Stimulation of glycogenolysis by beta adrenergic agonists in skeletal muscle of mice with the phosphorylase kinase deficiency mutation (I strain)

The mechanism by which beta adrenergic agonist stimulate glycogenolysis in intact skeletal muscle was investigated in mice with the phosphorylase kinase deficiency mutation (I strain). Although extracts of I strain diaphragm muscle had only 3.7% of the phosphorylase kinase activity found in extracts of the control strain (C57BL), incubation of I strain hemidiaphragms in Krebs-Ringer bicarbonate buffer with either isoproterenol or epinephrine resulted in a stimulation of the rate of glycogenolysis. In C57BL diaphragms, the EC50 values for isoproterenol and epinephrine were 2 and 14 nM, respectively. With I strain diaphragms, dl-isoproterenol or l-epinephrine stimulated glycogenolysis as a linear function of the log of the drug concentration with no apparent plateau of response up to concentrations of 30 to 40 mugM. For each 10-fold increase in drug concentration, isoproterenol and epinephrine stimulated glycogenolysis in I strain muscles an additional 0.37 to 0.42 mg/g/hr, a slope in the concentration-response relationship of 0.17 and 0.37, respectively, of that measured in C57BL diaphragms at concentrations around the EC50. The highest glycogenolytic response measured in I strain hemidiaphragms (at 40 mugM isoproterenol) was 80% of the maximal catecholamine-stimulated glycogenolysis in C57BL diaphragms. Both 4 nM and 4 mugM isoproterenol, in a concentration-dependent manner, stimulated phosphorylase b to a conversion in I and C57BL diaphragms and increased cyclic adenosine 3':5'-monophosphate (cyclic AMP) concentrations. The glycogenolytic response to 10.1 nM dl-isoproterenol in both I and C57BL diaphragms was blocked by 34 nM l-propranolol but not by 34 nM d-propranolol. The response to 4 mugM isoproterenol was enhanced by the cyclic nucleotide phosphodiesterase inhibitors papaverine (27 mugM) or dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724, 3 mugM). From the results of these studies, we conclude: 1) Catecholamines stimulate glycogenolysis in skeletal muscle of I mice, as in C57BL mice, by interacting with the beta adrenergic receptor, thereby increasing tissue cyclic AMP concentrations and stimulating phosphorylase b to a conversion. 2) alternative hypotheses for the mechanism of the catecholamine-stimulated decrease in glycogen concentration in I skeletal muscle-inhibition of glycogen synthesis, hyposia and 5'-AMP stimulation of phosphorylase b activity-have been ruled out. 3) the activity of the mutant phosphorylase kinase, although it is only 3.7% of that in extracts of C57BL muscle, is sufficient to produce phosphorylase b to a conversion and thereby account for the glycogenolytic response of I strain muscle to catecholamines.     

Utilization of 1-14C- glucose, 1-14C-acetate and 1-14C-palmitate during lipid synthesis in vitro in the liver and skeletal muscles of pigs at different ontogenic stages

The specific radioactivity of lipids synthesized in the liver and quadriceps muscle of 110-day embryos, and 1-, 30-, 60-, 90-day and 12-month pigs in vitro from [1-14C]acetate, [1-14C] glucose and [1-14C] palmitate was investigated. The lipid synthesis from most of the above compounds in the fetal liver developed at a greater rate and in the fetal skeletal muscles at a smaller rate than in livers and skeletal muscles of newborn piglets. During the first months after birth the lipid synthesis from [1-14C] acetate, [1-14C] glucose and [1-14C] palmitate in the liver increased gradually. At the same time the lipid synthesis in skeletal muscles from [1-14C]glucose and [1-14C]palmitate decreased and from [1-14C] acetate increased. The utilization of the above compounds in the lipid synthesis of the liver and quadriceps muscle of pigs follows the decreasing order: [1-14C] palmitate greater than [1-14C]acetate greater than [1-14C]glucose.     

The role of the growth hormone/insulin-like growth factor I axis in stimulation of protein synthesis in skeletal muscles following oral refeeding

The mechanisms behind stimulation of protein synthesis in skeletal muscles following oral feeding are not well understood. Previous research has not confirmed that insulin is a major factor behind this stimulation. In the present study we have used genetically altered mice, with either a lack of GH secretion due to a mutational gene inactivation [GH (-/-) dwarf, DW/JOrlBom-dw] or mice with a homozygous site-specific insertion mutation in the insulin-like growth factor-1 gene [IGF-I (m/m)], leading to a deficient IGF-I production. These gene knock-outs were used in comparison to their normal wild types for evaluation of the role that the GH/IGF-I axis may have in activation of nutritionally induced stimulation of protein synthesis in skeletal muscles during oral refeeding. Weight stable adult C57B16 mice served as an additional normal control group. Protein synthesis was measured by a modified flooding dose technique with radioactive L-[14C-U]phenylalanine incorporation into acid precipitated muscle proteins. Fractional protein synthesis in skeletal muscles after an overnight fast was comparable among C57B16 (0.076 +/- 0.009%/h), wild-type IGF-I(+/+) (0.061 +/- 0.008) and IGF-I(m/m) deficient mice (0.068 +/- 0.006%/h), whereas GH(-/-) incompetent mice had a lower fractional synthesis rate compared with GH(+/+) competent mice (0.045 +/- 0.006 vs. 0.068 +/- 0.007, P < 0.05). Refeeding with standard chow diet stimulated protein synthesis in muscles by more than 60% in all animal groups. This response was independent of circulating GH, total IGF-I concentrations in blood, as well as up-regulation of locally produced IGF-I messenger RNA (mRNA) in skeletal muscles.     

Effect of caloric restriction on basal insulin levels and the in vivo lipogenesis and glycogen synthesis from glucose in the Koletsky obese rat

Fasting plasma immunoreactive insulin levels increased with age in hyperinsulinemic Koletsky obese rats, being almost four times as high as in lean siblings at 3 mo (40 +/- 5 muU/ml) and rising steadily to 82 +/- 4 muU/ml at 6 mo (about seven times higher than lean siblings). Restricting the food intake of the obese rats markedly reduced but did not normalize the hyperinsulinemia, which in these rats was accompanied by normal plasma glucose concentrations. The incorporation in vivo of D-U-14C-glucose into tissue lipids and glycogen was measured 1 hr after the intravenous injection of 1 g glucose (containing 100 muDi D-U-14C-glucose) per kg body weight in obese rats eating ad libitum, obese rats after 3 mo on a restricted food intake, and lean siblings. All tissues (heart, diaphragm, skeletal muscle, and adipose tissues and liver) of obese rats exhibited a significantly greater lipogenesis from glucose than those of lean siblings. Dietary restriction of the obese rats reduced the 14C incorporation into lipid to levels not significantly different from lean controls in all tissues except skeletal muscle and liver, where, although greatly reduced, lipogenesis was still significantly higher than in lean rats. Glycogen synthesis tended to be greater in all tissues of obese rats than in lean animals. Dietary restriction of obese rats did not greatly affect glycogen synthesis.     

Glucosamine metabolism in regenerating rat liver

1. Glycoprotein synthesis was investigated with [1-14C]glucosamine in vivo. [14C]Glucosamine was administered intravenously 24h after hepatectomy to rats. 2. Incorporation into the acid-soluble fraction was maximum at 15 min after injection both in sham-operated and hepatectomized rats. 3. Enhancement of incorporation into UDP-N-acetylhexosamine in regenerating liver was observed. However, its specific activity was lower, because of a greater enhancement of synthesis de novo of the amino sugar. 4. In the liver acid-insoluble fraction, maximum incorporation of [14C]glucosamine was at 30 min in sham-operated rats and 2 h in hepatectomized rats respectively. 5. In sham-operated rats, incorporation into the plasma acid-insoluble fraction followed that of the liver acid-insoluble fraction, but hepatectomy resulted in a rapid enchancement of incorporation into plasma. 6. It is concluded that synthesis of liver glycoproteins is stimulated after partial hepatectomy and that glycoproteins synthesized are released rapidly into the plasma.     

A case of advanced gastric cancer that responded to long-term administration of low-dose 5''-DFUR]

Muscle glycogen synthesis is modulated by physiologically relevant changes in cell volume. We have investigated the possible involvement of integrin-extracellular matrix interactions in this process using primary cultures of rat skeletal muscle subject to hypo- or hyper-osmotic exposure with integrin binding peptide GRGDTP to disrupt integrin actions and the inactive analogue GRGESP as control. Osmotically induced increases (77%) and decreases (34%) in glycogen synthesis (D-[14C]glucose incorporation into glycogen) were prevented by GRGDTP (but not GRGESP) without affecting glucose transport. Cytoskeletal disruption with cytochalasin D or colchicine had similar effects to GRGDTP. Osmotically induced modulation of muscle glycogen synthesis involves integrin-extracellular matrix interactions and cytoskeletal elements, possibly as components of a cell-volume 'sensing' mechanism.     

Postmaturity in the rat: glucose metabolism in the fetus and the neonate

The present study was designed to investigate glucose metabolism in the postmature fetus and newborn. In the fetus, the decreased hepatic glycogen content together with the decrease by the same percentage of total hepatic glycogen radioactivity from directly injected [6-3H]glucose demonstrate that fetal glycogenolysis occurs during prolonged gestation. Moreover, fetal glycogen synthesis as tested by in vivo [6-3H]glucose incorporation experiments is inhibited. In vivo experiments with [14C]lactate are consistent with gluconeogenesis, being inactive in the postmature fetus as well as in the normal-term fetus. During the first hr after delivery, our in vivo data about conversion of [14C]lactate to glucose show that the gluconeogenic pathway is not functioning in spite of very high phosphoenolpyruvate carboxykinase activity in the postmature. By 3 hr postpartum, the phosphoenolpyruvate carboxykinase activity, the blood lactate level, the percentage of conversion, and the rate of gluconeogenesis are very elevated in the postmatures as compared to the term neonates. By 6 hr postpartum, despite maintained phosphoenolpyruvate carboxykinase activity, gluconeogenic rate becomes very weak in postmatures kept fasting. This is the time characterized by a profound hypoglycemia. In contrast, fed postmature neonates exhibit normal blood glucose levels by 6 and 12 hr postpartum as a result of sustained rate of gluconeogenesis.     

Peripheral but not hepatic insulin resistance in mice with one disrupted allele of the glucose transporter type 4 (GLUT4) gene

Glucose transporter type 4 (GLUT4) is insulin responsive and is expressed in striated muscle and adipose tissue. To investigate the impact of a partial deficiency in the level of GLUT4 on in vivo insulin action, we examined glucose disposal and hepatic glucose production (HGP) during hyperinsulinemic clamp studies in 4-5-mo-old conscious mice with one disrupted GLUT4 allele [GLUT4 (+/-)], compared with wild-type control mice [WT (+/+)]. GLUT4 (+/-) mice were studied before the onset of hyperglycemia and had normal plasma glucose levels and a 50% increase in the fasting (6 h) plasma insulin concentrations. GLUT4 protein in muscle was approximately 45% less in GLUT4 (+/-) than in WT (+/+). Euglycemic hyperinsulinemic clamp studies were performed in combination with [3-3H]glucose to measure the rate of appearance of glucose and HGP, with [U-14C]-2-deoxyglucose to estimate muscle glucose transport in vivo, and with [U-14C]lactate to assess hepatic glucose fluxes. During the clamp studies, the rates of glucose infusion, glucose disappearance, glycolysis, glycogen synthesis, and muscle glucose uptake were approximately 55% decreased in GLUT4 (+/-), compared with WT (+/+) mice. The decreased rate of in vivo glycogen synthesis was due to decreased stimulation of glucose transport since insulin's activation of muscle glycogen synthase was similar in GLUT4 (+/-) and in WT (+/+) mice. By contrast, the ability of hyperinsulinemia to inhibit HGP was unaffected in GLUT4 (+/-). The normal regulation of hepatic glucose metabolism in GLUT4 (+/-) mice was further supported by the similar intrahepatic distribution of liver glucose fluxes through glucose cycling, gluconeogenesis, and glycogenolysis. We conclude that the disruption of one allele of the GLUT4 gene leads to severe peripheral but not hepatic insulin resistance. Thus, varying levels of GLUT4 protein in striated muscle and adipose tissue can markedly alter whole body glucose disposal. These differences most likely account for the interindividual variations in peripheral insulin action.     

Leptin increases glucose transport and utilization in skeletal muscle in vitro

1. The present study examines the effect of leptin on glucose transport and metabolism in incubated soleus muscle from male lean albino rats. 2. Insulin (100 microU/ml) increased glucose uptake by twofold while the leptin group (100 nmol/l) reached 75% of the insulin response after 1 hr of incubation. However, leptin did not potentiate the insulin effect on glucose uptake in soleus muscle. 3. Leptin elicited a significant increase (27.7%) in total lactate production, accompanied by a three-fold increment in glycogen synthesis from [U-14C]D-glucose. 4. Insulin raised glycogen synthesis by sixfold. The leptin plus insulin group increased glycogen synthesis by eightfold, which is equivalent to the sum of the separated leptin and insulin groups. 5. Leptin per se exerts an insulin-like effect stimulating glucose uptake, glycogen synthesis, and lactate formation and also seems to potentiate the effect of insulin on glucose incorporation into glycogen in incubated soleus muscle.     

Islet amyloid polypeptide decreases the effects of insulin-like growth factor-I on glucose transport and glycogen synthesis in skeletal muscle

Previous studies have shown that islet amyloid polypeptide (IAPP) is co-secreted with insulin from the beta-cell. IAPP reduces insulin-stimulated rates of glycogen synthesis in skeletal muscle but the mechanisms are unclear. Insulin-like growth factor I (IGF-I) is an important regulator of glucose metabolism in skeletal muscle and acts through its own receptor, which has many structural and functional similarities with the insulin receptor. Despite this, the effects of IGF-I on glucose utilization are not identical to those of insulin. The aim of the study was to determine the effects of IAPP on IGF-I-stimulated rates of glucose transport and metabolism (measured by 3-O-methyl[3H]glucose and [U-14C]glucose, respectively) in rat soleus muscle, and compare them with those simulated by insulin. IAPP (10 nM) decreased the sensitivity of 3-O-methylglucose transport, the flux of glucose to hexosemonophosphate and the sensitivity of glycogen synthesis to IGF-I. In contrast, IAPP had no effect on IGF-I-stimulated rates of lactate formation (i.e., glycolysis). IAPP decreased the sensitivity of 3-O-methylglucose transport and glycogen synthesis to insulin. It is concluded that IAPP blunts the stimulation of glucose uptake and deposition by IGF-I or insulin in skeletal muscle. These observations expand those made initially for IAPP and insulin and suggest that IAPP affects IGF-I- or insulin-stimulated glucose metabolism in muscle by a mechanism which is common for both hormones. These experiments may serve as a framework for future studies in order to clarify the mechanisms by which IAPP affects glucose metabolism in skeletal muscle.     

The forensic medical assessment of the mechanisms of trauma to the large joints of the upper extremity

Glycogen synthesis and degradation were studied in cultured rat hepatocytes prelabeled by incubation with [14C]glucose or [14C]galactose. During prelabeling about 75% of the accumulated glycogen was synthesized from glucose and about 25% from gluconeogenic precursors. Following the labeling period, glycogen synthesis and degradation were estimated at 5 and 12.5 mM glucose and varying concentrations of insulin and glucagon. At 12.5 mM glucose and 10 nM insulin the accumulation of glycogen was comparable to in vivo values, whereas the level of radioactivity in prelabeled glycogen remained constant. Further addition of 0.1 nM glucagon resulted in constant values of both content and radioactivity of glycogen. Increasing the concentration of glucagon to 10 nM resulted in a parallel decrease of content and radioactivity in glycogen. At 5 mM glucose, 10 nM insulin, and 0.1 nM glucagon both the content and the radioactivity of glycogen were constant, whereas addition of 10 nM glucagon resulted in a parallel decrease of content and radioactivity of glycogen, which was 64% higher than that observed with 12.5 mM glucose. In the absence of insulin, prostaglandin D2 had effects similar to those of 10 nM glucagon, whereas no effects was observed in the presence of insulin. From these results and from calculated rates of glucose 6-phosphate formation, it is concluded that the rate of glycogen degradation is less than 10% of the rate of synthesis under conditions favoring glycogen accumulation. At conditions favoring glycogen degradation (10 nM insulin plus 10 nM glucagon or prostaglandin in the absence of insulin) no synthesis could be detected. Results from cells prelabeled with [14C]galactose suggested that glycogen degradation is not an absolutely ordered process, but that some random degradation takes place.     

False-positive gray-scale ultrasonography for intra-abdominal abscesses

Rats were subjected to a strenuous program of treadmill running for 2.5--3 months. Ubiquinone content was determined in the skeletal muscles and the muscle mitochondria; at the same time incorporation of labeled acetate-1-14C precursor into ubiquinone and sterols by thin muscle slices was studied. Ubiquinone and mitochondrial protein content increased in the muscles of trained rats. Incorporation of 14C into ubiquinone of trained rats increased; as to sterols, their incorporation remained unchanged.     

Mechanisms of insulin resistance during acute endotoxemia

We characterized the mechanisms underlying acute endotoxin-induced alterations in glucose metabolism and determined the extent to which catecholamines mediate these changes. Acute endotoxemia was induced in chronically catheterized awake rats by a bolus injection of lipopolysaccharide (LPS; 1 mg/kg; LD10). Basal glucose turnover (Rt; infusion of [5-3H]glucose), in vivo insulin action on overall glucose utilization (euglycemic clamp), glycolysis, and glycogen synthesis were determined in four groups of rats. These groups received 1) LPS (LPS rats; n = 6), 2) saline (control rats; n = 6), 3) LPS and alpha beta-blockade (alpha beta-blockade and LPS rats; n = 9), or 4) saline and alpha beta-blockade (alpha beta-blockade control rats; n = 9). In the basal state, LPS induced hypotension and transient hyperglycemia. These changes were associated with glycogen depletion in both skeletal muscle and liver, and increased Rt. During hyperinsulinemia, whole body glucose disposal was 37% decreased (105 vs. 166 mumol/kg.min; P < 0.01). This whole body insulin resistance was characterized by decreased glycogen synthesis and glycogen synthase activity, but not by altered whole body glycolysis. alpha beta-Blockade abolished transient hyperglycemia, increased Rt, and accelerated basal liver glycogen depletion (45 vs. 105 mmol/kg dry, LPS and alpha beta-blockade rats vs. LPS rats; P < 0.05), but inhibited muscle glycogenolysis. alpha beta-Blockade did not reverse the insulin resistance induced by endotoxin. These data suggest that catecholamines counteract the LPS-induced increase in basal glucose turnover and stimulate muscle glycogenolysis during acute endotoxemia. These effects might explain the better preservation of hepatic glycogen in the absence than in the presence of alpha beta-blockade and serve as a defense mechanism against hypoglycemia. Catecholamines do not seem to be the immediate causes of insulin resistance during acute endotoxemia.     

Skeletal muscle metabolism in two Pavulon treated tetanus patients

In two patients with severe tetanus the skeletal muscle metabolism was studied. Muscle biopsies were taken 4-6 weeks after the development of tetanus. The patients were treated during this time with a neuromuscular blocking agent. In one of the patients a biopsy was also taken before treatment, 2 days after the first symptoms of the disease. Enzyme activities representative for glycogen synthesis, glycogen greakdown, glycolysis, fatty acid beta-oxidation and respiratory chain, and fluxes of glucose, palmitate and leusine in vitro, and the concentrations of glycogen, triglycerides, phospholipids, cholesterol, proteins and RNA were determined in the muscle tissue. The enzyme activities in the muscle tissue examined 2 days after the development of tetanus were close to normal, excep for decreased activities of phosphofructokinase and glycogen synthase. After 4-6 weeks of treatment the fluxes of glucose and palmitate and the corresponding enzyme activities were low in both patients. These findings are consistent with muscular inactivity. In contrast the activity of glucose-6-phosphate dehydrogenase, the incorporation rate of leucine-carbon into proteins and the RNA content were high, indicating a high protein synthesis as a probable manifestation of active repairative processes. It is concluded that the skeletal muscle metabolism in these patients was affected in a degenerative manner and that this effect was attributed more to the treatment than to the disease per se. Dynamic physical training under supervision is recommended for the period after the generalized spasms to overcome the "disuse" effects in the skeletal muscles.     

Stress urinary incontinence in women. II. Abnormalities of glycogenolysis in tissues related to the lower urinary tract

The aim of the study was the investigation of the biochemical condition of elements likely to directly participate in active closing of the urethral lumen. We estimated glycogenolysis in urinary bladder, perivesical connective tissue and levator ani muscle (LAM) samples obtained intraoperatively from 80 stress incontinent women. Glycogen content as well as activities of active and total glycogen phosphorylase and acid exo-1,4-alpha-glucosidase were measured. Material from the urinary bladder and perivesical connective tissue was insignificantly altered, and glycogen contents in the bladder (2.03 +/- 1.38 g/100 g wet tissue) were considered to be normal. In the LAM glycogenolysis was much more activated than in other tissues (p < 0.001 by Fischer's exact test). Of LAM specimens 78% (22/28) revealed imbalanced biochemistry of glycogen with activation of hydrolytic decomposition. We conclude that stress urinary incontinence in women is frequently associated with metabolic alterations in the periurethral striated fibres. This study indirectly supports our recent hypothesis on the pathogenesis of the disease in terms of muscle fibre type transitions.     

Prolonged exposure to halothane and associated changes in carbohydrate metabolism in rat muscles in vivo

Halothane, an anesthetic presently used in animal experimentation, is reported to stimulate glycogen breakdown in isolated preparations of rat skeletal muscles, suggesting that it may not be a suitable anesthetic for the study of glycogen metabolism in rats in vivo. The purpose of this study was to establish whether prolonged exposure to halothane in rats in vivo is associated with accelerated glycogenolysis. Exposure of rats to halothane for up to 1 h was not accompanied by either any change in the levels of glycogen or increase in activity ratios of glycogen phosphorylase in muscles, irrespective of their fiber compositions. In marked contrast, the levels of lactate, inorganic phosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-bisphosphate, and fructose 2, 6-bisphosphate changed progressively during anesthesia. Accordingly, the interpretation of muscle metabolite levels must be performed with caution in experiments involving prolonged exposure to halothane. Overall, our findings indicate that the reported halothane-mediated stimulation of glycogen breakdown in vitro is likely to be an artifact and that halothane is a suitable anesthetic for experiments concerned with glycogen metabolism in rats.     

Effect of endurance exercise training on muscle glycogen supercompensation in rats

The purpose of this study was to test the hypothesis that the rate and extent of glycogen supercompensation in skeletal muscle are increased by endurance exercise training. Rats were trained by using a 5-wk-long swimming program in which the duration of swimming was gradually increased to 6 h/day over 3 wk and then maintained at 6 h/day for an additional 2 wk. Glycogen repletion was measured in trained and untrained rats after a glycogen-depleting bout of exercise. The rats were given a rodent chow diet plus 5% sucrose in their drinking water and libitum during the recovery period. There were remarkable differences in both the rates of glycogen accumulation and the glycogen concentrations attained in the two groups. The concentration of glycogen in epitrochlearis muscle averaged 13.1 +/- 0.9 mg/g wet wt in the untrained group and 31.7 +/- 2.7 mg/g in the trained group (P < 0.001) 24 h after the exercise. This difference could not be explained by a training effect on glycogen synthase. The training induced approximately 50% increases in muscle GLUT-4 glucose transporter protein and in hexokinase activity in epitrochlearis muscles. We conclude that endurance exercise training results in increases in both the rate and magnitude of muscle glycogen supercompensation in rats.     

Significant amounts of glycogen are synthesized from 3-carbon compounds in astroglial primary cultures from mice with participation of the mitochondrial phosphoenolpyruvate carboxykinase isoenzyme

The incorporation was studied of the gluconeogenic substrates lactate, alanine, aspartate and glutamate into glycogen of astroglial primary cultures derived from mouse brain. The incorporation was inhibited by 3-mercaptopicolinate, an inhibitor of one of the characteristic gluconeogenic enzymes, phosphoenolpyruvate carboxykinase. Only the mitochondrial isoenzyme of phosphoenolpyruvate carboxykinase was detectable in the astroglial primary cultures. After the incubation of glucose-starved cells with medium containing a mixture of [6-3H]glucose and [U-14C]glucose, the newly synthesized glycogen showed a 3H/14C ratio which was approximately 15% less than the isotope ratio for the medium. The decrease of the isotope ratio was not significantly inhibited by 3-mercaptopicolinate, indicating a cycling of approximately 15% of the glucose to the level of the triose phosphates before its incorporation into astroglial glycogen. During the initial phase of glycogen resynthesis, the contribution of the gluconeogenic substrates appeared to be higher. This was in agreement with the accumulation of fructose 2,6-bisphosphate during refeeding. A participation of gluconeogenic substrates in glycogen metabolism was also detectable when the glycogen content was not changing significantly.     

Interleukin 1beta and interleukin 6, but not tumor necrosis factor alpha, inhibit insulin-stimulated glycogen synthesis in rat hepatocytes

Recent evidence indicates that inflammatory cytokines are involved in changes of blood glucose concentrations and hepatic glucose metabolism in infectious diseases, including sepsis. However, little is known regarding how cytokines interact with glucoregulatory hormones such as insulin. The objective of the present study is to investigate if and how cytokines influence insulin-stimulated glycogen metabolism in the liver. Interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) markedly inhibited the increase of glycogen deposition stimulated by insulin in primary rat hepatocyte cultures; however, tumor necrosis factor alpha had no effect. Labeling experiments revealed that both cytokines counteracted insulin action by decreasing [14C]-glucose incorporation into glycogen and by increasing [14C]-glycogen degradation. Furthermore, it was discovered that IL-1beta and IL-6 inhibited glycogen synthase activity and, in contrast, accelerated glycogen phosphorylase activity. In experiments with kinase inhibitors, serine/threonine kinase inhibitor K252a blocked IL-1beta- and IL-6-induced inhibitions of glycogen deposition, as well as glycogen synthase activity, whereas another kinase inhibitor staurosporine blocked only IL-6-induced inhibition. Tyrosine kinase inhibitor herbimycin A blocked only IL-1beta-induced inhibition. These results indicate that IL-1beta and IL-6 regulate insulin-stimulated glycogen synthesis through different pathways involving protein phosphorylation in hepatocytes. They may mediate the change of hepatic glucose metabolism under pathological and even physiological conditions by modifying insulin action in vivo.     

Ryanodine binding sites measured in small skeletal muscle biopsies

A method allowing measurement of the concentration of [3H]ryanodine binding sites in small skeletal muscle specimens (> 10-20 mg) was developed. A membrane fraction containing 87% of the [3H]ryanodine binding sites of the tissue and exhibiting one single KD of 18-27 nmol l-1 in rat and 8 nmol l-1 in human muscles (p < 0.05) was obtained. Maximum binding to rat EDL and soleus muscles equalled 59.1 and 16.2 pmol g-1 wet wt, whereas in human gluteus muscles binding was 12.3 pmol g-1 wet wt. The [3H]ryanodine binding showed a dependency on Mg2+ and pH similar to previously published results. As measured by Ca2+ selective mini-electrodes, the [Ca2+] causing 50% of maximum [3H]ryanodine binding (K0.5) was 200-400 nmol l-1 for different muscles. [Ca2+] higher than 1 mmol l-1 caused strong inhibition of the [3H]ryanodine binding, and both high and low [Ca2+] caused rapid dissociation of the complex. At ionic strength lower than 100 mmol l-1, more than 50% of the [3H]ryanodine was bound to particles with size less than 1.2 microns which were not retained by GF/C filters. Thus, we have obtained an almost complete quantitative recovery of functional RyRs from small muscle specimens exhibiting high affinity for Ca2+, which stimulated ligand binding.