Post mortem glycogenolysis is a combination of phosphorolysis and hydrolysis

1. Glycogen, glucose, lactate and glycogen phosphorylase concentrations and the activities of glycogen phosphorylase a and acid 1,4-alpha-glucosidase were measured at various times up to 120 min after death in the liver and skeletal muscle of Wistar and gsd/gsd (phosphorylase b kinase deficient) rats and Wistar rats treated with the acid alpha-glucosidase inhibitor acarbose. 2. In all tissues glycogen was degraded rapidly and was accompanied by an increase in tissue glucose and lactate concentrations and a lowering of tissue pH. In the liver of Wistar and acarbose-treated Wistar rats and in the skeletal muscle of all rats glycogen loss proceeded initially very rapidly before slowing. In the gsd/gsd rat liver glycogenolysis proceeded at a linear rate throughout the incubation period. Over 120 min 60, 20 and 50% of the hepatic glycogen store was degraded in the livers of Wistar, gsd/gsd and acarbose-treated Wistar rats, respectively. All 3 types of rat degraded skeletal muscle glycogen at the same rate and to the same extent (82% degraded over 2 hr). 3. In Wistar rat liver and skeletal muscle glycogen phosphorylase was activated soon after death and the activity of phosphorylase a remained well above the zero-time level at all later time points, even when the rate of glycogenolysis had slowed significantly. Liver and skeletal muscle acid alpha-glucosidase activities were unchanged after death. 4. The decreased rate and extent of hepatic glycogenolysis in both the gsd/gsd and acarbose-treated rats suggests that this process is a combination of phosphorolysis and hydrolysis. 5. Glycogen was purified from Wistar liver and skeletal muscle at various times post mortem and its structure investigated. Fine structural analysis revealed progressive shortening of the outer chains of the glycogen from both tissues, indicative of random, lysosomal hydrolysis. Analysis of molecular weight distributions showed inhomogeneity in the glycogen loss; in both tissues high molecular weight glycogen was preferentially degraded. This material is concentrated in lysosomes of both skeletal muscle and liver. These results are consistent with a role for lysosomal hydrolysis in glycogen degradation.     

Skeletal muscle glycogen content, structure, and metabolism are normal in rats with hepatic glycogen phosphorylase kinase deficiency

Skeletal muscle glycogen content and structure, and the activities of several enzymes of glycogen metabolism are reported for the hepatic glycogen phosphorylase b kinase deficient (gsd/gsd) rat. The skeletal muscle glycogen content of the fed gsd/gsd rat is 0.50 +/- 0.11% tissue wet weight, and after 40 hours of starvation this value is lowered 40% to 0.30 +/- 0.05% tissue wet weight. In contrast the gsd/gsd rat liver has an elevated glycogen content which remains high after starvation. The skeletal muscle phosphorylase b kinase, glycogen phosphorylase, glycogen synthase and acid alpha-glucosidase activities are 17.2 +/- 2.9 units/g tissue, 119.9 +/- 6.4 units/g tissue, 12.2 +/- 0.4 units/g tissue and 1.4 +/- 0.4 milliunits/g tissue, respectively, with approx. 20% of phosphorylase and approx. 24% of synthase in the active form (at rest). These enzyme activities resemble those of Wistar skeletal muscle, and again this contrasts with the situation in the liver where there are marked differences between the Wistar and the gsd/gsd rat. Fine structural analysis of the purified glycogen showed resemblance to other glycogens in branching pattern. Analysis of the molecular weight distribution of the purified glycogen indicated polydispersity with approx. 66% of the glycogen having a molecular weight of less than 250 X 10(6) daltons and approx. 25% greater than 500 X 10(6) daltons. This molecular weight distribution resembles those of purified Wistar liver and skeletal muscle glycogens and differs from that of the gsd/gsd liver glycogen which has an increased proportion of the low molecular weight material.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of glycogen synthesis upon refeeding following starvation.

1. Starvation of rats for 40 hr decreased the body weight, liver weight and blood glucose concentration. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% (from 410 mumol/g tissue to 16 mumol/g tissue) and 55% (from 40 mumol/g tissue to 18.5 mumol/g tissue), respectively. 2. Fine structural analysis of glycogen purified from the liver and skeletal muscle of starved rats suggested that the glycogenolysis included a lysosomal component, in addition to the conventional phosphorolytic pathway. In support of this the hepatic acid alpha-glucosidase activity increased 1.8-fold following starvation. 3. Refeeding resulted in liver glycogen synthesis at a linear rate of 40 mumol/g tissue per hr over the first 13 hr of refeeding. The hepatic glycogen store were replenished by 8 hr of refeeding, but synthesis continued and the hepatic glycogen content peaked at 24 hr (approximately 670 mumol/g tissue). 4. Refeeding resulted in skeletal muscle glycogen synthesis at an initial rate of 40 mumol/g tissue per hr. The muscle glycogen store was replenished by 30 min of refeeding, but synthesis continued and the glycogen content peaked at 13 hr (approximately 50 mumol/g tissue). 5. Both liver and skeletal muscle glycogen synthesis were inhomogeneous with respect to molecular size; high molecular weight glycogen was initially synthesised at a faster rate than low molecular weight glycogen. These observations support suggestions that there is more than a single site of glycogen synthesis.     

Adrenergic and serotonergic regulation of skeletal muscle metabolism in rat. I. The effects of adrenergic and serotonergic antagonists on the regulation of muscle amino acid release, glycogenolysis, and cyclic nucleotide levels

The biochemical mechanisms of serotonergic and adrenergic action on skeletal muscle cyclic nucleotide, glycogen, and amino acid metabolism have been investigated in intact rat epitrochlaris skeletal muscle preparations. Endogenous catecholamine levels in these preparations were 28.6 +/- 2.1 pg/mg of muscle. Release of these catecholamines by tyramine produced a 25% inhibition of alanine and glutamine release. Pretreatment of animals in vivo with 6-hydroxydopamine depleted catecholamine content by 85%. On incubation, preparations from these pretreated animals showed no effect of tyramine on amino acid metabolism. Serotonin (10(-5) M) and epinephrine (10(-5) M) inhibited alanine and glutamine release equally in preparations from 6-hydroxydopamine-pretreated as compared to control rats. Adrenergic antagonists such as dl-propranolol (10(-8)-10(-6) M), oxprenolol (10(-8)-10(-6) M), and practolol (10(-6)-10(-4) M) blocked equally the inhibition of alanine and glutamine release, prevented the stimulations of muscle cAMP levels, phosphosphorylase a formation, and the depletion of muscle glycogen produced by either epinephrine or serotonin. In contrast, serotonergic antagonists such as methysergide (10(-8)-10(-6) M) and cyproheptadine (10(-8)-10(-6) M) blocked the inhibition of alanine and glutamine release, the stimulations of muscle cAMP levels and phosphorylase a formation, and the decreased muscle glycogen content effected by serotonin but not by epinephrine. Incubation of muscles with both epinephrine and serotonin together produced additive stimulation of muscle cAMP levels, but not of the inhibition of alanine and glutamine release. These data indicate that the action of these agonists on skeletal muscle protein and amino acid, glycogen, and cyclic nucleotide metabolism proceeds directly via separate and discrete serotonergic and adrenergic receptor-adenylyl cyclase mechanisms in skeletal muscle.     

Glycogen synthase kinases. Distribution in mammalian tissues of forms that are independent of cyclic AMP.

Extracts of rat tissues contain kinases which catalyze the conversion of glycogen synthease from the glucose 6-phosphate-independent (I) form to the glucose 6-phosphatate-dependent (D) form. These kinases were stimulated by adenosine 3':5' monophosphate (cyclic AMP). The glycogen synthase kinase activity ratio (activity in the absence of cyclic AMP divided by activity in the presence of cyclic AMP) varied from 0.28 to 0.97. The activity ratio for histone kinase in the same extracts ranged from 0.11 to 0.29. The levels of glycogen synthase kinase varied by a factor of 80 in the following rat tissues (given in order of decreasing enzyme activity): kidney, liver, stomach mucosa, lung, brain, heart, skeletal muscle, and adipose tissue. In the same tissues the levels of histone kinase varied by only a factor of 6 and did not correlate with the levels of glycogen synthase kinase. A modification of the method of Walsh et al. ((1971) J. Biol. Chem. 246, 1977-1985) was developed for purification of the heat-stable inhibitor of cyclic AMP-dependent protein kinases (inhibitor). The modified procedure resulted in good yields of highly purified inhibitor and was much simpler than the previously described procedure. This inhibitor completely inhibited cyclic AMP-dependent histone kinase activity of the extracts but much of the glycogen synthase kinase activity was not inhibited. The portion of glycogen synthase kinase that was insensitive to the inhibitor was: stomach mucosa, 95%; brain, 90%; liver, 82%; kidney, 81%; lung, 68%; adipose tissue, 65%; skeletal muscle, 63%; and heart, 54%. This histone kinase activity in the extracts and hte ratio of glycogen synthase kinase to histone kinase activity of purified catalytic subunit of the cyclic AMP-dependent protein kinase was used to calculate for each extract the glycogen synthase kinase activity contributed by the cyclic AMP-dependent protein kinase. Based on these calculations, the portion of the glycogen synthase kinase which was due to kinases independent of cyclic AMP was: kidney, 97%; liver, 91%; lung, 89%; brain, 87%, heart, 85%; stomach mucosa, 84%; adipose tissue, 38%; and skeletal muscle, 33%. A significant portion of the glycogen synthase kinase activity, but virtually none of the cyclic AMP-dependent histone kinase activity, of these extracts could be adsorbed to phosphocellulose columns. Liver extracts contained, in addition, a form of glycogen synthase kinase which was not adsorbed to phosphocellulose and which could be separated from the cyclic AMP-dependent protein kinase by additional chromatography. These studies demonstrate that kinases independent of cyclic AMP account for most of the glycogen synthase kinase activity of many tissues. The widespread distribution and high concentrations of these enzymes suggest that they are of physiological importance.     

L-type glycogen synthase. Tissue distribution and electrophoretic mobility

We previously reported (Kaslow, H.R., and Lesikar, D.D.FEBS Lett. (1984) 172, 294-298) the generation of antisera against rat skeletal muscle glycogen synthase. Using immunoblot analysis, the antisera recognized the enzyme in crude extracts from rat skeletal muscle, heart, fat, kidney, and brain, but not liver. These results suggested that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type, referred to as "M-type" glycogen synthase. We have now used an antiserum specific for the enzyme from liver, termed "L-type" glycogen synthase, to study its distribution and electrophoretic mobility. Immunoblot analysis using this antiserum indicates that L-type glycogen synthase is found in liver, but not skeletal muscle, heart, fat, kidney, or brain. In sodium dodecyl sulfate-polyacrylamide gels of crude liver extracts prepared with protease inhibitors, rat L-type synthase was detected with electrophoretic mobility Mapp = 85,000. In contrast, the M-type enzyme in crude skeletal muscle extracts with protease inhibitors was detected with Mapp = 86,000 and 89,000. During purification of L-type synthase, apparent proteolysis can generate forms with increased electrophoretic mobility (Mapp = 75,000), still recognized by the antiserum. These M-type and L-type antisera did not recognize a protein with Mapp greater than phosphorylase. The anti-rat L-type antisera recognized glycogen synthase in blots of crude extracts of rabbit liver, but with Mapp = 88,000, a value 3,000 greater than that found for the rat liver enzyme. The anti-rat M-type antisera failed to recognize the enzyme in blots of crude extracts of rabbit muscle. Thus, in both muscle and liver, the corresponding rat and rabbit enzymes are structurally different. Because the differences described above persist after resolving these proteins by denaturing sodium dodecyl sulfate electrophoresis, these differences reside in the structure of the proteins themselves, not in some factor bound to the protein in crude extracts.     

Regulation of lysosomal glycogen metabolism: studies of the actions of mammalian acid alpha-glucosidases

1. Acid alpha-glucosidases were purified to homogeneity from rat liver, rat skeletal muscle and human placenta. The properties of these enzymes were investigated. 2. Their pH optima for activity toward various substrates were in the range 4-5. 3. Time course and pH dependence experiments revealed that all glycogen substrates were not hydrolysed at the same rate; the rate of hydrolysis was inversely related to the molecular size of the substrate. The most rapidly hydrolysed glycogen substrate was the smallest (commercial oyster) while the least rapidly hydrolysed was the largest (native rat or rabbit liver). Intermediate sized glycogens were hydrolysed at intermediate rates. 4. Glycogen hydrolysis was stimulated by added sodium ions; this stimulation was pH dependent. 5. It is suggested that lysosomal glycogen metabolism may be controlled by pH, salt concentration and the size of the glycogen substrate. 6. Since the high molecular weight glycogen associated with lysosomes is formed by disulphide bridges between lower molecular weight material it is proposed that an important step of lysosomal glycogen degradation is disulphide bond reduction.     

Partial structure and hormonal regulation of rabbit liver inhibitor-1; distribution of inhibitor-1 and inhibitor-2 in rabbit and rat tissues

Inhibitor-1 purified from rabbit liver could not be distinguished from the skeletal muscle protein by chromatographic, electrophoretic and immunological criteria. Amino acid sequences comprising 68% of rabbit liver inhibitor-1 were identical to the skeletal muscle protein indicating that they are products of a single gene. Total inhibitor-1 activity in heat-treated rabbit liver extracts was similar to that in skeletal muscle extracts, and the phosphorylation state of inhibitor-1 increased from 14% to 42% in rabbit liver in vivo after an intravenous injection of glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-1 recognised a single major protein of identical electrophoretic mobility (26 kDa) in each rabbit tissue examined (skeletal muscle, liver, brain, heart, kidney, uterus and adipose). The antibodies also recognised a single major (30 kDa) protein in the same rat tissues, except liver. The results show that while there are interspecies differences in apparent molecular mass, inhibitor-1 is likely to be the same gene product in each mammalian tissue. Inhibitor-1 was not detected in rat liver, either by activity measurements or immunoblotting, irrespective of the age, sex or strain of the animals. Immunoblotting also failed to detect inhibitor-1 in mouse liver, although it was present in guinea pig, porcine and sheep liver. The absence of inhibitor-1 in rat liver indicates that phosphorylation of this protein cannot underlie the increased phosphorylation of hydroxymethylglutaryl-CoA reductase observed after stimulation by glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-2 recognised a 31 kDa protein in each rabbit tissue, and a 33 kDa protein in all rat tissues including liver. The results suggest that inhibitor-2 is the same gene product in each mammalian tissue.     

Glycogen synthase (casein) kinase-1: tissue distribution and subcellular localization

The distribution of glycogen synthase (casein) kinase-1 (CK-1) among different rat tissues and subcellular fractions was investigated. Using casein, glycogen synthase and phosphorylase kinase as substrates, CK-1 activity was detected in kidney, spleen, liver, testis, lung, brain, heart, skeletal muscle and adipose tissue. The distribution of CK-1 among different subcellular fractions of rat liver was; cytosol (72.1%), microsome (17.6%), mitochondria (9.6%) and nuclei (0.7%). CK-1 from rat tissues was shown to have a similarly wide substrate specificity as highly purified CK-1 from rabbit skeletal muscle. Such wide substrate specificity and distribution among different mammalian tissues and subcellular organelles indicate that CK-1 may be involved in the regulation of diverse cellular functions.     

Inhibition of rabbit muscle isozymes by vitamin C

The ability of vitamins C, E and K to inhibit enzymes directly has been investigated. It was found that vitamin E and some analogs and menadione (vitamin K3) inhibited several enzymes irreversibility at concentrations below one millimolar. Ascorbate inhibits rabbit muscle 6-phosphofructokinase (MPFK-1; EC 2.7.1.11), muscle type LDH (EC 1.1.1.27), and muscle AK (EC 2.7.4.3) at low concentrations that do not inhibit equivalent liver isozymes. Ascorbate Ki values for muscle-type LDH and heart-type LDH isozymes are 0.007 and 3 mM, respectively. The ascorbate Ki value for rabbit skeletal muscle PFK-1 is 0.16 mM; liver PFK-I is not inhibited by ascorbate. Dehydroascorbate does not inhibit any enzyme at ascorbate concentrations normally found in cells. All ascorbate inhibitions are completely reactivated or nearly so by L-ascorbate oxidase, CYS, GSH, or DTT. We propose a hypothesis that ascorbate facilitates glycogen storage in muscle by inhibiting glycolysis. The relationship between ascorbate metabolism and diabetes is discussed.     

The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1

Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions.     

肌酸对游泳大鼠乳酸、糖原含量和乳酸脱氢酶活性的影响

为探讨肌酸对提高大鼠运动能力的作用 ,观察了肌酸对游泳大鼠血清、心肌和骨骼肌乳酸、糖原含量和乳酸脱氢酶 (LDH)活性的影响。实验用雄性wistar大鼠 2 4只 ,随机分为正常组、游泳对照组和游泳补充肌酸组。两个游泳组每天游泳训练 1h,9天后 ,游泳 4h ,测定血清、心肌和骨骼肌乳酸水平 ,测定血清和骨骼肌乳酸脱氢酶活性以及心肌与骨骼肌糖原含量。结果显示 :肌酸可抑制游泳运动后大鼠血清、心肌和骨骼肌乳酸浓度以及血清LDH活性的升高幅度 ,抑制心肌和骨骼肌糖原含量及骨骼肌LDH活力的下降。以上结果表明 ,肌酸可改善运动后机体乳酸和糖原的代谢 ,降低运动性疲劳 ,提高大鼠的运动能力     

The inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.

The activity of glycogen synthase phosphatase in rat liver stems from the co-operation of two proteins, a cytosolic S-component and a glycogen-bound G-component. It is shown that both components possess synthase phosphatase activity. The G-component was partially purified from the enzyme-glycogen complex. Dissociative treatments, which increase the activity of phosphorylase phosphatase manyfold, substantially decrease the synthase phosphatase activity of the purified G-component. The specific inhibition of glycogen synthase phosphatase by phosphorylase a, originally observed in crude liver extracts, was investigated with purified liver synthase b and purified phosphorylase a. Synthase phosphatase is strongly inhibited, whether present in a dilute liver extract, in an isolated enzyme-glycogen complex, or as G-component purified therefrom. In contrast, the cytosolic S-component is insensitive to phosphorylase a. The activation of glycogen synthase in crude extracts of skeletal muscle is not affected by phosphorylase a from muscle or liver. Consequently we have studied the dephosphorylation of purified muscle glycogen synthase, previously phosphorylated with any of three protein kinases. Phosphorylase a strongly inhibits the dephosphorylation by the hepatic G-component, but not by the hepatic S-component or by a muscle extract. These observations show that the inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.     

Role of the sympathoadrenal system in the regulation of glycogen metabolism in resting and exercising skeletal muscles.

The purpose of the present study was to characterize the role of catecholamines in the regulation of skeletal muscle glycogen metabolism during exercise. Using the rat hindlimb perfusion technique we have measured skeletal muscle glycogen content, glycogen phosphorylase and synthase activities in sympathectomized and/or demedullated rats under epinephrine treatment (10(-7) M) at rest and during muscle contraction. When epinephrine and/or norepinephrine deficiency was induced, muscle contraction resulted in a decrease in glycogen content (-63%) despite a decrease in glycogen phosphorylase activity ratio (0.25 to 0.11; p less than 0.001) and an increase in glycogen synthase activity ratio (0.13 to 0.27; p less than 0.001). Under these conditions, epinephrine treatment further reduced glycogen content while blunting the changes in the activity ratio of the rate-limiting enzymes. These data indicate that catecholamines do not play a primary role in skeletal muscle glycogen breakdown during acute exercise and suggest that allosteric regulators may be of prime importance.     

Comparative structural analyses of purified glycogen particles from rat liver,human skeletal muscle and commercial preparations

Glycogen is a cellular energy store that is crucial for whole body energy metabolism, metabolic regulation and exercise performance. To understand glycogen structure we have purified glycogen particles from rat liver and human skeletal muscle tissues and compared their biophysical properties with those found in commercial glycogen preparations. Ultrastructural analysis of commercial liver glycogens fails to reveal the classical α-rosette structure but small irregularly shaped particles. In contrast, commercial slipper limpet glycogen consists of β-particles with similar branching and chain lengths to purified rat liver glycogen together with a tendency to form small α-particles, and suggest it should be used as a source of glycogen for all future studies requiring a substitute for mammalian liver glycogen.     

Tissue kallikrein and bradykinin do not have direct insulin-like actions on skeletal muscle glucose utilization

Studies suggest that the actions of insulin on glucose metabolism may be mediated through activation of a membrane-bound serine protease with properties similar to a kallikrein-like enzyme. Also, bradykinin, a vasoactive product of kallikrein's action upon kininogen substrates, increases glucose uptake when infused into the human forearm. To determine whether a kallikrein or a kinin directly affects cellular glucose metabolism or participates in mediating insulin's actions, we studied their effects on isolated rat soleus muscle. Although trypsin (1.34 microM) increased incorporation of glucose into muscle glycogen to the same extent as insulin (200 mu units/ml), a purified rat tissue (urinary) kallikrein (0.4-1.34 microM) produced no such effect. Furthermore, the tissue kallikrein inhibitor, aprotinin, or a polyclonal kallikrein antiserum did not inhibit the action of insulin on incorporation of glucose into muscle glycogen. Treatment of the muscle preparation with bradykinin (1nM - 10 microM) did not result in any change in basal or insulin-stimulated (20 - 2000 mu units/ml) entry of glucose into glycogen or the glycolytic pathway. Bradykinin (1nM - 10 microM) also did not influence basal or insulin-stimulated (1000 mu units/ml) initial rates of glucose transport. These studies suggest that the previously observed in vivo effects of bradykinin on peripheral glucose uptake are probably mediated by changes in tissue perfusion rather than direct kinin effects on skeletal muscle, and that the putative membrane serine protease involved in the insulin-effector system is not tissue kallikrein.     

Carbohydrate energy reserves and effects of food deprivation in male and female rainbow trout

We investigated the effects of nutritional state on carbohydrate, lipid, and protein stores in the heart, liver, and white skeletal muscle of male and female rainbow trout. For fed animals we also partitioned glycogen into fractions based on acid solubility. Fish (10-14 months-old, ~400-500 g) were held at 14 °C and either fed (1% of body weight, every other day) or deprived of food for 14 days. Under fed conditions, glycogen was increased 54% in ventricles from males compared with females, and elevated in the liver (87%) and white muscle (70%) in sexually-maturing versus immature males. Acid soluble glycogen predominated over the acid insoluble fraction in all tissues and was similar between sexes. Food deprivation 1) selectively reduced glycogen and free glucose in male ventricles by ~30%, and 2) did not change glycogen in the liver or white muscle, or triglyceride, protein or water levels in any tissues for both sexes. These data highlight sex differences in teleost cardiac stores and the metabolism of carbohydrates, and contrast with mammals where cardiac glycogen increases during fasting and acid insoluble glycogen is a significant fraction. Increased glycogen in the hearts of male rainbow trout appears to pre-empt sex-specific cardiac growth while storage of acid soluble glycogen may reflect a novel strategy for efficient synthesis and mobilization of glycogen in fishes.     

Differential response of rat skeletal muscle glycogen metabolism to testosterone and estradiol.

Although reports on sex steroids have implicated them as promoting protein synthesis and also providing extra strength to the skeletal muscle, it remains unclear whether sex steroids affect glycogen metabolism to provide energy for skeletal muscle functions, since glycogen metabolism is one of the pathways that provides energy for the skeletal muscle contraction and relaxation cycle. The purpose of the current study was to show that testosterone and estradiol act differentially on skeletal muscles from different regions, differentially with reference to glycogen metabolism. To study this hypothesis, healthy mature male Wistar rats (90-120 days of age, weighing about 180-200 g) were castrated (a bilateral orchidectomy was performed to test the significance of skeletal muscle glycogen metabolism in the absence of testosterone). One group of castrated rats was supplemented with testosterone (100 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards). To test whether estradiol has any effect on male skeletal muscle glycogen metabolism 17beta-estradiol (5 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards) was administered to orchidectomized rats. To test whether these sex steroids have any differential effect on skeletal muscles from different regions, skeletal muscles from the temporal region (temporalis), muscle of mastication (masseter), forearm muscle (triceps and biceps), thigh muscle (vastus lateralis and gracilis), and calf muscle (gastrocnemius and soleus) were considered. Castration enhanced blood glucose levels and decreased glycogen stores in skeletal muscle from head, jaw, forearm, thigh, and leg regions. This was accompanied by diminished activity of glycogen synthetase and enhanced activity of muscle phosphorylase. Following testosterone supplementation to castrated rats, a normal pattern of all these parameters was maintained. Estradiol administration to castrated rats did not bring about any significant alteration in any of the parameters. The data obtained suggest a stimulatory effect of testosterone on skeletal muscle glycogenesis and an inhibitory effect on glycogenolysis. Estradiol did not play any significant role in the skeletal muscle glycogen metabolism of male rats.     

A novel glycogen-targeting subunit of protein phosphatase 1 that is regulated by insulin and shows differential tissue distribution in humans and rodents

Stimulation of glycogen-targeted protein phosphatase 1 (PP1) activity by insulin contributes to the dephosphorylation and activation of hepatic glycogen synthase (GS) leading to an increase in glycogen synthesis. The glycogen-targeting subunits of PP1, GL and R5/PTG, are downregulated in the livers of diabetic rodents and restored by insulin treatment. We show here that the mammalian gene PPP1R3E encodes a novel glycogen-targeting subunit of PP1 that is expressed in rodent liver. The phosphatase activity associated with R3E is slightly higher than that associated with R5/PTG and it is downregulated in streptozotocin-induced diabetes by 60-70% and restored by insulin treatment. Surprisingly, although mRNA for R3E is most highly expressed in rat liver and heart muscle, with only low levels in skeletal muscle, R3E mRNA is most abundant in human skeletal muscle and heart tissues with barely detectable levels in human liver. This species-specific difference in R3E mRNA expression has similarities to the high level of expression of GL mRNA in human but not rodent skeletal muscle. The observations imply that the mechanisms by which insulin regulates glycogen synthesis in liver and skeletal muscle are different in rodents and humans.