Carbohydrate Flux Research Articles

Microbial fluxes of free monosaccharides and total carbohydrates in freshwater determined by PAD-HPLC

A new sensitive pulsed amperometric detection (PAD) method for measurements of mono- and disaccharides in nM concentrations was used in combination with high performance liquid chromatography (HPLC) to study fluxes of dissolved free and combined carbohydrates (DFCHO and DCCHO) in lake water. In a diel study concentrations of individual free saccharides typically were 5–50 nM, while total DFCHO concentrations ranged from 67 to 224 nM. No diel trends in concentration changes were obvious. At in situ light-dark conditions, dominant DFCHO were galactose, glucose, fructose and mannose/xylose. In addition to these saccharides, an increased abundance of melibiose and arabinose was measured in a parallel dark incubation. In a 118 h laboratory incubation of 1.0 μm filtered lake water, concentrations of DFCHO decreased from 194 nM (at 12 h) to a minimum of 54 nM (at 73 h). Dominant DFCHO were glucose, fructose and cellobiose. During the incubation DCCHO varied from 1.27 to 2.20 μM. Glucose, galactose and cellobiose made up 40, 30 and 10 mol-%, respectively, of the DCCHO. Fructose was degraded during hydrolysis of the DCCHO. A decline of DCCHO at 55 h was reflected in a simultaneous increase of DFCHO, but otherwise no similarities between the two saccharide pools were found. Incrased DCCHO concentrations and a high assimilation of glucose and fructose that was not reflected in a decline of their concentrations, both indicate that carbohydrates were produced during the experiment. Polysaccharides were probably excreted by the bacteria. Net assimilation of glucose and fructose sustained 14–19% (diel study) and 32% (long-term study) of the net bacterial carbon requirement.

Microbial fluxes of free monosaccharides and total carbohydrates in freshwater determined by PAD-HPLC

Microbial fluxes of free monosaccharides and total carbohydrates in freshwater determined by PAD-HPLC

Diurnal changes in net uptake rate of nitrate are associated with changes in estimated export of carbohydrates to roots.

The rate of NO3- uptake by soybean (Glycine max [L.] Merrill) roots generally declines during the night in association with progressive depletion of the nonstructural carbohydrate pool in the shoot as well as the concentration of carbohydrates in roots. To determine if NO3- uptake rate changes in response to variations in translocation rate of carbohydrates from shoot to roots per se or to carbohydrate status of the roots, the night period was interrupted with a low light level from incandescent lamps to alter the diurnal pattern of NO3- uptake by roots and export of carbohydrate from shoots of nonnodulated soybean. Depletion of NO3- from replenished, complete nutrient solutions containing 1 mM NO3- was measured by ion chromatography and rates of NO3- uptake were calculated. Changes in export of carbohydrates from shoot to roots during intervals of the night period were calculated as the differences between rates of disappearance in contents of nonstructural carbohydrates and their estimated rates of utilization in shoot respiration and growth. A positive, significant correlation occurred between changes in calculated rates of carbohydrate export from shoots and NO3- uptake rates. Conversely, there was no significant correlation between concentrations of nonstructural carbohydrates in roots and NO3- uptake rates. These results support the hypothesis that carbohydrate flux from shoot to roots has a direct role in regulation of nitrogen uptake by the whole plant.

Composition and dynamics of dissolved carbohydrates and lignin-degradation products in two coniferous forests, N.E. Bavaria, Germany

In order to examine the biogeochemical control of dissolved carbohydrates and lignin-derived moieties at two Norway spruce sites, their chemical composition and dynamics were investigated in bulk precipitation, throughfall, forest floor solution, and in the mineral soil solution collected from two profile depths (30 and 90 cm). The series of sugars released by trifluoroacetic acid hydrolysis indicated that carbohydrates in the bulk and canopy precipitation were rich in hexoses and deoxysugars, and poor in pentoses. This indicates that microbial communities living on the needles and on or in the dust particles of the input are main sources of carbohydrates released in the forest canopy. In the forest floor preferentially carbohydrates rich in plant-derived pentoses were solubilized and translocated into the mineral soil. They are possibly of a lignocellulose nature. Annual flux of carbohydrates infiltrating the mineral soil amounted to ca 70kg ha −1. Carbohydrate flux in the mineral soil output was much lower ( ca 5 kg ha −1 yr −1), probably due to co-adsorption of carbohydrates associated with hydrophobic, recalcitrant compounds by the soil matrix, and biodegradation of free carbohydrates. CuO oxidation products served to characterize the lignin component. Compared with the remnant lignin in the forest floor preferentially highly carboxylated lignin-derived substances entered the forest floor solution. Thus, the solubilization of lignin-derived moieties is controlled by their degree of biooxidation. The amount of CuO-lignin percolating into the mineral soil was ca 30 kg ha −1 yr −1, and the annual CuO-lignin export from the solum amounted only to ca 0.2 kg ha −1. Adsorption, rather than biodegradation is more likely to be responsible for this low CuO-lignin flux in the mineral soil output.

Cloning and Analysis of the Gene Encoding Hummingbird Proinsulin

Because hummingbirds exhibit the highest mass-specific metabolic rates seen among vertebrates and rely on sugars as their main energy source, we have investigated the structure of hummingbird insulin ( Selaphorus rufus) to determine whether it possesses structural adaptations that increase its receptor binding affinity (potency). We report here the nucleotide sequence of hummingbird proinsulin determined from hummingbird genomic DNA. The predicted amino acid sequence of the A-chain of hummingbird insulin is identical to that of chickens and the B-chain differs by only one amino acid at a noncritical position, B2 (Val in hummingbird and Ala in chicken). These findings suggest that alterations in secretory and metabolic dynamics of insulin are of greater importance than changes in binding affinity in the adaptation to states of high carbohydrate flux in these very energetic organisms.

Amino acid, hexosamine and carbohydrate fluxes to the deep subarctic Pacific (Station P)

Sediment trap samples covering the period from September 1982 to September 1983 at Station P in the subarctic Pacific were analysed for organic carbon, nitrogen, amino acids, hexosamines and carbohydrates. One trap was deployed at 3800 m water depth for the whole period and a second trap was deployed at 1000 m from March to September 1983. Peaks of particle fluxes were observed in September–November 1982. May–June 1983 and July–September 1983. Organic compounds, used as indicators for organic matter sources and degradation, revealed that organic matter is generally least degraded during periods of maximum particle fluxes. During most of the year organic matter is derived from phytoplankton (mainly diatoms). In July to October concentrations of all measured organic compounds peak simultaneously at both trap depths, and amino acid and hexosamine fluxes are higher in the deeper trap. Spectral distributions of amino acids and hexosamines suggest that their increase is due to the addition of organic matter derived from copepods. In this case about 15% of the organic matter in the shallow trap and about 60% of the organic matter in the deep trap is contributed by copepods. The considerable enrichment of the more resistant hexosamines in the deep trap indicates that the copepods entering the traps are not active swimmers, but their decayed remains or molts.

Non‐invasive techniques for assessing carbohydrate flux:

Glycogen forms the smallest yet most labile energy substrate store. Therefore studying carbohydrate flux may be crucial to understanding the regulation of energy balance. Indirect calorimetry has been used to measure carbohydrate oxidation overnight and during exercise in nine fasted subjects. Overnight carbohydrate oxidation (averaging 2.85 +/- 0.8 g h-1) was assumed to be derived primarily from hepatic glycogen since subjects were inactive or asleep, and since glucose oxidized after gluconeogenesis from protein is measured as protein oxidation. Lower-limb muscle glycogen stores were depleted by repeated 30-min periods of cycle ergometry at 45% VO2max until exhaustion (8 +/- 1 periods). The carbohydrate oxidation rate decreased as exercise progressed. Quadratic curves yielded a close fit to each individual's exercise carbohydrate depletion data (mean multiple correlation r = 0.9996) and provided excellent inter-subject discrimination. Total (muscle plus liver) glycogen stores prior to exercise were estimated by extrapolation of the depletion curves to zero oxidation rate. This produced an extrapolation of the depletion curves to zero oxidation rate. This produced an estimate (174 +/- 61 g) which compared well with predictions (208 +/- 43 g) based on reference values for muscle mass and initial glycogen content. The results demonstrate that non-invasive estimates of glycogen status can be obtained from accurate respiratory exchange data.

Non‐invasive techniques for assessing carbohydrate flux

A non-invasive method for studying the dynamics of post-exercise carbohydrate storage by means of whole-body calorimetry and 13CO2 breath tests is described. Seven untrained glycogen-depleted subjects were offered naturally 13C-labelled high carbohydrate meals (97% by energy) at 30 min intervals for 5 h and asked to consume as much as possible. Mean intake averaged 757 +/- 211 (SD) g. Exogenous carbohydrate oxidation over 16 h calculated from gas exchange and isotope ratio measurements averaged 161 +/- 45 g, and endogenous carbohydrate oxidation averaged 31 +/- 25 g. Net carbohydrate storage, calculated as the difference between amount ingested and oxidized, was 563 g which was more than twice the measured hepatic and muscle carbohydrate oxidized during the depletion phase. After correction for body size the major determinant of glycogen storage was the amount of carbohydrate consumed (r = 0.97, P < 0.001) which in turn was determined by each subject's dietary tolerance. Post-repletion exercises (12 h after last meal) were used to remobilize freshly stored glycogen. 13CO2 enrichments indicated that a substantial part of the new glycogen was derived from the exogenous carbohydrate provided by the repletion meals.

Fatty acid homeostasis in the normoxic and ischemic heart

Fatty acid homeostasis in the normoxic and ischemic heart

Endotoxin stimulation of liver parenchymal cell phosphofructokinase activity requires nonparenchymal cells.

The rate of carbohydrate flux through phosphofructokinase (measured as the rate of [3-3H]glucose detritiation) was increased fourfold in rat liver parenchymal cells incubated with conditioned medium from lipopolysaccharide-stimulated adherent liver non-parenchymal cells. The rate was not affected in parenchymal cells incubated either with lipopolysaccharide directly or with conditioned medium from non-stimulated non-parenchymal cells. The stimulation of carbohydrate flux through phosphofructokinase by conditioned medium was not duplicated by peptide cytokines known to be released by lipopolysaccharide-activated liver non-parenchymal cells (interleukin-1, interleukin-6, tumor necrosis factor-alpha, and transforming growth factor-beta) or platelet activating factor. Furthermore, formation of the active conditioned medium was not prevented by inclusion of cycloheximide or dexamethasone to inhibit cytokine synthesis, or indomethacin or BW755c to inhibit arachidonic acid metabolism, during lipopolysaccharide-stimulation of the non-parenchymal cells. The results indicate that intercellular communication between lipopolysaccharide-stimulated liver non-parenchymal cells and parenchymal cells by soluble mediators is responsible for the stimulation of liver phosphofructokinase activity during endotoxin-induced shock. Studies to isolate and identify the factor(s) in the conditioned medium are currently in progress.

Growth and physiological characteristics of soybean in open-top chambers in response to ozone and increased atmospheric CO 2

Open-top chamber studies were conducted during 1989 at USDA-BARC to determine interactive effects of increased atmospheric CO 2 and O 3 air pollution on physiological and growth responses in soybeans. The plants were grown full-season in chambers supplied with charcoal-filtered air (CF), non-filtered (NF) air or NF+40 nl l −1 O 3 having CO 2 concentrations of ambient, +50 or +150 μl l −1 CO 2. The resultant seasonal 7 h O 3 concentrations for the three air quality regimes were 23.0 nl l −1, 40.3 nl l −1 and 66.4 nl l −1 O 3, respectively. Photosynthesis rates (PS) and stomata conductance (SC) values were measured on fully expanded leaves on 11 dates throughout the growing season and vegetative samples were collected for carbohydrate analyses and for plant biomass determinations. Seed weights, grain yields and quality measurements were made following harvest at physiological maturity. Photosynthesis rates were stimulated by the +50 and +150 μl −1 CO 2 treatments and reduced by the NF+40 nl l −1 O 3 treatment. Reductions in PS rates observed for the NF+O 3 treatment during preflowering were counteracted by the +150 μl l −1 CO 2 treatment. Wide variation in stomata conductance was observed throughout the study; however, the combined effects of CO 2 and O 3 on stomata conductance appeared additive. Leaf glucose and fructose concentrations were lower for the high O 3 exposures but were unchanged by CO 2 concentrations. Sucrose and starch concentrations were significantly increased by + 150 μl l −1 CO 2. Sucrose was unaffected by O 3 treatments, but starch was significantly increased by the NF+O 3 treatment at ambient and +50 μl l −1 CO 2 thus suggesting that O 3 injury reduced the flux of carbohydrates from leaves, especially at low CO 2 levels. Leaf N concentrations were significantly reduced by the NF+O 3 treatment. Significant changes in plant biomass, leaf area, specific leaf weight, pods and seeds per plant, and grains yield were observed among treatments. Maximum increases in plant growth and productivity were observed in response to CO 2 increases in the absence of O 3 air pollution. Changes observed in plant biomass, pods and seeds per plant and grain yields were consistent with the results for the PS and leaf carbohydrate data which suggest that the +50 μl l −1 CO 2 treatment had a protective role against adverse effects of O 3 exposure. Grain oil contents were increased and protein contents significantly reduced in response to CO 2; however, both quality features were unaffected by the O 3 treatments.

Current Concepts in Lactate Exchange

There are several goals to this introductory paper in the symposium proceedings, “Current Concepts in Lactate Exchange.” First, an attempt is made to set the historical context for the symposium and foreshadow how the paper of each participant contributes to our contemporary understanding of the field. As implied in the symposium title, an emphasis will be placed on the exchange of lactate for other metabolites and ions so that utilization can be temporally and spatially disassociated from formation. Thus, rather than a dead-end metabolite, which only accumulates during exercise, there appears to be great usefulness in the formation, exchange between cells, blood and organs, and utilization of lactic acid (lactate). Specific papers will deal with aspects of lactate release and uptake by skeletal muscle, hepatic lactate balance, the flux of dietary carbohydrate through various lactate pools in the synthesis of liver glycogen, lactate metabolism in the heart, properties of the sarcolemmal lactate transporter, and evolution of a model to predict lactate production from blood measurements. Second, in this review an attempt will be made to present and support a unifying hypothesis (the “lactate shuttle”) in which the various aspects of lactate exchange may be integrated and understood. Emphasis will be placed on showing several corollaries between muscle and whole-body lactate metabolism. These are: temporal dependence on lactate uptake and release, the effects of beta-adrenergic stimulation on lactate formation and release, the effect of prior endurance training on lactate metabolism, the effect of lactate on glucose uptake and utilization, and the role of low oxygen tension (hypoxia) in loosening the control of glycolysis. The formation, exchange, and utilization of lactate represents a central means by which the coordination of intermediary metabolism in diverse tissues and different cells within tissues can be accomplished.

Fetal Cerebral Blood Flow and Metabolism during Oligemia and Early Postoligemic Reperfusion

The early time period following ischemia may be of pathogenetic importance in hypoxic-ischemic brain injury. Global cerebral oligemia was induced in ten late gestation fetal sheep by inflation of a balloon occluder around the brachiocephalic artery. Cerebral blood flow, oxygen, glucose, and lactate net flux, and oxygen delivery were measured by the Fick principle following 1 h of oligemia and at 5, 30, and 60 min of postoligemic reperfusion. During oligemia, cerebral blood flow decreased by 74 +/- 10% (mean +/- SD) and oxygen consumption decreased by 34 +/- 24%. The glucose:oxygen quotient was elevated throughout the oligemic period. In the early (5 min) reperfusion period, blood flow and oxygen delivery were not different from control but oxygen consumption was persistently depressed by 27 +/- 32%; fractional extraction of oxygen was 0.38 +/- 0.10 during control and 0.24 +/- 0.09 during early reperfusion. The venous oxygen tension increased modestly from 15.2 +/- 2.4 to 18.0 +/- 1.7 mm Hg; the postoligemic venous pO2 was limited by the lack of reactive hyperemia combined with the low arterial pO2 of the intrauterine environment. Postoligemic carbohydrate fluxes could not be differentiated from control possibly due to blood-brain barrier limitations. These factors may be related to the relative resistance of the fetal brain to hypoxic-ischemic injury.

Nutritional Controls Over Nitrogen and Phosphorus Resorption From Alaskan Birch Leaves

Little is known about factors that control nutrient loss from plants. We measured N (nitrogen) and P (phosphorus) resorption from leaves of Alaskan birch (Betula papyrifera) trees that differed strongly in their tissue nutrient status. We then experimentally manipulated those factors that we thought were most likely to control nutrient resorption. Trees of high nutrient status had larger maximum leaf N and P pools, translocated more N and P out of leaves, and left larger N and P pools in abscised leaves than did trees on infertile soils. However, plant nutrient status had no effect on the efficiency of N or P resorption (i.e., the proportion of the maximum nutrient pool resorbed from leaves during autumn senescence) except for low efficiency of P resorption in the highly fertile lawn. Prolonging the time that a leaf remained attached to the tree had no effect on N and P resorption efficiency. Leaching by autumn rains accounted for 25% of the N disappearance and 55% of the P disappearance from senescing leaves in a high—fertility field. Weakening of source—sink interactions by shading senescing leaves in a high—fertility field. Weakening of source—sink interactions by shading senescing leaves or by removing catkins (a sink for carbohydrates and nutrients) significantly reduced resorption efficiency. On the basis of our results and previous work, we conclude that nutrient resorption efficiency is influenced more strongly by carbohydrate flux from leaves (i.e., source—sink interactions) than by factors governing hydrolysis of nutrient—containing fractions in leaves.

Maximal Activities of Enzymes of Energy Metabolism in Cephalopod Systemic and Branchial Hearts

The maximal in vitro activities of enzymes of energy metabolism were assessed in systemic ventricle and the branchial hearts of the cephalopods Loligo forbesi, Sepia officinalis, Eledone cirrhosa, and Octopus vulgaris. Within each species, activity levels of enzymes of glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, and octopine dehydrogenase), citrate synthase, cytochrome oxidase, and total ATPase were higher in the systemic ventricle than in branchial hearts. The greater metabolic potential is consistent with the higher levels of pressure development and subsequent energy demand in the ventricle than in branchial hearts. The systemic ventricle is nourished by oxygenated blood, whereas branchial hearts receive deoxygenated venous blood. Despite differences in oxygen delivery, there is no suggestion from the enzyme profile that anaerobic metabolism is enhanced in branchial hearts. Pairwise regressions were assessed for all marker enzymes from ventricles and branchial hearts of the four species. There are strong linear correlations between phosphofructokinase and citrate synthase activities, and between hexokinase and cytochrome oxidase activities. This suggests that, as interspecific demand for carbon to support the citric acid cycle is increased, there is a concomitant expansion of carbohydrate flux. Consideration of the ventricle alone reveals higher activity levels of phosphofructokinase, citrate synthase, and ATPase in decapods than in octopods. This is consistent with the greater sustainable swimming capability of the former group and may reflect maximum rates of cardiac energy metabolism.

2 CURRENT CONCEPTS IN LACTATE EXCHANGE

There are several goals to this introductory paper in the symposium proceedings, "Current Concepts in Lactate Exchange." First, an attempt is made to set the historical context for the symposium and foreshadow how the paper of each participant contributes to our contemporary understanding of the field. As implied in the symposium title, an emphasis will be placed on the exchange of lactate for other metabolites and ions so that utilization can be temporally and spatially disassociated from formation. Thus, rather than a dead-end metabolite, which only accumulates during exercise, there appears to be great usefulness in the formation, exchange between cells, blood and organs, and utilization of lactic acid (lactate). Specific papers will deal with aspects of lactate release and uptake by skeletal muscle, hepatic lactate balance, the flux of dietary carbohydrate through various lactate pools in the synthesis of liver glycogen, lactate metabolism in the heart, properties of the sarcolemmal lactate transporter, and evolution of a model to predict lactate production from blood measurements. Second, in this review an attempt will be made to present and support a unifying hypothesis (the "lactate shuttle") in which the various aspects of lactate exchange may be integrated and understood. Emphasis will be placed on showing several corollaries between muscle and whole-body lactate metabolism. These are: temporal dependence on lactate uptake and release, the effects of beta-adrenergic stimulation on lactate formation and release, the effect of prior endurance training on lactate metabolism, the effect of lactate on glucose uptake and utilization, and the role of low oxygen tension (hypoxia) in loosening the control of glycolysis. The formation, exchange, and utilization of lactate represents a central means by which the coordination of intermediary metabolism in diverse tissues and different cells within tissues can be accomplished.

Cyclic variations in nitrogen uptake rate in soybean plants: uptake during reproductive growth.

Net uptake of NO3- by non-nodulated soybean plants [Glycine max (L.) Merr. cv. Ransom] growing in flowing hydroponic culture was measured daily during a 63 d period of reproductive development between the first florally inductive photoperiod and [unknown word] seed growth. Removal of NO3- from a replenished solution containing 1.0 mol m-3 NO3- was determined by ion chromatography. Uptake of NO3- continued throughout reproductive development. The net uptake rate of NO3- cycled between maxima and minima with a periodicity of oscillation of 3 to 7 d during the floral stage and about 6 d during the fruiting stage. Coupled with increasing concentrations of carbon and C : N ratios in tissues, the oscillations in net uptake rates of NO3- are evidence that the demand for carbohydrate by reproductive organs is contingent on the availability of nitrogen in the shoot pool rather than that the demand for nitrogen follows the flux of carbohydrate into reproductive tissues.

Seasonal Flux of Nonstructural Carbohydrate in Five Species of Submerged Macrophytes in a Precambrian Shield Lake. Part 1: Effect of Light and Water Depth

AbstractSummary: Soluble carbohydrate and starch were examined in shoots of Ceratophyllum demersum, Elodea canadensis, Myriophyllum exalbescens, Potamogeton foliosus and P. zosteriformis in Shoal Lake (Manitoba–Ontario) during the 1985 season. A wide range of variation was found for the same species on any given day among different sampling stations at the same depth. Correlations of each of total soluble carbohydrate and starch with water depth were inconsistent on any given day, although inverse correlations were more common. In M. exalbescens, roots consistently contained more soluble carbohydrate than shoots. The soluble carbohydrate fraction of all species contained glucose, fructose and sucrose. However C. demersum also contained significant and relatively constant proportions of melibiose, raffinose and stachyose. Starch content decreased during the season in C. demersum but increased in P. foliosus and P. zosteriformis shoots. E. canadensis and M. exalbescens showed no significant seasonal trends. Starch comprised 30 … 85 % of the total nonstructural carbohydrate in the 5 species. Seasonal carbohydrate and starch concentrations were not significantly correlated with each other in any of the macrophytes examined. The proportion of starch relative to the total nonstructural carbohydrate increased during the season in P. foliosus and P. zosteriformis.

Seasonal Flux of Nonstructural Carbohydrate in Five Species of Submerged Macrophytes in a Precambrian Shield Lake. Part 2: Fluctuation in Relation to Chlorophyll Content, Temperature and Water Chemistry

AbstractTotal soluble carbohydrate, individual sugars and starch content in green shoots of Ceratophyllum demersum, Elodea canadensis, Myriophyllum exalbescens, Potamogeton foliosus and P. zosteriformis from Shoal Lake (Manitoba‐Ontario) were examined in relation to tissue chlorophyll content, water chemistry and temperature. The different species showed a variety of seasonal carbohydrate patterns. Seasonal concentrations of total soluble carbohydrate were significantly positively correlated with chlorophyll and total chlorophyll a + b in E. canadensis and P. foliosus. Seasonal starch content was inversely correlated with chlorophyll a and total chlorophyll a + b in C. demersum and P. foliosus. In C. demersum nonstructural carbohydrate was inversely correlated with total alkalinity, total molybdenum reactive phosphorus, total inorganic nitrogen and temperature of the water. Sucrose concentrations in P. foliosus in June were positively correlated with phosphorus levels in the water.

Metabolic correlates to glycerol biosynthesis in a freeze-avoiding insect,Epiblema scudderiana

The course of glycerol biosynthesis, initiated by exposure to −4°C, was monitored in larvae of the goldenrod gall moth,Epiblema scudderiana, and accompanying changes in the levels of intermediates of glycolysis, adenylates, glycogen, glucose, fructose-2,6-bisphosphate, and fermentative end products were characterized. Production of cryoprotectant was initiated within 6 h after a switch from +16° to −4°C, with halfmaximal levels reached in 30 h and maximal content, 450–500 μmol/g wet weight, achieved after 4 days. Changes in the levels of intermediates of the synthetic pathway within 2 h at −4°C indicated that the regulatory sites involved glycogen phosphorylase, phosphofructokinase, and glycerol-3-phosphatase. A rapid increase in fructose-2,6-bisphosphate, an activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase, appeared to have a role in maintaining flux in the direction of glycerol biosynthesis. Analysis of metabolite changes as glycerol production slowed suggested that the inhibitory restriction of the regulatory enzymes was slightly out of phase. Inhibition at the glycerol-3-phosphatase locus apparently occurred first and resulted in a build-up of glycolytic intermediates and an overflow accumulation of glucose. Glucose inhibition of phosphorylase, stimulating the conversion of the activea to the inactiveb forms, appears to be the mechanism that shuts off phosphorylase function, counteracting the effects of low temperature that are the basis of the initial enzyme activation. Equivalent experiments carried out under a nitrogen gas atmosphere suggested that the metabolic make-up of the larvae in autumn is one that obligately routes carbohydrate flux through the hexose monophosphate shunt. The consequence of this is that fermentative ATP production during anoxia is linked to the accumulation of large amounts of glycerol as the only means of maintaining redox balance.