtraction-enriched products were cloned, and clones were sequenced from both of the resulting libraries. When 27 of these genes were screened by semi-quantitative RT-PCR to identify candidates for differential expression based generally on 2-fold changes in expression, one encoding FK506-binding protein 5 was classified as up-regulated in response to seasonal change, another encoding the mitochondrial solute carrier 25 member 25 (ATP-Mg/Pi carrier) was similarly classified with seasonal change and low temperature shift, and the one encoding the 78 kDa glucose-regulated protein was provisionally classified as down-regulated with low temperature shift. Analysis of fall (warm) and winter (cold) seasonal samples by quantitative PCR (qPCR) revealed significant up-regulation of genes encoding FK506-binding protein 51 and the mitochondrial solute carrier, whereas the gene encoding the glucose-regulated protein showed no significant change in expression. The mitochondrial solute carrier and FK506-binding protein results may relate to changes in cortisol action, as both are regulated by cortisol in other species.

Relationship between food availability, glycerol and glycogen levels in low-temperature challenged rainbow smelt Osmerus mordax.

Rainbow smelt Osmerus modax accumulate glycerol in winter that serves as an antifreeze. Fish were held at 8 degrees C, or subjected to a decrease in water temperature to -1 degrees C over a 19 day period, and subsequently maintained at -1 degrees C from 15 January to 11 May 2004. Starved fish did not survive the challenge of temperature decrease, with death ensuing above the typical freeze point for marine teleosts (-0.8 degrees C). A decrease in temperature activates the glycerol accumulation mechanism at about 5 degrees C with peak plasma levels exceeding 300 micromol ml(-1). Glycerol levels begin to decrease in late February even at water temperatures below -1 degrees C, suggesting either an inherent circannual or photoperiod trigger, possibly in association with sufficiently high levels of antifreeze protein. Glycogen levels in liver did not change significantly in starved fish maintained at 8 degrees C. However, liver glycogen was depleted in fish subjected to the low-temperature challenge and at a faster rate in starved than in fed fish. Stored glycogen in liver and other tissues can account for only a small amount of the total glycerol production, suggesting a strong requirement for food during accelerated glycerol production. Liver glycogen levels increased in April and May in association with the decrease in glycerol. Levels of glycerol in liver, kidney, spleen, gill, intestine, heart, muscle and brain follow the same pattern as that in plasma. During the early part of the glycerol accumulation phase, all tissues except for liver have lower levels of glycerol in the intracellular space than the levels in plasma. In liver, glycerol is in equilibrium between the two compartments.

Sequence of Atlantic cod (Gadus morhua) GLUT4, GLUT2 and GPDH: Developmental stage expression, tissue expression and relationship to starvation-induced changes in blood glucose.

cDNAs of putative glucose transporters, GLUT4 and GLUT2, were cloned from Atlantic cod (Gadus morhua). The GLUT4 cDNA encodes a 503 amino acid and the GLUT2 cDNA a 506 amino acid protein. Phylogenetic analysis, amino acid sequence alignment, and tissue distribution support categorizing them as homologues of mammalian GLUT4 and 2. GLUT4 clusters with GLUT4s from fish and other vertebrates. It shows 84% amino acid identity to GLUT4 from coho salmon and brown trout and 65% identity with other vertebrates. It is most highly expressed in heart, strongly expressed in red and white skeletal muscle and present at lower levels in gill, gonad, intestine, and kidney. GLUT2 clusters with GLUT2 from rainbow trout and other vertebrates. It shows 75% amino acid identity with rainbow trout and 62% identity with chicken GLUT2. In Atlantic cod, GLUT2 is most highly expressed in liver with lower levels noted in intestine and kidney. Food deprivation for 2 months was used as a vehicle to monitor GLUT expression at different blood glucose levels. Starvation resulted in a decrease in blood glucose and liver glycogen that recovered following 20 days of re-feeding. GLUT4 expression in heart was decreased with starvation and increased with re-feeding. GLUT4 mRNA level in heart correlated with blood glucose. It is suggested that this relationship is related to insulin responsiveness. GLUT4 expression in white muscle increased with starvation and decreased with re-feeding. It is proposed that this is due to the necessity to maintain high levels of the glucose transporter protein in the face of starvation-associated proteolysis. GLUT2 expression in liver correlated with blood glucose, consistent with higher rates of glucose transport from liver to blood in the fed state than in the food-deprived state. Glycerol-3-phosphate dehydrogenase (GPDH) cDNA was also cloned. It encodes a 351 amino acid protein, which is 73-90% identical to GPDH from numerous other fish species. GPDH is ubiquitously expressed. Expression in heart decreased with starvation and increased with refeeding, whereas expression in liver did not change with starvation. In other studies, gene expression was monitored at nine time points from fertilization of eggs to larval development. GLUT4 is detectable in fertilized eggs and is fully expressed by the halfway to hatching point. GLUT2 is not evident at fertilization, is detectable at halfway to hatching, and fully expressed at hatching. GPDH expression was evident from fertilization.

Glycerol production in rainbow smelt (Osmerus mordax) may be triggered by low temperature alone and is associated with the activation of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase.

Rainbow smelt (Osmerus mordax) accumulate high levels of glycerol in winter that serves as an antifreeze. Fish were subjected to controlled decreases in water temperature and levels of plasma glycerol, liver metabolites and liver enzymes were determined in order to identify control mechanisms for the initiation of glycerol synthesis. In two separate experiments, decreases in temperature from 8 degrees C to 0 degrees C over a period of 10-11 days resulted in increases in plasma glycerol from levels of less than 4 mmol l(-1) to approximate mean levels of 40 (first experiment) and 150 mmol l(-1) (second experiment). In a third experiment, decreases in temperature to -1 degrees C resulted in plasma glycerol levels approaching 500 mmol l(-1). The accumulation of glycerol could be driven in either December or March, thus eliminating decreasing photoperiod as a necessary cue for glycerol accumulation. Glycerol accumulation in plasma was associated with changes in metabolites in liver leading to increases in the mass action ratio across the reactions catalyzed by glycerol-3-phosphate dehydrogenase (GPDH) and glycerol-3-phosphatase (G3Pase). The maximal, in vitro activity of GPDH, increased twofold in association with a sharp increase in plasma glycerol level. The metabolite levels and enzyme activities provide complementary evidence that GPDH is a regulatory site in the low temperature triggered synthesis of glycerol. Indirect evidence, based on calculated rates of in vivo glycerol production by liver, suggests that G3Pase is a potential rate-limiting step. As well, transient increases in glyceraldehyde-3-phosphate dehydrogenase and alanine aminotransferase suggest that these sites are components of a suite of responses, in rainbow smelt liver, induced by low temperature.

Accelerated hepatic glycerol synthesis in rainbow smelt (Osmerus mordax) is fuelled directly by glucose and alanine: a 1H and 13C nuclear magnetic resonance study.

At seawater temperatures below 1 degrees C, rainbow smelt (Osmerus mordax) accumulate plasma levels of glycerol up to 400 mM. Aspects of the synthesis of glycerol in liver and its regulation were previously investigated, but the pathways leading to glycerol synthesis remained unconfirmed. Here, we report nuclear magnetic resonance (NMR) studies which elucidate, in more detail, the fuel sources for rapid glycerol synthesis in rainbow smelt. Initial NMR analysis of liver homogenates from fish held at cold (-1 degrees C) temperatures and from fish transferred from 8 degrees C to -1 degrees C showed elevated glycerol, whereas those from fish held at 8 degrees C had far lower glycerol levels. These results confirm a temperature-responsive glycerol synthesis and show that NMR is a suitable approach to investigate the phenomenon. Further studies with fish held at low temperature and injected with labelled L-[2,3-(13)C(2)] alanine or D-[U-(13)C(6)]glucose revealed conversion of both alanine and glucose to glycerol. (13)C spectra showed satellites ((1)J(CC)=41.1 Hz) about the glycerol resonances indicating intact incorporation of a (13)C-(13)C unit in liver glycerol of fish injected with L-[2,3-(13)C(2)]alanine and a (13)C-(13)C-(13)C unit in liver glycerol of fish injected with D[U-(13)C(6)]glucose. Thus, glycerol can be efficiently produced directly from amino acid precursors by glyceroneogenesis, which is an abbreviated gluconeogenesis process leading to glycerol through dihydroxyacetone phosphate (DHAP). Glucose can also be metabolised to glycerol via an abbreviated form of glycolysis that similarly leads to glycerol through DHAP.

Seasonal freeze resistance of rainbow smelt (Osmerus mordax) is generated by differential expression of glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, and antifreeze protein genes.

In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol-3-phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall-winter-spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression.