Glycogen Formation Research Articles

A Repeated GGA Motif Is Critical for the Activity and Stability of the Riboregulator RsmY of Pseudomonas fluorescens

The riboregulator RsmY of Pseudomonas fluorescens strain CHA0 is an example of small regulatory RNAs belonging to the global Rsm/Csr regulatory systems controlling diverse cellular processes such as glycogen accumulation, motility, or formation of extracellular products in various bacteria. By binding multiple molecules of the small regulatory protein RsmA, RsmY relieves the negative effect of RsmA on the translation of several target genes involved in the biocontrol properties of strain CHA0. RsmY and functionally related riboregulators have repeated GGA motifs predicted to be exposed in single-stranded regions, notably in the loops of hairpins. The secondary structure of RsmY was corroborated by in vivo cleavage with lead acetate. RsmY mutants lacking three or five (out of six) of the GGA motifs showed reduced ability to derepress the expression of target genes in vivo and failed to bind the RsmA protein efficiently in vitro. The absence of GGA motifs in RsmY mutants resulted in reduced abundance of these transcripts and in a shorter half-life (< or = 6 min as compared with 27 min for wild type RsmY). These results suggest that both the interaction of RsmY with RsmA and the stability of RsmY strongly depend on the GGA repeats and that the ability of RsmY to interact with small regulatory proteins such as RsmA may protect this RNA from degradation.

My Love Affair with Insulin: JBC Centennial 1905-2005 100 Years of Biochemistry and Molecular Biology

My Love Affair with Insulin: JBC Centennial 1905-2005 100 Years of Biochemistry and Molecular Biology

Muscle triglyceride and glycogen in endurance exercise: implications for performance.

The importance of muscle glycogen as a metabolic substrate in sustaining prolonged exercise is well acknowledged. Being stored in proximity to the site of contraction and able to sustain high rates of adenosine diphosphate (ADP) phosphorylation, glycogen is viewed as the primary fuel for the maintenance of exercise of a moderate to intense nature. As such, to ensure optimal exercise performance, endurance athletes are encouraged to maximise the availability of muscle glycogen through the ingestion of a high carbohydrate (CHO) diet prior to competition. The skeletal muscle cell also contains significant quantities of triglyceride. Recent improvements in the ability to measure these intramyocellular triglyceride (IMTG) stores have confirmed that IMTG acts as a significant fuel substrate during prolonged exercise. While early research of the role of muscle glycogen in endurance exercise provided clear prescriptive information for the endurance-trained athlete, no such direction for optimising exercise performance is yet apparent from research concerning IMTG. In this article, we review the processes of muscle glycogen and triglyceride storage and metabolism. Attention is given to the effects of short-term alterations in diet on muscle substrate, particularly IMTG storage, and the implications of this to endurance exercise performance and competition preparation. We demonstrate that like glycogen, IMTG formation may be relatively rapid, and its storage predominates under conditions that promote minimal glycogen formation. This observation suggests that the role of IMTG is to maintain a readily available substrate to ensure that physical activity of a moderate nature can be performed when glycogen availability is not optimal. Under these conditions, IMTG may offer a similar availability of energy as glycogen in the endurance-trained athlete. Given the potential value of this substrate, the possibility of maximising IMTG storage without compromising glycogen availability prior to competition is considered.

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells.

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyl-transferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARbeta/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

Impact of the reg1 mutation glycocen accumulation and glucose consumption rates in Saccharomyces cerevisiae cells based on a macrokinetic model

In S. cerevisiae, catabolite repression controls glycogen accumulation and glucose consumption. Glycogen is responsible for stress resistance, and its accumulation in derepression conditions results in a yeast with good quality. In yeast cells, catabolite repression also named glucose effect takes place at the transcriptional levels, decreasing enzyme respiration and causing the cells to enter a fermentative metabolism, low cell mass yield and yeast with poor quality. Since glucose is always present in molasses the glucose effect occurs in industrial media. A quantitative characterization of cell growth, substrate consumption and glycogen formation was undertaken based on an unstructured macrokinetic model for a reg1/hex2 mutant, capable of the respiration while growing on glucose, and its isogenic repressible strain (REG1/HEX2). The results show that the estimated value to maximum specific glycogen accumulation rate (muG,MAX) is eight times greater in the reg1/hex2 mutant than its isogenic strain, and the glucose affinity constant (K SS) is fifth times greater in reg1/hex2 mutant than in its isogenic strain with less glucose uptake by the former channeling glucose into cell mass growth and glycogen accumulation simultaneously. This approach may be one more tool to improve the glucose removal in yeast production. Thus, disruption of the REG1/HEX2 gene may constitute an important strategy for producing commercial yeast.

Fat metabolism and diabetes: 2003 American Diabetes Association postgraduate course.

At the American Diabetes Association Postgraduate Course in New York, New York, on 10 January 2003, David E. Kelley (Pittsburgh, PA) discussed the relationship between fat metabolism and diabetes. Over the past few decades, he noted, the prevalence of obesity has doubled in adults and has quadrupled in teenagers, with more than half of U.S. adults now being overweight or obese. This is a manifestation of a much more longstanding trend. Data from military recruitment records, for example, show that the average weight, adjusted for height, has been increasing over the past 150 years. Sixty-one percent of the prevalence of diabetes can be attributed to obesity. There is a strong correlation between BMI and body fat. One of the correlates of insulin resistance is the blood fatty acid (FA) level. In 1963, Randle (1) initially suggested that FAs compete with glucose for metabolism. Kelley showed that, in a euglycemic clamp, when comparing subjects with and without lipid infusion, the former have marked resistance to muscle glucose uptake, with increased glycolysis and decreased glycogen formation—“precisely the profile we get in type 2 diabetes.” FA levels are strong predictors of muscle insulin resistance (2). Muscle fat content is increased in obesity and more so in type 2 diabetes. Electron microscopy shows decreased mitochondrial size in muscle from individuals with type 2 diabetes. Fats may not directly mediate insulin resistance, Kelly noted, but FA-derived muscle diacylglycerol (DAG) and fatty acyl CoA lead to increased intramuscular ceramide, which may directly affect muscle glucose uptake and metabolism. FA oxidation is greater and storage is lower in lean than in obese persons, but Kelley described an “inefficient use of fat” in persons with obesity: the normal insulin-induced increase in FA storage is attenuated and fails to improve with weight loss (3). He noted that while muscle …

Acetylcholine affects rat liver metabolism via type 3 muscarinic receptors in hepatocytes

Although the role of acetylcholine (Ach) in hepatic glucose metabolism is well elucidated, it is still unclear if it influences gluconeogenesis, glycogenolysis and high-energy phosphate metabolism, and if it does what the mechanisms of this influence are. Therefore, using isolated perfused rat liver as a model, we have studied the effect of Ach on oxygen consumption, synthesis of glucose from lactate and pyruvate, glycogen formation, mitochondrial oxidative phosphorylation and ATP-synthesis. We have established that effects of Ach on oxygen consumption depend on its concentration. When used at a concentration of 10 −7 M, Ach exerts maximum stimulatory effect, while its infusion at 10 −6 M causes a decrease of oxygen consumption by the liver. Moreover, when used at a concentration of 10 −6 M or 10 −7 M, Ach increases rates of glucose production from the gluconeogenic substrates lactate and pyruvate, leading to enhanced glycogen content in perfused liver. It was also shown that Ach possesses a stimulating effect on alanine and aspartate aminotransferases. As detected by 31P NMR spectroscopy, continuous liver perfusion with pyruvate and lactate in the presence of Ach leads to a significant decrease of ATP level, implying enhanced energy requirements for gluconeogenesis under these conditions. Elimination of the described effects of Ach by atropine, the antagonist of muscarinic receptors, and identification of the type 3 muscarinic receptors (m3) in isolated hepatocytes as well as in whole liver, imply that Ach may exert its effect on liver metabolism through m3 receptors.

Pathways for glucose disposal after meal ingestion in humans.

To characterize postprandial glucose disposal more completely, we used the tritiated water technique, a triple-isotope approach (intravenous [3-H(3)]glucose and [(14)C]bicarbonate and oral [6,6-(2)H(2)]glucose) and indirect calorimetry to assess splanchnic and peripheral glucose disposal, direct and indirect glucose storage, oxidative and nonoxidative glycolysis, and the glucose entering plasma via gluconeogenesis after ingestion of a meal in 11 normal volunteers. During a 6-h postprandial period, a total of approximately 98 g of glucose were disposed of. This was more than the glucose contained in the meal ( approximately 78 g) due to persistent endogenous glucose release ( approximately 21 g): splanchnic tissues initially took up approximately 23 g, and an additional approximately 75 g were removed from the systemic circulation. Direct glucose storage accounted for approximately 32 g and glycolysis for approximately 66 g (oxidative approximately 43 g and nonoxidative approximately 23 g). About 11 g of glucose appeared in plasma as a result of gluconeogenesis. If these carbons were wholly from glucose undergoing glycolysis, only approximately 12 g would be available for indirect pathway glycogen formation. Our results thus indicate that glycolysis is the main initial postprandial fate of glucose, accounting for approximately 66% of overall disposal; oxidation and storage each account for approximately 45%. The majority of glycogen is formed via the direct pathway ( approximately 73%).

Modelling the start-up of a full-scale biological phosphorous and nitrogen removing WWTP

In this paper we simulated the start-up of a full-scale biological phosphorus and nitrogen removing (BPNR) wastewater treatment plant (wwtp). For the simulation we used a metabolic phosphorus model integrated in ASM2d, referred to as the Technical University Delft Phosphorus (TUDP) model. In a previous study it was shown that under steady state conditions the model is determined by stoichiometry rather than kinetics. To evaluate the kinetics of the metabolic biological phosphorus model, we recorded and simulated the start-up of a full-scale upgraded BPNR process. The initial state of the start-up was the simulated steady state of the process prior to the start-up. We could evaluate the SRT during the start-up on the basis of the accumulation of total-phosphorus in the sludge. During the start-up, the process changed from partly nitrifying to fully nitrifying, denitrifying and phosphate removing. Growth of PAO only showed sensitive for the glycogen formation rate k GLY. Disregarding a 20% in- or decrease of the kinetic rates of biological phosphorus removal (BPR), all start-up simulations converged to comparable steady states. This underlines that BPR is determined by stoichiometry rather than kinetics. Previous simulation studies with the TUDP-model showed that glycogen accumulated to unrealistic high concentrations with an increasing sludge retention time (SRT). We modelled the glycogen kinetics with a maximum glycogen fraction, which proved an effective and straightforward method to avoid unrealistic glycogen accumulation. In the steady state simulations, the glycogen concentration was determined by the value of the maximum glycogen fraction ( f GLY max), whereas the overall sludge composition showed insensitive towards this parameter. Temperature appeared highly sensitive and therefore should not be neglected when modelling BPR.

Surprises and revelations in biochemistry: 1950–2000

Surprises and revelations in biochemistry: 1950–2000

Hepatic glucose uptake, gluconeogenesis and the regulation of glycogen synthesis.

Hepatic glycogen is replenished during the absorptive period postprandially. This repletion is prompted partly by an increased hepatic uptake of glucose by the liver, partly by metabolite and hormonal signals in the portal vein, and partly by an increased gluconeogenic flux to glycogen (glyconeogenesis). There is some evidence that the direct formation of glycogen from glucose and that formed by gluconeogenic pathways is linked. This includes: (i) the inhibition of all glycogen synthesis, in vivo, when gluconeogenic flux is blocked by inhibitors; (ii) a dual relationship between glucose concentrations, lactate uptake by the liver and glycogen synthesis (by both pathways) which indicates that glucose sets the maximal rates of glycogen synthesis while lactate uptake determines the actual flux rate to glycogen; (iii) the decrease of both gluconeogenesis and glycogen synthesis by the biguanide, metformin; and (iv) correlations between increased gluconeogenesis and liver glycogen in obese patients and animal models. The degree to which the liver extracts portal glucose is not entirely agreed upon although a preponderance of evidence points to about a 5% extraction rate, following meals, which is dependent on a stimulation of glucokinase. This enzyme may be linked to the expression of other enzymes in the gluconeogenic pathway. Perivenous cells in the liver may induce additional gluconeogenesis in the periportal cells by increasing glycolytically produced lactate. A number of potential mechanisms therefore exist which could link glycogen synthesis from glucose and gluconeogenic substrate.

Glycogen metabolism in aerobic mixed cultures.

In this study, the metabolism of glycogen storage and consumption in mixed cultures under aerobic conditions is described. The experimental results are used to calibrate a metabolic model, which as sole stoichiometric variables has the efficiency of oxidative phosphorylation (delta) and maintenance requirement in units of adenosine triphosphate (m(ATP)). Using the experimental data and values from the literature we show that delta and m(ATP) are strongly coupled and that the values determined for glycogen and poly-beta-hydroxybutyrate (PHB) metabolism are similar. We also demonstrate that storage of glycogen and subsequent growth occur without significant loss of energy, as compared with direct growth on glucose. For kinetic modeling, Monod kinetics is used most commonly in activated sludge models to describe the rate of microbial transformation. Monod kinetics, however, does not provide a good description of the data obtained. Second-order kinetics gives a better description of the rate of glycogen degradation. Formation and consumption of glycogen appears to be much faster than for PHB.

Overexpression of 1-acyl-glycerol-3-phosphate acyltransferase-alpha enhances lipid storage in cellular models of adipose tissue and skeletal muscle.

Plasma nonesterified fatty acids (NEFA) at elevated concentrations antagonize insulin action and thus may play a critical role in the development of insulin resistance in type 2 diabetes. Plasma NEFA and glucose concentrations are regulated, in part, by their uptake into peripheral tissues. Cellular energy uptake can be increased by enhancing either energy transport or metabolism. The effects of overexpression of 1-acylglycerol-3-phosphate acyltransferase (AGAT)-alpha, which catalyzes the second step in triglyceride formation from glycerol-3-phosphate, was studied in 3T3-L1 adipocytes and C2C12 myotubes. In myotubes, overexpression of AGAT-alpha did not affect total [14C]glucose uptake in the presence or absence of insulin, whereas insulin-stimulated [14C]glucose conversion to cellular lipids increased significantly (33%, P = 0.004) with a concomitant decrease (-30%, P = 0.005) in glycogen formation. [3H]oleic acid (OA) uptake in AGAT-overexpressing myotubes increased 34% (P = 0.027) upon insulin stimulation. AGAT-alpha overexpression in adipocytes increased basal (130%, P = 0.04) and insulin-stimulated (27%, P = 0.01) [3H]OA uptake, increased insulin-stimulated glucose uptake (56%, P = 0.04) and conversion to cellular lipids (85%, P = 0.007), and suppressed basal (-44%, P = 0.01) and isoproterenol-stimulated OA release (-45%, P = 0.03) but not glycerol release. Our data indicate that an increase in metabolic flow to triglyceride synthesis can inhibit NEFA release, increase NEFA uptake, and promote insulin-mediated glucose utilization in 3T3-L1 adipocytes. In myotubes, however, AGAT-alpha overexpression does not increase basal cellular energy uptake, but can enhance NEFA uptake and divert glucose from glycogen synthesis to lipogenesis upon insulin stimulation.

Intrauterine Growth Retardation in Experimental Diabetes: Possible Role of the Placenta

Fetal growth disorders are common in pregnancy complicated by diabetes. Whereas macrosomia often occurs in infants of diabetic women, growth retardation is almost a rule in spontaneous and experimental diabetes in animals. However, it is not clear when during development growth inhibition starts and how placental pathology might affect fetal growth in maternal diabetes. In this study pregnant Wistar rats were injected (ip) with a single dose of 50 mg/kg of streptozotocin (STZ) on gestation day (GD) 2 and a blood glucose level of 200 mg/dl or more determined 24 hrs later indicated diabetes. The controls were non-treated, buffer treated or, following confirmation of diabetes, injected with a single dose of 2–6 IU of insulin (Novo Ultralente) once daily. Fetuses and placentae were collected from GD 14–20. Intrauterine growth retardation (IUGR) in STZ group was significant as early as GD 15 and persisted to GD 20. Insulin produced a significant recovery in fetal weight gain. The placentas of STZ-treated group were significantly heavier than those of the control groups. The reduction in cord length of the STZ group became apparent on GD 16 and remained so to term. The placenta of GD 14 STZ group had a thicker decidua basalis and dilated maternal sinusoids. By GD 16, the decidua basalis contained glycogen-containing decidual cells and scattered glycogen cells confirmed by Best' carmine with or without diastase. The glycogen cells of the basal zone were more abundant, and had degenerated in some sites leaving behind cysts with eosinophilic mass. The giant cells had proliferated enormously. The labyrinthine zone appeared spongy with persistent fetal mesenchyme, peri-vascular fibrosis, and enhanced placental barrier. The trophoblasts of the labyrinths also contained traces of glycogen unlike the controls. By GD 18, the decidua basalis of the STZ group was thinner than that of the controls and contained necrotic giant cells and lymphocytic aggregations. In the basal zone, the giant cells had proliferated further; more glycogen cells had degenerated. Perivascular fibrosis was still extensive in the labyrinthine zone. Bloodless maternal sinusoids, extensive vacuolization, degeneration of glycogen islands and formation of cysts characterized the labyrinthine zone. These changes varied in intensity from one area to another in the same placenta and between placentas of the same and of different litters. The development of the upper and lower jaws, elevation and fusion of palatal shelves, reduction of physiological umbilical hernia, descent of the testes, fusion of the urethral folds and separation of digits of the paws were significantly delayed in the STZ group. The consistent association of placental pathology with fetal growth retardation is suggestive of an alteration in placental function possibly contributing to IUGR in STZ-induced diabetes in rats.

GLP-l effectively reverts insulin resistance in rats with CCL 4-induced liver cirrhosis

Introduction: Insulin resistance is a characteristic feature of patients with liver cirrhosis. To characterize and quantify the extent as well as the site of impaired glucose utilization and to elucidate the possible potency of GLP-l to revert the impaired glucose utilization, both healthy and cirrhotic rats underwent euglycemic hyperinsulinemic damp studies. Methods: In male Lewis rats, liver cirrhosis was induced by inhalation of CCI4 for 10 weeks and phenobarbital (PB)-enriched drinking water. Using the euglycemic hyperinsulinemic clamp technique coupled with isotopic measurements of glucose uptake in individual tissues, insulin action was studied in the presence and absence of 3.0 pmol GLP-l/kgBW/min during clamping. Clamp studies were performed by continuous insulin infusion in doses ranging from 1 to 10 nmol/kg BW/h. Blood samples were obtained and glucose infusion was adjusted accordingly to maintain euglycemia allowing determination of insulin action on whole body glucose disposal. During euglycemia, insulin action on individual tissues was measured with 2-deoxy-D-[1-3H]glucose and [U-1 Results: After 10 weeks, a CCli PB-induced and histologically-validated liver cirrhosis was found in 98 % of treated rats. In cirrhotic rats, glucose utilization was impaired and resulted in a significant reduction of total body glucose disposal by 30 %. In these animals, glycogen formation was significantly reduced in the diaphragm and red skeletal muscle. Administration of GLP-I fully reverted the impaired glucose utilization. Conclusion: In rats with a CCI4/PBinduced liver cirrhosis, insulin resistance is predominantly marked by an impaired glucose utilization located in the diaphragm and red skeletal muscle which can be completely reverted by GLP-l. These results stress the potential pharmacological properties of GLP-l in the treatment of NIDDM in humans.

Gluconeogenesis in the fetus and neonate

Gluconeogenesis (GNG), a key metabolic process, involves the formation of glucose and glycogen from non-glucose precursors via pyruvate. In the strict sense, it also includes the contribution of glycerol as well as recycled glucose carbon (Cori's cycle). The developmental expression of GNG in the fetus and newborn and the quantitative contribution of GNG to glucose has been extensively investigated in humans and other mammalian species. Data from studies in rodents, rabbits, and sheep fetuses show that the development of GNG is a well-orchestrated process that is regulated by the expression of specific factors involved in the transcription of the genes for specific regulating enzymes, which catalyze GNG. These transcription factors and the genes for gluconeogenic enzymes are expressed at specific time periods during development. Although the fetus has the potential for GNG, the actual formation of glucose from pyruvate is not apparent until after birth because the rate limiting enzyme phosphoenolpyruvate carboxykinase appears only after birth in the immediate newborn period. Several tracer isotope methods have been employed to quantify the contribution of GNG to glucose. Of these, the recently developed stable isotope techniques with deuterium labeled water and the mass isotopomer distribution analysis appear to be the most precise and easily applicable in human studies. The available data show that in the human newborn, GNG appears soon after birth and contributes 30% to 70% to glucose produced. Application of new molecular biology techniques, in combination with sensitive tracer isotopic methods, will allow us to identify and examine metabolic disorders that impact GNG and help develop intervention strategies.

Assessment of human muscle glycogen synthesis and total glucose content by in vivo 13C MRS.

Obesity is often accompanied by a decreased ability of insulin to stimulate glucose uptake and glycogenesis in skeletal muscle. The aim of this study was to investigate the rate of glycogen formation and of muscular glucose content in relation to insulin sensitivity under euglycemic conditions. We applied a hyperinsulinemic (430 pmol m-2 min-1) euglycemic clamp with infusion of 20% glucose (30% enriched with 13C-1-glucose) to 8 subjects with a wide range of insulin sensitivities. Glycogen and glucose levels were monitored simultaneously by in vivo 13C MRS of the calf muscle on a clinical MR system at 1.5T field strength. Glycogen synthesis rate showed a strong correlation with whole body glucose uptake during the clamp (r = 0.93, P < 0.01). With the use of 13C MRS, total muscular glucose content could be determined in vivo, and showed a positive, linear correlation with glycogen synthesis rate (r = 0.85, P < 0.01). 13C MRS provides important information regarding in vivo insulin action. Preliminary results indicate that the glycogen synthesis rate improves after treatment with troglitazone.

Skeletal muscle metabolic response to exercise in horses with 'tying-up' due to polysaccharide storage myopathy.

Polysaccharide storage myopathy (PSSM) is a distinct cause of exertional rhabdomyolysis in Quarter Horses that results in glycogen and abnormal polysaccharide accumulation. The purpose of this study was to determine if excessive glycogen storage in PSSM is due to a glycolytic defect that impairs utilisation of this substrate during exercise. Muscle biopsies, blood lactates and serum CK were obtained 1) at rest from 5 PSSM Quarter Horses, 4 normal Quarter Horses (QH controls) and 6 Thoroughbreds with recurrent exertional rhabdomyolysis (TB RER) and 2) after a maximal treadmill exercise test in PSSM and QH controls. In addition, 3 PSSM horses performed a submaximal exercise test. At rest, muscle glycogen concentrations were 2.4x and 1.9x higher in PSSM vs. QH controls or TB RER, respectively. Muscle lactates at rest were similar between PSSM and QH controls but significantly higher in PSSM vs. TB RER. Muscle glucose-6-phosphate concentrations were also higher in PSSM horses than controls combined. During maximal exercise, mean muscle glycogen concentrations declined 2.7x more and mean lactate increased 2x more in PSSM vs. QH controls; however, differences were not statistically significant. Blood lactate concentrations after maximal exercise did not reflect generally higher muscle lactate in PSSM vs. QH controls. No change in blood lactate concentrations occurred in PSSM horses with submaximal exercise. Serum CK activity increased significantly 4 h after maximal and submaximal exercise and was significantly higher in PSSM vs. QH controls. These results show that during maximal exercise, PSSM horses utilised muscle glycogen and produce lactic acid via a functional glycolytic pathway and that during submaximal exercise oxidative metabolism was unimpaired. The excessive glycogen storage and formation of abnormal polysaccharide in PSSM horses therefore appear to reflect increased glycogen synthesis rather than decreased utilisation. The specific subset of horses with exertional rhabdomyolysis due to PSSM would likely benefit clinically from a diet low in soluble carbohydrates like grain with fat added as well as gradually increasing daily exercise to reduce excessive glycogen accumulation and enhance utilisation.

Short-Term Insulin-Induced Glycogen Formation in Primary Hepatocytes as a Screening Bioassay for Insulin Action

We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat hepatocytes by determination of [3H]glycogen fromd-[6-3H]glucose. The dose–response curve of insulin in this assay exhibited an EC50of 0.42 (±0.04) nM, which is comparable to the dissociation constant of insulin from its receptor in hepatocytes. We used this assay to examine possible residual insulin-like activity of the four major fragments formed upon insulin degradation by insulin protease. Fragments A1–13B1–9, A1–14B1–9,and A14–21B14–30showed no measurable activity. Although preparations of fragment A14–21B10–30displayed dose-dependent agonist activity with an EC50of 380 (±40) nM, we conclude that this was due to an insulin-like impurity since the chemically synthesized fragment showed no such activity. In summary, this bioassay demonstrates the action of insulin on glycogen formation in hepatocytes and provides a rapid and sensitive measurement of insulin-like activity which could facilitate screening studies.

Time course of insulin action on tissue-specific intracellular glucose metabolism in normal rats.

We investigated the time course of insulin action in conscious rats exposed to constant physiological hyperinsulinemia (approximately 100 mU/l) while maintaining euglycemia (approximately 100 mg/dl) for 0, 0.5, 2, 4, 8, or 12 h. [3-3H]glucose was infused to quantitate whole body glucose disposal (rate of disappearance, Rd), glycolysis (generation of 3H2O in plasma), hepatic glucoses production (HGP), and skeletal muscle and liver glycogen synthesis ([3-3H]glucose incorporation into glycogen and time-dependent change in tissue glycogen concentration). The basal Rd, which equals HGP, was 6.0 +/- 0.3 mg.kg-1.min-1. With increased duration of hyperinsulinemia from 0 to 0.5 to 2 to 4 h, Rd increased from 6.0 +/- 0.3 to 21.0 +/- 1.1 to 24.1 +/- 1.5 to 26.6 +/- 0.6 mg.kg-1.min-1 (P < 0.05 for 2 and 4 h vs. 0.5 h). During the first 2 h the increase in Rd was explained by parallel increases in glycolysis and glycogen synthesis. From 2 to 4 h the further increase in Rd was entirely due to an increase in glycolysis without change in glycogen synthesis. From 4 to 8 to 12 h of hyperinsulinemia, Rd decreased by 19% from 26.6 +/- 0.6 to 24.1 +/- 1.1 to 21.6 +/- 1.8 mg.kg-1.min-1 (P < 0.05 for 8 h vs. 4 h and 12 h vs. 8 h). The progressive decline in Rd, in the face of constant hyperinsulinemia, occurred despite a slight increase (8-14%) in glycolysis and was completely explained by a marked decrease (64%) in muscle glycogen synthesis. In contrast, liver glycogen synthesis increased fourfold, indicating an independent regulation of muscle and liver glycogen synthesis by long-term hyperinsulinemia. In the liver, during the entire 12-h period of insulin stimulation, the contribution of the direct (from glucose) and the indirect (from C-3 fragments) pathways to net glycogen formation remained constant at 77 +/- 5 and 23 +/- 5%, respectively. HGP remained suppressed throughout the 12-h period of hyperinsulinemia.